Development of a novel recombinant biotherapeutic with applications in targeted therapy of human arthritis.
ABSTRACT To isolate recombinant antibodies with specificity for human arthritic synovium and to develop targeting reagents with joint-specific delivery capacity for therapeutic and/or diagnostic applications.
In vivo single-chain Fv (scFv) antibody phage display screening using a human synovial xenograft model was used to isolate antibodies specific to the microvasculature of human arthritic synovium. Single-chain Fv antibody tissue-specific reactivity was assessed by immunostaining of synovial tissues from normal controls and from patients with rheumatoid arthritis and osteoarthritis, normal human tissue arrays, and tissues from other patients with inflammatory diseases displaying neovasculogenesis. In vivo scFv antibody tissue-specific targeting capacity was examined in the human synovial xenograft model using both (125)I-labeled and biotinylated antibody.
We isolated a novel recombinant human antibody, scFv A7, with specificity for the microvasculature of human arthritic synovium. We showed that in vivo, this antibody could efficiently target human synovial microvasculature in SCID mice transplanted with human arthritic synovial xenografts. Our results demonstrated that scFv A7 antibody had no reactivity with the microvasculature or with other cellular components found in a comprehensive range of normal human tissues including normal human synovium. Further, we showed that the reactivity of the scFv A7 antibody was not a common feature of neovasculogenesis associated with chronic inflammatory conditions.
Here we report for the first time the identification of an scFv antibody, A7, that specifically recognizes an epitope expressed in the microvasculature of human arthritic synovium and that has the potential to be developed as a joint-specific pharmaceutical.
- SourceAvailable from: Yuti Chernajovsky[Show abstract] [Hide abstract]
ABSTRACT: OBJECTIVES: The synovial endothelium targeting peptide (SyETP) CKSTHDRLC has been identified previously and was shown to preferentially localise to synovial xenografts in the human/severe combined immunodeficient (SCID) mouse chimera model of rheumatoid arthritis (RA). The objective of the current work was to generate SyETP-anti-inflammatory-cytokine fusion proteins that would deliver bioactive cytokines specifically to human synovial tissue. METHODS: Fusion proteins consisting of human interleukin (IL)-4 linked via a matrix metalloproteinase (MMP)-cleavable sequence to multiple copies of either SyETP or scrambled control peptide were expressed in insect cells, purified by Ni-chelate chromatography and bioactivity tested in vitro. The ability of SyETP to retain bioactive cytokine in synovial but not control skin xenografts in SCID mice was determined by in vivo imaging using nano-single-photon emission computed tomography-computed tomography (nano-SPECT-CT) and measuring signal transducer and activator of transcription 6 (STAT6) phosphorylation in synovial grafts following intravenous administration of the fusion protein. RESULTS: In vitro assays confirmed that IL-4 and the MMP-cleavable sequence were functional. IL-4-SyETP augmented production of IL-1 receptor antagonist (IL-1ra) by fibroblast-like synoviocytes (FLS) stimulated with IL-1β in a dose-dependent manner. In vivo imaging showed that IL-4-SyETP was retained in synovial but not in skin tissue grafts and the period of retention was significantly enhanced through increasing the number of SyETP copies from one to three. Finally, retention correlated with increased bioactivity of the cytokine as quantified by STAT6 phosphorylation in synovial grafts. CONCLUSIONS: The present work demonstrates that SyETP specifically delivers fused IL-4 to human rheumatoid synovium transplanted into SCID mice, thus providing a proof of concept for peptide-targeted tissue-specific immunotherapy in RA. This technology is potentially applicable to other biological treatments providing enhanced potency to inflammatory sites and reducing systemic toxicity.Annals of the rheumatic diseases 07/2012; · 8.11 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.PLoS ONE 01/2013; 8(9):e75772. · 3.73 Impact Factor
ARTHRITIS & RHEUMATISM
Vol. 63, No. 12, December 2011, pp 3758–3767
© 2011, American College of Rheumatology
Development of a Novel Recombinant Biotherapeutic With
Applications in Targeted Therapy of Human Arthritis
Panagiotis Kamperidis,1Tahereh Kamalati,1Mathieu Ferrari,1Margaret Jones,1Toby Garrood,1
Malcolm D. Smith,2Soraya Diez-Posada,3Chris Hughes,1Ciara Finucane,1Stephen Mather,1
Ahuva Nissim,1Andrew J. T. George,4and Costantino Pitzalis1
Objective. To isolate recombinant antibodies with
specificity for human arthritic synovium and to develop
targeting reagents with joint-specific delivery capacity
for therapeutic and/or diagnostic applications.
