Eur J Nucl Med Mol Imaging (2010) 37:386–398
DOI 10.1007/s00259-009-1272-0
REVIEW ARTICLE
Molecular imaging of rheumatoid arthritis by radiolabelled
monoclonal antibodies: new imaging strategies to guide
molecular therapies
G. Malviya & F. Conti & M. Chianelli & F. Scopinaro &
R. A. Dierckx & A. Signore
Received: 11 August 2009 / Accepted: 25 August 2009 / Published online: 24 September 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract The closing of the last century opened a wide
variety of approaches for inflammation imaging and
treatment of patients with rheumatoid arthritis (RA). The
introduction of biological therapies for the management of
RA started a revolution in the therapeutic armamentarium
with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised
and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion
molecules, activation markers, antigens or receptors, to
interfere with specific inflammation pathways at the
molecular level, leading to immune-modulation of the
underlying pathogenic process. These new generation of
mAbs can also be radiolabelled by using direct or
indirect method, with a variety of nuclides, depending
upon the specific diagnostic application. For studying
rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, antiG. Malviya : M. Chianelli : R. A. Dierckx : A. Signore
Department of Nuclear Medicine and Molecular Imaging,
University Medical Centre Groningen, University of Groningen,
Groningen, The Netherlands
F. Conti
Rheumatology Unit, I Faculty of Medicine and Surgery,
“Sapienza” University of Rome,
Rome, Italy
M. Chianelli
Unit of Nuclear Medicine, Regina apostolorum Hospital,
Albano, Rome, Italy
F. Scopinaro : A. Signore (*)
Nuclear Medicine Department, “Sapienza” University of Rome,
St. Andrea Hospital, Via di Grottarossa 1035,
00189 Rome, Italy
e-mail: alberto.signore@uniroma1.it
CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody,
have been radiolabelled mainly with 99mTc or 111In.
Scintigraphy with these radiolabelled antibodies may offer
an exciting possibility for the study of RA patients and
holds two types of information: (1) it allows better staging
of the disease and diagnosis of the state of activity by
early detection of inflamed joints that might be difficult to
assess; (2) it might provide a possibility to perform
‘evidence-based biological therapy’ of arthritis with a view
to assessing whether an antibody will localise in an inflamed
joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection
of patients to be treated since biological therapies can be
associated with severe side-effects and are considerably
expensive. This article reviews the use of radiolabelled mAbs
in the study of RA with particular emphasis on the use of
different radiolabelled monoclonal antibodies for therapy
decision-making and follow-up.
Keywords Monoclonal antibodies . Rheumatoid arthritis .
Molecular imaging . Biological therapies .
Therapy decision-making
New molecular therapies for rheumatoid arthritis
Rheumatoid arthritis (RA) is a chronic inflammatory
autoimmune disease of unknown aetiology that leads to
progressive joint destruction, functional disability and extra
articular complications. RA is a systemic disease that
affects many organ systems and is associated with
abnormally high rates of other associated diseases including
malignancies, infections and cardiovascular diseases [1–5].
The genetic background predisposes to the disease, but also
person-related and environmental factors (like age, gender,
Eur J Nucl Med Mol Imaging (2010) 37:386–398
387
infectious agents, smoking and dietary factors) are thought
to play a role in disease pathogenesis. The prevalence of
RA globally is around 1%. Recently, it has been shown that
50% of patients with RA are disabled within 10 years of
onset of disease and survival is reduced [6–8]. Over the last
decades the strategy of treating RA has changed, and now
the mainstays of RA therapy are disease modifying antirheumatic drugs (DMARDs) such as azathioprine, hydroxychloroquine, sulphasalazine, cyclosporin A, leflunomide
and methotrexate; the last of these has generally become the
standard of care. Many of these medications reduce
radiological progression of structural damage [9]. An earlier
use of DMARDs reduces the risk of joint damage in future
[10]. Several studies demonstrated that an imbalance occurs
in cytokine cascade resulting in presence of proinflammatory cytokines in synovium and plasma, which
leads to joint inflammation and cartilage destruction
characteristic of RA. Attention has therefore been given to
develop inhibitors of pro-inflammatory cytokines and their
receptors. The clinical management of RA has been
revolutionised by introduction of the ‘biological agents’,
including monoclonal antibody, fusion protein and Fab’
fragment of monoclonal antibody. These biologicals target
pro-inflammatory cytokines (e.g., TNF-α, IL-1 and IL-6)
and membrane-bound receptors (e.g., CD3 and CD4) for
treatment of patients [11, 12]. This new class of drugs has
been termed ‘biologic response modifiers’. Nevertheless,
treatment with biologicals is expensive, especially in the
case of long treatments such as in RA.
A pre-therapy scintigraphic approach with radiolabelled
monoclonal antibodies (mAbs) may allow us to evaluate the
presence of the target molecules in the inflammatory lesion,
thus helping in the selection of the most efficient therapy
and predicting therapy response; overall, they may provide
a cost-effective solution [13]. Additionally, for scintigraphic
purpose, patients receive a tracer (non-pharmacological)
dose of radiolabelled mAb, which does not induce any
clinical response or side-effect.
This article will review the use of radiolabelled mAbs in
the study of RA (Table 1) with particular emphasis on the
use of different radiolabelled monoclonal antibodies for
therapy decision-making and follow-up.
Human antibodies
Antibodies are composed by light (L) and heavy (H) chains.
These chains are covalently linked via interchain disulfide
(S-S) bonds between Cysteine residues and by non-covalent
interactions such as hydrogen bonding, hydrophobic interactions and salt linkage. Similar non-covalent interactions
and disulphide bridges have also been linked to two
identical H and L chain combinations to each other,
forming the basic four-chain (H-L)2 structure of an
antibody. Each antibody has either kappa (κ) or lambda
(1) L chains. Different antibody isotypes IgA, IgG, IgD,
IgE and IgM are named according to their H chain types
(α, γ, δ, ε or μ), which influences the effecter functions of
the antibody molecules. IgG are most frequent monoclonal
antibodies and have a Y-shaped structure with a molecular
weight of approximately 150,000 Daltons (Da).
Therapeutic antibodies
In 1975, Kohler and Milstein first reported production of
murine monoclonal antibodies using hybridoma technology
[14], and by 1980 mAbs were already involved in the
human studies. OKT3 (Orthoclone, Muromonab) is one of
the first drug in the class of monoclonal antibodies
indicated for the treatment of autoimmune diseases. Murine
monoclonal antibodies are 100% murine protein therefore
they can induce several major side-effects. Mouse antibodies are recognised as an antigen by the human immune
system that generates its own human anti-mouse antibodies
(HAMA). These HAMA may inactivate and eliminate
murine antibodies after repeated administration [15]. For
this reason, therapeutic benefits of murine mAbs could be
limited by their side-effect profile and short serum half-life
[16]. In 1984, Morrison et al. first demonstrated the
production of chimeric antibody composed of mouse
antigen-binding domain and human constant region domain
[17]. A chimeric monoclonal antibody is a combination of
mouse and human gene sequences, roughly 25 and 75%,
respectively. These mAbs had minimized immunogenic
content, triggered the immunologic efficiency and allowed
a prolonged serum half-life in comparison with murine
Table 1 Molecular imaging of rheumatoid arthritis by radiolabelled mAbs
Monoclonal antibody
Company
Type
Class
Isotope
Target
Ref.
Infliximab (Remicade)
Adalimumab (Humira)
Rituximab (Rituxan/Mabthera)
MAX.16H5
1.2B6
OKT-3 (Muromonab)
Centocor, Inc.
Abbott Labs
Genentech/Roche
–
–
Ortho Pharma
Chimeric
Fully human
Chimeric
Murine
Murine
Murine
IgG1
IgG1
IgG1
IgG1
IgG1
IgG2
99m
TNF-α
TNF-α
CD20
CD4
E-selectin
CD3
13, 34, 35
40, 41
49, 51, 52
57–59
67–69, 73, 74
83, 84
Tc
Tc
99m
Tc
99m
Tc
111
In
99m
Tc
99m
388
mAbs. Chimeric mAbs such as infliximab (Remicade),
however, also have problems of triggering human antichimeric antibodies (HACA) response [18, 19], that may
reduce the therapeutic benefit and effectiveness of the
therapy. In 1986, Jones et al. reported the production of
humanised monoclonal antibody that could help to overcome the problems of HAMA and HACA responses
discussed above [20, 21]. These genetically engineered
humanised mAbs have approximately 95% of human
sequence that results in minimal or no immunogenic
response [22]. Fully human antibodies have eventually
been produced that contain almost 100% human protein.
The key techniques for the development of fully human
mAbs are the phage display technology and genetically
engineered mice [23–25]. These antibodies, however,
have been considered as ‘fully human’ in the sense that
coding genes do not contain any part from other species.
These antibodies are expected to have a lower therapeutic dose and dosing frequency with a better side-effect
profile. Fab’ fragments have also recently been introduced for the treatment of RA. To obtain a Fab’
fragment, the Fc portion of mAb, which serves to bind
various effecter molecules of immune system, is completely removed. Fab’ fragments cannot induce, therefore, antibody-dependent cellular cytotoxicity (ADCC) or
apoptosis in T lymphocytes or macrophages compared to
complete antibodies [26, 27] and should have, therefore, a
better safety profile.
New imaging strategies as a guide to molecular
therapies
Several monoclonal antibodies have been radiolabelled for
diagnostic purposes in patients with RA and in particular
for therapy decision-making and follow-up. These are
reported below.
Anti-TNF-α monoclonal antibody (infliximab)
In August of 1998, the United States Food and Drug
Administration (FDA) approved infliximab (Remicade) for
the treatment of moderate to severe active rheumatoid
arthritis. Infliximab is a chimeric IgG1κ mAb with a murine
variable (Fv) domain of mouse anti-human TNF-α antibody and constant (Fc) sequences of human IgG1. It is
produced by recombinant cell culture technique. Infliximab
specifically targets and binds with both soluble and
membrane-bound TNF-α with high avidity and affinity
(Kd=1010 M–1) and forms a stable non-dissociating immune
complex [28]. This binding neutralizes the biological
activity of TNF-α by inhibiting the binding of TNF-α to
its receptor [29]. Infliximab does not neutralize TNF-β
Eur J Nucl Med Mol Imaging (2010) 37:386–398
(lymphotoxin α). Infliximab has a median terminal half-life
of 9.5 days. In brief, TNF-α induces the pro-inflammatory
cytokines (such as IL-1 and IL-6) and acute-phase
reactants, activates the function of eosinophils and neutrophils, and enhances the migration of leukocytes by
increasing permeability of endothelial layer along with the
expression of adhesion molecules in synovitis [30]. Studies
have been demonstrated that infliximab may induce cell
lysis in transmembrane TNF-α expressing cells in vitro [29]
and in vivo [31], mediated by ADCC or complementdependent cytotoxicity (CDA) [32, 33].
Molecular imaging with
(infliximab)
99m
Tc-anti-TNF-α mAb
Infliximab was radiolabelled with 99mTc using direct radiolabelling method [34]. In brief, disulfide bridges present in
mAb were reduced by 2-mercaptoethanol (2-ME) and
radiolabelling of activated mAb was performed by using
methylene diphosphonic acid (MDP), as a weak competitive ligand. By using this method, a high labelling
efficiency (LE) of more than 97% was achieved.