Methods. In vivo single-chain Fv (scFv) antibody
phage display screening using a human synovial xeno-
graft model was used to isolate antibodies specific to the
microvasculature of human arthritic synovium. Single-
chain Fv antibody tissue-specific reactivity was assessed
by immunostaining of synovial tissues from normal
controls and from patients with rheumatoid arthritis
and osteoarthritis, normal human tissue arrays, and
tissues from other patients with inflammatory diseases
displaying neovasculogenesis. In vivo scFv antibody
tissue-specific targeting capacity was examined in the
human synovial xenograft model using both125I-labeled
and biotinylated antibody.
Results. We isolated a novel recombinant human
antibody, scFv A7, with specificity for the microvascu-
lature of human arthritic synovium. We showed that
in vivo, this antibody could efficiently target human
synovial microvasculature in SCID mice transplanted
with human arthritic synovial xenografts. Our results
demonstrated that scFv A7 antibody had no reactivity
with the microvasculature or with other cellular compo-
nents found in a comprehensive range of normal human
tissues including normal human synovium. Further, we
showed that the reactivity of the scFv A7 antibody was
not a common feature of neovasculogenesis associated
with chronic inflammatory conditions.
Conclusion. Here we report for the first time the
identification of an scFv antibody, A7, that specifically
recognizes an epitope expressed in the microvasculature
of human arthritic synovium and that has the potential
to be developed as a joint-specific pharmaceutical.
Rheumatoid arthritis (RA) is a chronic inflam-
matory disease that principally affects synovial joints,
causing disability with significant associated morbidity
and mortality (1,2). In RA, the synovium becomes
hyperplastic and locally invasive at the interface between
the cartilage and bone, resulting in the destruction of
articular cartilage and subchondral bone, leading to joint
damage and disability. Notably, in RA, the synovium
becomes heavily infiltrated by T and B cells, plasma
cells, and monocytes through the development of new
blood vessels (angiogenesis) (3,4). It is now clear that
synovial angiogenesis contributes significantly to disease
pathogenesis and progression (5,6) and may precede
other pathologic features of RA, since synovial hyper-
cellularity is sustained by an increase in the number
and density of synovial blood vessels (6–8). Further, not
only angiogenesis but also vasculogenesis may contrib-
ute to the increased vascularity observed in the RA
synovium (9), making the newly formed blood vessels a
particularly attractive therapeutic target for the manage-
Supported by the Nuffield Foundation (an Oliver Bird Rheu-
matism Programme PhD studentship to Dr. Kamperidis; Dr. Pitzalis
Programme Principal Investigator) and Arthritis Research UK (grant
1Panagiotis Kamperidis, PhD, Tahereh Kamalati, PhD, Ma-
thieu Ferrari, MSc, Margaret Jones, MIAT, Toby Garrood, MD,
MRCP, PhD, Chris Hughes, PhD, Ciara Finucane, PhD, Stephen
Mather, PhD, Ahuva Nissim, PhD, Costantino Pitzalis, MD, PhD,
FRCP: Barts and the London School of Medicine and Dentistry,
Queen Mary University of London, London, UK;2Malcolm D. Smith,
MBBS, FRACP: Repatriation General Hospital, Daw Park, Adelaide,
South Australia, Australia;
College London, London, UK;4Andrew J. T. George, PhD, FRCPath,
FRSA, FHEA: Imperial College London, Hammersmith Campus,
Drs. Kamperidis and Kamalati contributed equally to this
Address correspondence to Costantino Pitzalis, MD, PhD,
FRCP, William Harvey Research Institute, Barts and the London
School of Medicine and Dentistry, Charterhouse Square, London
EC1M 6BQ, UK. E-mail: firstname.lastname@example.org.
Submitted for publication April 1, 2011; accepted in revised
form August 23, 2011.
3Soraya Diez-Posada, PhD: University
ment of inflammatory arthritis (10). In support of this,
blockade of inflammatory neovascularization has been
shown to lead to the suppression of synovial inflamma-
tion and proliferation and to an attenuation of synovitis
in RA (11).
The treatment of this condition has been trans-
formed in the last decade by the use of recombinant
antibodies targeting proinflammatory cytokines such as
tumor necrosis factor ? (TNF?) (12). However, despite
the obvious impact of such therapies, 20–40% of pa-
tients do not respond (13), sustained and high-
magnitude clinical response is achieved only in a minor-
ity of cases (14), and prolonged treatment-free remission
has not been obtained. Additionally, these therapies
exhibit several adverse side effects that make persistent
administration undesirable (14–16). Therefore, the de-
velopment of new agents that offer greater efficacy and
improved safety profiles remains an important goal for
the treatment of RA. In this context, tissue-specific drug
delivery systems for targeting and improving the reten-
tion of bioactive agents are particularly important, as
they could be used to achieve higher levels of pharma-
ceuticals at the site of therapeutic interference and thus
prolong local activity within the joint, thereby reducing
systemic exposure and toxicity. Recombinant, single-
chain antibodies lend themselves well to the develop-
ment of such targeted pharmacodelivery strategies.
To date, joint-specific targeting for the treatment
of arthritic disease remains an unmet clinical goal. In
order to address this, we have developed a synovial
xenograft model in SCID mice where functional vascular
anastomoses allow the delivery of agents targeting hu-
man synovial tissue to be assessed in vivo (17). Previ-
ously, we successfully used this model for in vivo peptide
phage display and imaging of synovial tissue (18,19).
Here, we have extended the use of this model system to
carry out in vivo single-chain Fv (scFv) antibody phage
display screening, in order to identify scFv antibody
clones with specificity for the human synovial vascula-
ture of the xenografts. We report for the first time the
isolation and characterization of an scFv antibody (A7)
that exhibits specificity for the microvasculature of hu-
man arthritic synovial tissue, and we discuss its potential
application as an innovative recombinant pharmaceuti-
cal agent for the treatment of arthritic diseases.
MATERIALS AND METHODS
Human tissue transplantation into SCID mice. Beige
SCID CB17 mice ages 4–10 weeks were used in this study.
Human tissues (synovium and skin) were transplanted sub-
cutaneously in a dorsal position distal to the shoulder joints
(2 transplants per animal) as previously described (20). Mice
were inspected daily, and animal work was performed under
a Project License (PPL 70-6109). Human synovial tissue was
obtained from RA patients or osteoarthritis (OA) patients
undergoing joint replacement. Human skin tissue was obtained
from patients undergoing cosmetic surgery. Informed consent
was obtained from all patients. Additionally, ethical approval
to use human synovial and skin tissue for research purposes
was obtained from the Ethics Committee (Local Research
Ethics Committee no. 05/Q0703/198).
In vivo selection of synovium-specific scFv phage. The
human scFv libraries I ? J (Tomlinson I ? J) (21) were kindly
provided by Dr. Greg Winter (Medical Research Council
Centre for Protein Engineering, Cambridge, UK). Synovium-
specific phage was isolated following 4 rounds of enrichment in
SCID mice carrying human arthritic synovial tissue and skin
tissue xenografts (18, 19). Selection and enrichment were
monitored by phage titration, and the integrity of scFv coding
regions from phage from the final round of selection was
assessed by polymerase chain reaction using LMB3 and pHEN
seq primers (see below). Clones that showed expression of
full-length scFv fragments were used to infect the nonsuppres-
sor strain Escherichia coli HB2151 for the production of
soluble scFv protein. One hundred colonies were selected and
analyzed for scFv expression by enzyme-linked immunosor-
bent assay for protein A and protein L binding (22).