Conti et al. successfully performed a scintigraphic study in
a patient with arthritis to assess the degree of TNF-α mediated
inflammation in the affected knee [35]. The patient underwent
scintigraphic examination with 99mTc-labelled infliximab
before and 4 months after the intra-articular infliximab
therapy. After injection of 99mTc-infliximab (555 MBq),
planner images of the inflamed joint were acquired at 6
and 24 h. Scintigraphy showed intense accumulation of
99m
Tc-infliximab in the affected knee that represents the
presence of high level of intra-lesional TNF-α. Interestingly, 4 months after the intra-articular infliximab therapy,
there was no uptake found in the inflamed joint (Fig. 1).
Clinical parameters including visual analogue scale (VAS),
erythrocyte sedimentation rate (ESR) and C-reactive protein
(CRP) level confirmed the complete remission of the patient.
A pilot study was performed in seven RA patients (nine
inflamed joints) using scintigraphy with 99mTc-infliximab
[13]. RA patients underwent 99m-technetium labelled
infliximab scintigraphy before and 3 months after the
intra-articular infliximab therapy. Planar images of arthritic
joints were acquired at 3, 6, and 24 h after 555 MBq
(200 μg) injection of 99mTc-infliximab. In this study, 99mTcinfliximab demonstrated very specific uptake in the
inflamed joints, whereas normal joints did not show any
uptake. Post-treatment scintigraphy examinations demonstrated different amounts of radiopharmaceutical uptake in
inflamed joints. Three inflamed joints showed significant
changes in uptake whereas in other four joints uptake was
only slightly reduced, and it was unchanged in two joints.
Clinical improvement of symptoms and reduction of
swelling were higher in patients with the higher uptake of
Eur J Nucl Med Mol Imaging (2010) 37:386–398
389
Fig. 1 Scintigraphy with
99m
Tc-infliximab before (a) and
4 months after (b) intra-articular
administration of infliximab.
Red colour represents uptake of
the 99mTc-infliximab in
scintigraph. (From Conti F et al.
Arthritis Rheum 2005; 52 (4):
1224–1226, with permission)
99m
Tc-infliximab pre-therapy and decrease of uptake after
therapy.
These preliminary studies in humans have demonstrated
specific targeting of this radiopharmaceutical in inflamed
joints. These studies also showed that the selection of
candidates for unlabelled anti-TNF-α therapy and prediction of therapy response could be possible by using
99m
Tc-infliximab scintigraphy. The mechanism of accumulation at the site of inflammation, however, remains
speculative until now, and more studies are required to
investigate the specific uptake of this radiopharmaceutical.
Moreover, these scintigraphic studies have been performed in a very small number of highly selected patients,
and therefore need to be analysed in a larger series of
patients in multi-centre studies to evaluate the potential
use of this technique in a clinical setting.
Anti-TNF-α monoclonal antibody (adalimumab)
In December of 2002, the FDA approved adalimumab for the
management of moderate to severe active rheumatoid arthritis
and psoriatic arthritis. As the first ‘fully human’ antibody
against TNF-α, adalimumab (Humira) is engineered through
phase display technology. Adalimumab is a recombinant
human monoclonal IgG1 antibody, composed of two
kappa light chains and two IgG1 heavy chains, expressed in
Chinese Hamster Ovary cells. Interestingly, it is less immunogenic than chimeric mAbs such as infliximab [36]. It
recognizes both soluble and membrane-bound TNF-α
with high specificity and high affinity (Kd=6×10-10 M)
and inhibits its biological activity by blocking interaction
of TNF-α with p55 and p75 receptors [37]. Adalimumab
treatment exerts down-regulation of expression of other
pro-inflammatory cytokines, such as IL-6, IL-8 and GMCSF (granulocyte-macrophage colony-stimulating factor)
[38]. This antibody has minimised potential side-effects
and antigenicity of previous chimeric and humanised
mAbs.
Molecular imaging with
(adalimumab)
99m
Tc-anti-TNF-α mAb
Adalimumab was radiolabelled with 99mTc via an indirect
radiolabelling method as described by Abrams et al. [39]. In
brief, succinimidyl-hydrazino nicotinamide (S-HYNIC), a
bifunctional chelator, was conjugated with native mAb,
subsequently, labelling of the conjugated mAb was performed by using tricine as a co-ligand and stannous
chloride as a reducing agent. A high labelling efficiency
(greater than 95%) was achieved by using this method. HPLC
analysis demonstrated only a minimal release of <3% of the
radiolabel after 24 h at 37°C [40]. Adalimumab was also
radiolabelled with 99m-technetium by direct method using
2-ME reduction with a high LE (greater the 95%) [41].
Barrera et al. performed a scintigraphic imaging study in
ten patients with active RA to assess the sensitivity and
biodistribution of i.v. administered 99mTc-adalimumab [40].
Each patient underwent two scintigraphic examinations,
first to assess biodistribution of the radiolabelled antibody
and second after 2 weeks to assess specificity for TNF-α
targeting and sensitivity for changes in inflammation. Each
patient received a sub-therapeutic intravenous dose of
0.1 mg (740 MBq) of 99mTc-anti-TNF-α mAb. Wholebody and joint-specific images were acquired at 5 min, 4 h,
and 24 h after administration of the radiopharmaceutical.
The results of the first scintigraphy demonstrated that
inflamed joints were clearly visualised at 4 and 24 h after
injection and median increased uptake at 24 h was 30%.