DNA sequence analysis of scFv coding regions. The
DNA sequences encoding the scFv inserts of phage clones
from the final round of selection were determined using the
vector primers LMB3 (CAGGAAACAGCTATGAC) and
pHEN seq (CTATGCGGCCCCATTCA) (21,23). Sequencing
was performed using the Big Dye Terminator v3.1 Cycle
Sequencing kit (Applied Biosystems) on an ABI PRISM 3130
Production of soluble scFv antibodies. Single-chain Fv
fragments encoded by phage from the final round of selection
were expressed in E coli HB2151 as soluble, secreted pro-
teins and purified from bacterial culture supernatants by
affinity chromatography using protein A–Sepharose Fast
Flow Resin (GE Healthcare), as described previously (24).
Purified antibodies were analyzed by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and size-exclusion chroma-
tography on Superdex 75 HR10/30 columns (Amersham Bio-
Biotinylation of scFv antibodies. Single-chain Fv anti-
bodies were biotinylated using the EZ-Link Sulfo-NHS-SS-
Biotinylation kit (Perbio Science). Briefly, purified scFv pro-
tein was diluted in 0.5–2 ml phosphate buffered saline (PBS),
added to a 20-fold molar excess of 10 mM Sulfo-NHS-SS-
Biotin, and incubated on ice for 1 hour. Biotinylated proteins
were subsequently purified using spin-column chromatography
(Perbio Science) according to the manufacturer’s instructions.
Iodination of scFv antibodies. Single-chain Fv antibody
fragments were radiolabeled with Na125I using the iodogen
method (25). Iodination reaction tubes precoated with iodogen
were used according to the manufacturer’s instructions (Perbio
Science). Typically, 25 ?g of purified scFv in 150 ?l of PBS
was radiolabeled to specific activities of 0.15–0.2 MBq/?g. The
efficiency of iodination was evaluated by instant thin-layer
chromatography and typically found to be ?90%. The purity
TARGETED THERAPY OF HUMAN ARTHRITIS3759
of the labeled scFv was determined by size-exclusion high-
performance liquid chromatography.
In vivo localization of soluble scFv A7 antibody. Two
SCID mice bearing double xenografts of human arthritic
synovial and skin tissues (2 arthritic synovium and 2 human
skin grafts per animal) were injected with 6 ?g of biotinylated
scFv A7 four weeks after transplantation. Biotinylated anti–
hen egg lysozyme antibody, scFv HEL (22), was used as a
negative control. The biotinylated antibody fragments were
administered via the tail vein in a total volume of 200 ?l and
were allowed to circulate for 15 minutes, after which time the
mice were perfused under terminal anesthesia. The human
grafts along with murine tissues were excised and immediately
snap-frozen in liquid nitrogen for histologic examination. The
tissue-specific localization of soluble scFv A7 was examined by
immunohistochemical detection of biotinylated antibody using
avidin–biotin–horseradish peroxidase complex (Dako).
In vivo targeting capacity of iodinated scFv A7 anti-
body. Five double-transplanted SCID mice (2 arthritic syno-
vium and 2 human skin grafts per animal) were injected with
iodinated scFv antibody 4 weeks following transplantation.
Each animal was administered an injection, via the tail vein, of
200 ?l sterile saline containing 1.25 ?g labeled scFv with a
specific activity of 0.16 MBq/?g. Mice were killed 4 hours or 24
hours after injection, and grafts as well as mouse organs were
collected for gamma counting. The results were corrected for
tissue weight and background radioactivity in the blood pool
and expressed as a percentage of the total injected dose.
Iodinated scFv HEL was used as an untargeted scFv control.
Immunohistochemical analysis. Slide-mounted frozen
tissue sections were fixed in ice-cold acetone. Paraffin-
embedded tissues were dewaxed and subsequently treated with
proteinase K (Dako) for 4 minutes at room temperature for
antigen retrieval. Slides were stained with 1 ?g of biotinylated
scFv A7 and visualized with avidin–biotin–horseradish perox-
idase complex using 3,3?-diaminobenzidine chromogen. The
presence of human blood vessels in tissue sections was visual-
ized using mouse anti-human von Willebrand factor (vWF)
(Dako), followed by a horseradish peroxidase–conjugated anti-
mouse antibody (Dako). Rabbit anti-mouse CD31 (BD Bio-
sciences) and rabbit anti-mouse CD34 (Cambridge Bioscience)
were used to detect mouse endothelial cells in murine tissues.
An anti–?-smooth muscle actin antibody (Sigma) was used to
visualize the stromal component of the microvasculature.
Sections were counterstained with hematoxylin, mounted with
Depex mounting medium (Dako), and analyzed using a light
microscope (Olympus). Images were acquired with CellP Soft
Imaging System version 1.2 (Olympus).
Immunofluorescence analysis. Slide-mounted frozen
tissue sections were fixed in ice-cold acetone prior to antibody
staining. Biotinylated scFv A7 reactivity was detected with
Texas Red–conjugated NeutrAvidin (Invitrogen). Mouse anti-
human vWF, mouse anti-human CD31 (Sigma), and rabbit
anti-NG2 antibody (Millipore) reactivity was detected using
goat anti-mouse and goat anti-rabbit antibodies conjugated to
Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Sections were
subsequently mounted in fluorescent mounting media
(Vectashield) with DAPI nuclear counterstain (Vector) and
Figure 1. Immunohistochemical analysis of single-chain Fv (scFv) antibody reactivity with
human arthritic synovial tissue. The reactivity of scFv antibody (scFv A7) with sections of
human arthritic synovial and skin tissue was examined using biotinylated scFv A7. Biotinylated
scFv HEL was used as an antibody negative control. The presence of blood vessels in tissue
samples was visualized with anti-human von Willebrand factor (vWF) antibody. Biotinylated
scFv antibodies were detected with avidin–biotin–horseradish peroxidase (HRP) complex, while
anti-vWF antibody reactivity was detected using an HRP-labeled antibody. RA ? rheumatoid
arthritis; OA ? osteoarthritis. Bar ? 100 ?m.
3760KAMPERIDIS ET AL
examined using an Axioskop 2 microscope (Carl Zeiss). Im-
ages were captured by an AxioCam digital color camera using
KS300 image analysis software (Carl Zeiss).
Pearson’s correlation coefficient analysis. Velocity 5.5
imaging software (PerkinElmer) was used to perform thresh-
olded Pearson’s correlation coefficient analysis of images in
order to accurately quantify and correlate overlap of image
pixels from 2 different channels (26). A value of ?1 indicates
complete pixel-to-pixel overlap of the pixels from the 2 chosen
channels. A value of 0 indicates no overlap or correlation of
pixels from 2 different channels, and a value of ?1 indicates
complete disparity/exclusion of pixels from the 2 channels that
have been compared.
Statistical analysis. Results are expressed as the
mean ? SEM. Parametric analyses were performed by unpaired
2-tailed t-test using GraphPad Prism software.