However, not all clinically affected joints (particularly
small) showed uptake of 99mTc-adalimumab, which may be
explained by the absence of this cytokine because TNF-α
may not be always present in inflamed joints. Importantly,
however, no uptake was seen in normal joints. For the
second scintigraphic examination, patients were divided
into two groups. One group received a therapeutic dose
(10 mg/kg) of unlabelled anti-TNF-α mAb immediately
before the 99mTc-anti-TNF-α mAb administration for
390
competition study. Another group received an intramuscular injection of corticosteroid (120 mg) 2 days before the
second scintigraphy to check the sensitivity to reflect
decreased inflammation. Simultaneous injection of unlabelled TNF-α mAb has reduced the joint uptake of 99mTcTNF-α mAb by a median of 25% as a percentage of
injected doses after 24 h. This approach has proved the in
vivo specificity of this radiolabelled mAb to target TNF-α
in arthritic joints. In another group of patients, systemic
corticosteroid administration may reduce the disease activity,
which caused decreased uptake of the radiopharmaceutical.
This well-designed study clearly demonstrated that this
radiopharmaceutical may also be used for the detection of
clinically relevant changes in disease activity.
A pilot study was also performed by our group for therapy
decision-making and follow-up with 99m-technetium
labelled infliximab and adalimumab mAbs in RA patients
[41]. Twelve and nine patients with active RA underwent
scintigraphic examination with 99m Tc-infliximab and
99m
Tc-adalimumab, respectively. Imaging was performed
before and 3 months after intra-articular therapy with
infliximab or systemic therapy with adalimumab. After
injection of 370 MBq 99mTc-infliximab or 99mTc-adalimumab, planar anterior and posterior images of arthritic
joints were acquired at 6 and 20 h p.i., and target-tobackground (T/B) ratio was calculated in all affected joints
(Fig. 2). In RA patients, no differences of biodistribution
were observed between these two radiopharmaceuticals. A
variable degree of joint uptake (T/B ratio ranged from 1 to 4)
has been observed, which was not always correlated with
joint pain or swelling. After the therapy with unlabelled antiTNF-α mAb, if the patient demonstrated reduction in the
joint uptake of 99mTc-anti-TNF-α mAb, it was also correlated with the reduction of clinical symptoms. Interestingly,
patients that showed high pre-therapy uptake had more
therapeutic benefit than patients who showed less uptake in
the inflamed joints before therapy. Scintigraphic scores
assigned to the patients with active RA on the basis of
99m
Tc-anti-TNF-α mAb scintigraphy were found to be very
reliable for disease monitoring and therapy decision-making.
In the same study, we were also able to perform scintigraphy
with 99m-technetium labelled non-specific immunoglobulin
(HIG) in two patients, to compare it to 99mTc-anti-TNF-α
mAb scintigraphy for follow-up of the disease activity.
99m
Tc-HIG scintigraphy was not able to demonstrate the
reduction in the joints uptake after the treatment with cold
anti-TNF-α mAb, which was confirmed by the clinical
data. This study concluded that 99mTc-TNF-α antibodies
could be used for therapy decision-making in patients with
active RA being predictive of success of therapy with
same unlabelled mAb.
In both of the above studies, very low (nonpharmacological) doses of the radiolabelled mAbs were
Eur J Nucl Med Mol Imaging (2010) 37:386–398
used for imaging purposes and no side-effects were
observed in any RA patient. However, the complete
mechanism of uptake of this radiopharmaceutical has not
been revealed until now, means whether these 99mTc-antiTNF-α mAbs target to the membrane bound TNF-α or to
the soluble TNF-α. Moreover, a comparative analysis of
this new technique with conventionally used diagnostic
methods for RA, like ultrasound, X-ray and MRI, may
provide a rationale for its possible use for early diagnosis
and therapy decision-making in RA patients.
Anti-CD20 monoclonal antibody (rituximab)
Rituximab (Rituxan) was the first chimeric monoclonal
antibody approved in 1997 for the treatment of malignancy
and recently, in February of 2006, the FDA approved it for
the treatment of patients with active RA who do not
respond to one or more TNF antagonist therapies. Rituximab is a genetically engineered chimeric murine/human
monoclonal antibody to CD20 antigen found on the surface
of normal and malignant B lymphocyte [42]. CD20
antigens are involved in production of auto-antibodies,
rheumatoid factor (RF), T cell activation, and proinflammatory cytokine production, and therefore plays an
important role in the pathogenesis of RA [43–45].
Rituximab consists of IgG1κ immunoglobulin containing
murine variable and human constant region sequences [42].
Rituximab binds to the CD20 antigen expressed on B
lymphocyte with Fab domain, and Fc domain recruits
immune effecter functions to mediate B-cell lysis in vitro
[46]. Rituximab cytotoxicity is mediated by different
mechanisms; ADCC, CDC, direct disruption of signalling
pathways and the triggering of apoptosis. These different
mechanisms predominate in the treatment of different
diseases [47, 48].
Molecular imaging with
99m
Tc-anti-CD20 mAb (rituximab)
Rituximab was radiolabelled with 99mTc using a photoactivation method developed by Stalteri et al. [49, 50], and
by using a direct radiolabelling method for different
inflammatory and autoimmune diseases including RA [51,
52]. Briefly, in the photoactivation method, rituximab was
purified from Mabthera solution (Roche) by ultrafiltration
using a Centricon YM-10 tube. The same amount of
rituximab and Amerscan Medronate II Kit solution (containing medronate, stannous fluoride and p-aminobenzoic
acid), were exposed to UV light for 20 min at 302 nm.
Radiolabelling of photo-reduced mAb was performed by
incubating it with 99mTc at room temperature for 1 h. A
high radiolabelling yield of >95% was achieved using this
method. Under the conditions employed, an average of 4.4
free thiol groups per antibody were obtained, which was
Eur J Nucl Med Mol Imaging (2010) 37:386–398
391
Fig. 2 Scintigraphic images of
wrists of a RA patient injected
with 99mTc-adalimumab
(anti-TNF-α mAb) before
(a and b; dorsal images after 6
and 20 h p.i., respectively) and
3 months after systemic therapy
with adalimumab
(c and d; dorsal images after 6
and 20 h p.i., respectively)
less than the total number of available thiol groups in mAb
but sufficient to provide efficient labelling. Rituximab
(Mabthera) has also been radiolabelled by direct radiolabelling method with 99mTc using 2-ME reduction, with a
high LE of approximately 95% and specific activity
(3,500–3,700 MBq/mg) [51].
Malviya et al. performed a pilot study in ten patients
with different autoimmune diseases including RA, for
scintigraphic imaging of B lymphocytes; all patients
underwent immunoscintigraphy before treatment with unlabelled rituximab to assess accumulation in affected joints
for possible selection of patients to be treated [52]. In order
to test the best time for scintigraphic imaging, whole-body
images were acquired 1, 2, 4, 6 and 20 h p.i.; anterior and
posterior images of regions of interest were acquired at 6
and 20 h after injection of 370 MBq of 99mTc-rituximab. No
adverse or allergic reactions were observed in patients
studied with 99mTc-rituximab. Patients showed rapid and
persistent spleen uptake. The best imaging time was
obtained at 6 h p.i., although after 4 h it was already
possible to see accumulations in the inflamed regions that
correlated with the clinical data, in particular with DAS28
score. Interestingly, in one patient, when 99mTc labelled
anti-CD20 mAb scintigraphy was compared with anti-TNFα scintigraphy, a different degree of uptake was observed in
different joints with either tracer (Fig. 3); this might
indicate selective inflammatory pathways in different joints
that might benefit from targeted therapies. In conclusion,
biological therapies with mAbs may have severe sideeffects and are very expensive; a pre-therapy scintigraphic
approach with radiolabelled mAbs for therapy decisionmaking may provide a cost-effective solution. Studies in a
larger series of patients are needed to assess the usefulness
of this radiopharmaceutical in RA patients.
Anti-CD4 monoclonal antibody
CD4 is a 55-kDa monomeric membrane glycoprotein
expressed on T lineage cells, including majority of thymocytes and a subset of peripheral T cells and monocytes. The
extra-cellular domain of CD4 binds to the conserved regions
of MHC II molecules on antigen-presenting cells (APCs).
CD4+ T cells constitute the helper subset that regulates T and
B cell function during T cell-dependent responses. CD4+ T
392
Eur J Nucl Med Mol Imaging (2010) 37:386–398
Fig. 3 Scintigraphic images of
wrists of a RA patient injected
with 99mTc-rituximab
(anti-CD20 mAb) (a and b; after
6 h p.i. dorsal and ventral
images, respectively) and 1
week later scintigraphic images
with 99mTc-adalimumab
(anti-TNF-α mAb) (c and
d; after 6 h p.i. dorsal and
ventral images, respectively), in
the same patient. This study
clearly indicates that different
joints may have a different kind
of inflammation and it is therefore desirable to perform a
targeted individualised therapy
based on scintigraphic
evaluation of joint activity
cells and their cytokine products may play an important role
in RA [53]. A number of anti-CD4 monoclonal antibodies
have been available for the management of RA and other
autoimmune disease patients, including murine and
primatized anti-CD4 mAbs [54].
Keliximab (IDEC-CE9.1, IDEC) is an IgG1 primatized
macaque-human anti-CD4 monoclonal antibody, which
reduces the surface density of CD4 and inhibits the
CD4-HLA-II interaction to decrease the disease activity in
RA [55]. It also causes ADCC of T cells following binding
to FcγR that depletes the CD4+ T cell population.
Another, chimeric macaque-human anti-CD4, IgG4
mAb, clenoliximab (IDEC-151, IDEC) is similar to
keliximab with ablated FcγR binding activity. It was
developed by substitution of some key amino acids in the
constant heavy domain of IgG. Ablation of FcγR binding
by IgG4 is a critical factor, since in vivo studies have been
demonstrated that IgG4 monoclonal antibodies can bind to
FcγRIIIA and induce substantial lymphocyte depletion in
patients [56].
Molecular imaging with
99m
Tc-CD4 mAb
Anti-CD4 monoclonal antibody was radiolabelled with
Tc by direct-labelling method using 2-mercaptoethanol
99m
reduction. A high labelling efficiency, greater than 95%,
was achieved by using this method. Several studies
performed in RA patients using anti-CD4 monoclonal
antibody demonstrated its specific targeting in inflamed
joints.
A study was performed by Kinne et al. to detect the in
vivo specificity of 99m-technetium labelled anti-CD4 mAb
to its target molecule. Direct comparison was performed
between radiolabelled murine IgG1anti-CD4 specific mAb
(MAX.16H5) and non-specific human polyclonal immunoglobulin (HIG) for imaging inflamed joints [57]. Two
normal volunteers and eight patients with active or severe
RA were intravenously injected with a sub-therapeutic dose
of 200–300 µg (370–550 MBq) of 99mTc-MAX.16H5 or
1 mg (370 MBq) of 99mTc-HIG. Whole-body and jointspecific scintigraphic images were acquired at 1, 4 and
24 h post-injection. 99mTc-CD4 mAb demonstrated a higher
T/B ratio in arthritic joints of RA patients in comparison to
99m
Tc-HIG, just after 4 h injection. In two patients, an
arthroscopic synovectomy of inflamed knee was also
performed to obtain synovial tissue. In immunohistological
detection, a high number of specific target molecule
(macrophages, T-cells) of anti-CD4 mAbs were identified
in inflamed synovial membrane of patients. These experiments are an excellent approach for verifying the specific
Eur J Nucl Med Mol Imaging (2010) 37:386–398
binding of radiolabelled anti-CD4 mAb in the inflamed
joint, moreover, on the basis of their data authors concluded
that the radiolabelled anti-CD4 mAb allows more specific
detection of inflammatory infiltrates, which are rich in
CD4-positive cells. However, authors propose that this
radiopharmaceutical may also be used to differentiate chronic
joint inflammation from septic arthritis and other joint
disorders, because of the fact that anti-CD4 mAb preferentially bind to mononuclear cells infiltrating chronically
inflamed joints, but not to granulocytes characteristics of
acute inflammation.
Another interesting study was performed by the same
authors for the direct comparison of joint-imaging characteristics of inflammation specific and non-specific mAbs in
a long-standing severe RA patient [58]. In two scintigraphic
examinations, patients received an inflammation-specific
99m-technetium labelled MAX.16H5 (murine IgG1 antiCD4 mAb) and non-specific 99m-technetium labelled antiCEA (anti-carcinoembryonic) mAb at a 9-day time interval.
Although, for the reasons of diagnosis (anti-CEA mAb) or
compatibility (anti-CD4 mAb), the doses of the two
radiopharmaceuticals were different (2 and 0.25 mg,
respectively). Anterior and posterior whole-body and
joint-specific images were acquired at 2, 4 and 24 h after
radiopharmaceutical injection. At all time points, the level
of circulating anti-CD4 was lower than the anti-CEA mAb,
which causes reduced background activity, providing an
improved joint image. Scintigraphic images showed that the
uptake was much clearer at 4 h p.i. of the anti-CD4 mAb in
comparison with anti-CEA mAb. In quantitative evaluation at 4 h p.i. of anti-CD4 mAb, the ratio of average
counts/pixel in synovial membrane region to those in
adjacent blood vessels was 1.22, whereas, it was less
than 1 (0.53) in anti-CEA mAb case. Therefore, this
study evidently showed that an inflammation-specific
mAb could undoubtedly allow more specific detection of
inflammatory infiltrates in RA.
Becker et al. performed scintigraphy with 99mTc-labelled
CD4 specific antibody (MAX.16H5) to study six patients
with active and severe RA [59]. Five out of six patients
received a sub-therapeutic dose of 200–300 µg of 99mTclabelled CD4 specific antibody (555 MBq), and three-phase
bone imaging was performed. Lymphocytes of one patient
were isolated and in vitro labelled with mAb. Seven days
prior to radiolabelled mAb scintigraphy the patients were
injected with 555 MBq of 99m-technetium labelled HDP
(hydroxymethylendiphosphonic acid) and, early (5 min)
and late (2 h) scans were already performed. In both scans
with in vivo and in vitro labelled lymphocytes, anterior and
posterior images clearly visualised the inflamed joints at
1.5 h p.i. and joints showed delineation at 4 to 6 h p.i. The
localisation of diseased joints correlates with clinical signs
(P<0.01), early HDP scan (P<0.01) and late bone scan
393
(P>0.05). Different patterns of joint accumulation, negative
late bone scan with high uptake in mAb scan and high
bone scan without positive mAb scan were observed in
patients. However, authors concluded that the 99mTclabelled CD4 specific antibody can specifically detect
diseased joints in patients with active RA and the localisation
of diseased joints was superior to with 99mTc-HDP, because of
higher specificity and sensitivity in early joint disease.
Moreover, this study demonstrated the possible labelling
of human lymphocytes for specific immunoscintigraphic
examination, and the method was also comparable with
clinical diagnosis.
The above results indeed demonstrate that radiolabelled
anti-CD4 mAbs may provide a valuable tool for early and
specific diagnosis of the inflamed joints, which could be
superior to 99mTc-HIG or 99mTc-HDP scan, but in this case
also the available data is limited by a very low sample size.
Further studies are required in a large patient population to
verify the efficacy and potential use of this technique in
inflammatory disorders.
Anti-E-selectin monoclonal antibody
E-selectin is an endothelial-specific, cytokine-inducible
adhesion molecule [60] that is exclusively expressed on
the luminal surface of vascular endothelium during the
inflammatory response. Its expression has been demonstrated by immunohistochemistry in a variety of acute
and chronic inflammatory diseases, including RA [61].
E-selectin plays a key role in the inflammatory process; it
mediates neutrophil, monocyte and eosinophil adhesion to
activated vascular endothelium via carbohydrate ligands
such as sialyl Lewis X [62, 63].
It is well known that the expression of E-selectin on
endothelial cells is induced after stimulation by interleukin-1
(IL-1), TNF or lipopolysaccharide. It is not expressed by
resting endothelial cells [64, 65]. Moreover, the increased
expression of E-selectin has been detected in several
inflammatory disorders [60, 66]. Therefore, for imaging
inflammation in RA, a monoclonal antibody against
E-selectin radiolabelled with 111In and was successfully
tested previously in an animal model [67] and thereafter
also in humans [68, 69].
Molecular imaging with
111
In labelled anti-E-selectin mAb
The F(ab’)2 fragments of the anti-E-selectin mAb were
generated by pepsin digestion and labelled with 111In after
diethyltriaminepentaacetic acid (DTPA) coupling. A high
labelling efficiency greater than 97% was achieved by
using this method [69].
A comparative study was performed by Jamar et al.
between 99mTc-HIG (an established tracer for arthritis
394
imaging) [70–72] and 111In-labelled 1.2B6 (F(ab’)2 fragments of anti-E-selectin mAb), in 11 patients with active
RA [69]. Each patient underwent 99mTc-HIG scintigraphy
followed by 111In-labelled anti-E-selectin scintigraphy
within 5 days. Scintigraphic imaging was performed at 4
and 24 h post-injection of 555 MBq of 99mTc-HIG or
15 MBq of 111In-anti-E-selectin. Scintigraphic results were
compared with clinical scores of joint involvement. In this
study, net 111In counts over joints were increased significantly between 4 and 24 h. Using joint tenderness or
swelling as an evidence of clinical activity, the sensitivity of
111
In-anti-E-selectin at 4 and 24 h was 69 and 82%,
respectively, compared to 69 and 62% for 99mTc-HIG.
Moreover, the images obtained from 111In-labelled anti-Eselectin demonstrated much less vascular activity than with
99m
Tc-labelled non-specific immunoglobulin. This study
revealed that radioimmunoscintigraphy using 111In-labelled
anti-E-selectin is more sensitive, effective and specific than
99m
Tc-HIG scintigraphy to assess RA activity and identify
active synovitis. Furthermore, another study published by
the same authors demonstrated the significant advantage of
using radiolabelled anti-E-selectin (1.2B6) antibody fragment over conventional 99mTc-HDP bone scanning to
image synovitis [73].
Keelan et al. performed a study with 111In-labelled antiE-selectin monoclonal antibody (1.2B6) in an animal model
to assess the imaging potential of the antibody [74]. In the
pig arthritis model, 111In-1.2B6 mAb or 111In-control
antibody were given intravenously, 3 h after the intraarticular injection of phytoheamagglutinin (PHA) and the
uptake measured by counting the tissues 25 h post-mortem,
which was an excellent approach to direct monitoring of the
radioactivity. Scintigraphic images acquired at 24 h showed
localisation of activity in the inflamed knee, and the
accumulation of intravenously injected 111In-1.2B6 mAb
was significantly greater in comparison to that of 111Incontrol antibody. This study demonstrated that 111In-anti-Eseletin mAb specifically accumulates in the synovitis and
the authors concluded that radiolabelled anti-E-selectin
mAb could be used to image localised inflammatory tissue.
This scintigraphic methodology has proved to be more
reliable than commercially available 99mTc-HIG and conventional 99mTc-HDP bone scan, and showed potential
results for the diagnosis of inflammatory lesions but,
unfortunately, no further studies were performed to establish this radiopharmaceutical and this approach remains in
the research field only.
Anti-CD3 monoclonal antibody
A humanised non-FcR binding derivative of the anti-human
CD3 monoclonal antibody, OKT3, (hOKT3γ1[Ala–Ala]),
can induce generalised immunosuppression in patients with
Eur J Nucl Med Mol Imaging (2010) 37:386–398
psoriatic arthritis [75–77]. In this mAb, two additional
alanine mutations (amino acids 234, 235) were introduced
to prevent FcR binding. The complementarity determining
region (CDR) of OKT3 was engrafted onto a human IgG1
backbone, for the low immunogenicity profile. This
antibody could cause modulation of the CD3-T-cell
receptor complex, induction of clonal anergy and/or
induction of regulatory T cells. Also, theoretically, imaging
with 99mTc-OKT-3 mAb is advantageous over 99mTc-antiCD4 mAb, since it recognizes CD4+ and CD8+ subtypes of
T lymphocytes, which are both present in the inflamed
synovium.
Visilizumab (Nuvion), a new generation of genetically
engineered anti-CD3 mAbs, has been developed by grafting
murine CDR derived from M291 hybridoma into human
non-CDR region of IgG2 and introducing non-FcR-binding
mutations at amino acid residues 234 and 237 (Val → Ala)
into the IgG2 Fc portion [78–80]. This non-FcR binding
humanised mAb binds with human CD3-ε chain with high
specificity and high avidity (Ka=0.5×109 M-1) [81]. An
early study has demonstrated that visilizumab induces
selective apoptosis of activated T cells in vitro, but this
did not appear in resting T cells, which may provide
therapeutic benefit in several autoimmune diseases [82].
Molecular imaging with
99m
Tc labelled anti-CD3 mAb
An anti-CD3 IgG2 murine mAb, muromonab (OKT-3), was
labelled with 99m-technetium by reducing disulphide
bonds, a high labelling efficiency (LE) of more than 95%
was assessed by ITLC and HPLC with consistently less
than 5% colloid formation [83].
A study was performed in 7 RA and 2 psoriatic arthritis
patients with radiolabelled anti-CD3 mAb [83]. Anterior
and posterior whole body scan and specific regional
imaging was commenced 20 min after the intravenous
injection of 99mTc-OKT-3 (185 MBq). In eight out of nine
of these patients, 99mTc-OKT-3 scans were positive even
when potent immunosuppressive and anti-inflammatory
drugs induced a good clinical response to pain. The study
demonstrated that all 34 joints with moderate to severe pain
had moderate to marked uptake of radioactivity. Unexpectedly, two out of a total of nine patients experienced shaking
chills, one with associated neck pain, within an hour of
radiopharmaceutical infusion. Both cases resolved spontaneously after 20–30 min with or without Benadryl
injection. These adverse reactions were surprising, when
the injected dose ranging from 5 to 10 μg only, which was
very less than therapeutic dose (5 mg q.d. for 10–14 days in
acute renal transplant rejection). The cause of these adverse
reactions could be related to the cytokine release syndrome,
which is well described in renal transplanted patients. The
authors concluded that 99mTc-anti-CD3 mAb imaging could
Eur J Nucl Med Mol Imaging (2010) 37:386–398
be useful in measuring the therapeutic effectiveness in RA,
but the side-effect profile limits the potential use of this
radiolabelled mAb in scintigraphic detection of RA.
Recently, Martins et al. performed a study in 38 patients
who met the American College of Rheumatology (ACR)
criteria for RA [84], with 99mTc labelled OKT-3 mAb, but
using a different radiolabelling technique than Marcus et al.
In this study, the mAb was labelled using a different chelant
and a reductor agent, in place of previously used ascorbic
acid reduction method. Each patient received an injection of
185 MBq (150 μg) of 99m Tc-OKT-3 mAb and all
scintigraphic images were acquired at 1 and 3 h p.i. for
5 min. Two nuclear medicine specialists evaluated scintigraphic scans independently and results were correlated
with clinical parameters. Interestingly, no side-effect of this
radiolabelled mAb was observed in any patient. The sideeffect with the same 99mTc labelled mAb in the above study
therefore could be associated with different radiolabelling
method of antibody, but it remains speculative. In this
study, 99mTc-OKT-3 uptake demonstrated a significant
correlation (P<0.05) with swollen joints, tender joints and
the VAS score. Scintigraphic results were also able to
follow-up the disease activity in inflamed joints and also
correlated with the DAS28 score. Moreover, the study
concluded that 99mTc-OKT-3 is a reliable and objective
method for detecting synovial activity and can be used for
disease prognosis.
However, as it is well known that a humanised mAb has
very less side-effect profile in comparison to a murine or
chimeric mAb, therefore, a 99m-technetium labelled
humanised anti-CD3 mAb may provide an effective tracer
for CD3-positive cell imaging with superior profile. Our
group recently radiolabelled visilizumab (Nuvion), with
99m-technetium by using a hetero bifunctional linker
SHNH/S-HYNIC (succinimidyl-6-hydrazinonicotinate
hydrochloride) with a high labelling efficiency (>90%) and
high specific activity (9,990–11,100 MBq/mg). The in vitro
and in vivo results in the animal model demonstrated very
potential results for the targeting of CD3 positive human
lymphocytes [85, 86]. The effectiveness of 99mTc-labelled
visilizumab, however, has yet to be checked in humans, but
this new radiopharmaceutical may provide a useful tool for
in vivo imaging of several immune-mediated inflammations
as well as a rationale for therapy with unlabelled anti-CD3
mAb and follow-up of the disease activity.
New molecules/receptors are regularly identified as
possible therapeutic targets and also radiolabelled to
generate a radiopharmaceutical that can identify a stagespecific inflammation. The possibility to image in vivo
location and quantify extent of these target molecules in
inflamed tissues is therefore extremely important in
planning an appropriate therapy and to follow-up the
efficacy of therapy. Moreover, the radiolabelled mAb
395
scintigraphy approach may also provide an explanation
for the failure of any targeted therapy and/or the justification to select specific targeted therapy for a patient with
inflammatory disease. However, these new-generation
radiopharmaceuticals still need to be evaluated carefully on
a large-scale trial before including in the list of routinely
used nuclear medicine tools. Some commercial drug
companies, however, are already in the process of preparing
commercial kits for scintigraphy with anti-TNF-α antibodies
and hopefully these will soon be available on the market.
Concluding remarks
In this new century we are slowly but continuously shifting
to the molecular level of nuclear medicine in the search of
high affinity and more specific radiopharmaceuticals. This
continuous development has explored a variety of intelligent approaches to establish the foundation of future
molecular nuclear medicine imaging modalities. The
conventional imaging technique using radiolabelled autologous leukocytes is still the gold standard in nuclear
medicine for the diagnosis of infection/inflammation. New
multidisciplinary modalities, however, are being developed
for more specific diagnostic imaging, targeted molecular
therapy, simplicity, cost and safety reasons. These techniques can detect the pathological changes at a very early
stage and are helpful in understanding the pathophysiology
of different diseases.
Unfortunately, even after numerous studies in the last
two decades that proved sensitivity and specificity of a
wide range of radiolabelled mAbs for immunoscintigraphy,
this useful technique is not used much in clinical practice
and until now has remained a research tool inside selected
laboratories. The safety profile of murine and chimeric
mAbs, unavailability of therapeutic application of mAb in
RA patients, and the high cost of therapy were some
reasons that have restricted the further development and
utilization of immunoscintigraphy in routine clinical practice. Several radiolabelled mAbs (such as anti-E-selectin
and anti-CD4) demonstrated their excellent capability for
the localization of inflammatory regions, but lack of their
use for the therapeutic purposes in RA patients limit their
further development and use for immunoscintigraphy. The
production of fully human mAbs (such as adalimumab)
with no side-effect profile, however, increased the safety
profile of immunoscintigraphy and approval of mAbs (such
as anti-TNF-α and anti-CD20) for treatment of RA patients
also provide us an opportunity to select the patients through
this technique.
Previous studies, particularly with radiolabelled polyclonal IgG (HIG), were focused on the detection of the state
of activity of the disease; although this is an important
396
application, no real clinical advantages have been found
compared to other available radiopharmaceuticals with
respect to the clinical management of patients.
The real breakthrough in targeted immunoscintigraphy in
rheumatoid arthritis activity is the possibility to highlight
the presence of the relevant receptors involved in the
pathophysiology of the disease directly by means of the
specific radiolabelled mAb that will eventually be used for
treatment. To this aim, results obtained so far are highly
encouraging and hold promise for therapy decision-making
and follow-up, with a view to assessing whether an
antibody will accumulate in an inflamed joint before using
the same unlabelled antibody for therapeutic purposes. This
kind of information can only be obtained with this new
imaging approach based on new radiopharmaceuticals that
provide a solid basis for the further development and
clinical use of immunoscintigraphy in RA.
In the future, we will need to carefully evaluate the clinical
role of targeted scintigraphy in RA; different biological
treatments are now available and will be developed and we
should play a role in selecting the best therapeutic option for
the individual patient; if we succeed in doing this, we will not
only improve the clinical care of these patients but also prove
cost-effective in doing so. It is foreseen that the development
of fully human mAbs will further improve the application and
development of these techniques, the high costs of clinical
developments of experimental radiopharmaceuticals being the
major problem to solve.
Acknowledgements GM acknowledges ISORBE (International
Society of Radiolabelled Blood Elements) for providing scientific
materials for the preparation of this manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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