Isolation of an scFv antibody with binding spec-
ificity for human arthritic synovial tissue. In order to
select scFv fragments targeting the human synovial
microvasculature, 4 cycles of in vivo selection using the
human scFv libraries I ? J (Tomlinson I ? J) (21) were
conducted using mice with dual xenografts of synovium
(target) and skin (control) tissues. The composition of
recovered scFv fragments in the final round of in vivo
selection was assessed by examining the ability of 100
phage-encoding full-length inserts to transduce bacteria
for secreted antibody protein expression. The function-
ality of these clones was further confirmed by demon-
strating secreted scFv binding to protein A and protein
L. Of these 100 clones, 24 expressed secreted scFv
antibody at high levels and were subsequently shown
to encode the same scFv sequence. Notably, this scFv
sequence had already been identified in a previous,
independent, in vivo screen of the Tomlinson library,
using the same synovial xenograft model system (results
not shown). Thus, clone A7, an scFv from the group of
24 identical clones from the final screen, which showed
robust soluble antibody expression (?1 mg/100 ml bac-
terial culture), was chosen for further studies.
Soluble scFv A7 protein was purified as a mono-
meric protein and used in immunohistochemical analysis
to assess binding specificity in human RA and OA
synovial tissues in comparison to that of skin, the control
tissue used in the in vivo selection process. We examined
15 OA and 8 RA synovial tissue samples and 5 skin
samples. As shown in Figure 1, scFv A7 exhibited
specific and strong reactivity with the microvasculature
of OA and RA synovial tissue. Importantly, scFv A7
exhibited no detectable reactivity with control human
skin tissue. The control, nontargeted antibody scFv HEL
did not exhibit binding to either synovium or control skin
Single-chain Fv A7 antibody specifically binds to
arthritic synovial microvasculature. Following the dem-
onstration that scFv A7 exhibits strong reactivity with
the microvasculature of arthritic synovial tissue, we
sought to determine which cell types within the micro-
vasculature were recognized by this antibody. To do this,
costaining of RA synovial tissues was performed using
scFv A7 and the 2 endothelial markers vWF and CD31,
and the pericyte-specific marker NG2. As shown in
Figure 2, there was no overlap in the pattern of staining
observed in RA tissue stained with scFv A7 and vWF or
CD31. However, costaining with scFv A7 and the peri-
cyte marker NG2 showed complete overlap in the pat-
Figure 2. Characterization of cellular reactivity of scFv A7 within
synovial microvasculature. The reactivity of biotinylated scFv A7 with
cellular components of synovial microvasculature was examined by
dual staining of frozen human synovial tissue from RA patients and
compared with staining for the endothelial markers vWF and CD31
and the pericyte-specific marker NG2. Bound scFv A7 was detected
using Texas Red–avidin conjugate. Binding of vWF, CD31, and NG2
was detected using Alexa Fluor 488– or Alexa Fluor 594–labeled
secondary antibodies (green and red, respectively). Bar ? 20 ?m. See
Figure 1 for definitions.
TARGETED THERAPY OF HUMAN ARTHRITIS 3761
tern of cellular staining observed. This demonstrates
that scFv A7 recognizes an epitope localized to pericytes
and the stromal component of the microvasculature of
In order to increase the accuracy and level of
confidence about the degree of colocalization, we used
Pearson’s correlation coefficient to provide a numeric
and nonsubjective analysis (27). The Pearson correlation
ranges from ?1 to ?1, whereby a correlation of ?1
indicates complete overlap of pixels from 2 different
channels. A value of 0 indicates no overlap, and a
correlation of ?1 indicates complete pixel disparity/
exclusion between the 2 channels being compared. Our
Pearson’s colocalization analysis of scFv A7 reactivity
(red pixels) and CD31 reactivity (green pixels) resulted
in a Pearson’s correlation of 0.07, demonstrating no
colocalization of scFv A7 and CD31 reactivity. Similarly,
Pearson’s colocalization analysis of NG2 reactivity
(green pixels) and vWF reactivity (red pixels) resulted in
a Pearson’s correlation of 0.01, demonstrating no colo-
calization of NG2 and vWF reactivity. However, Pear-
son’s colocalization analysis of scFv A7 reactivity (red
pixels) and NG2 reactivity (green pixels) resulted in a
Pearson’s correlation of 0.6, demonstrating significant
colocalization of scFv A7 and NG2 reactivity.
Single-chain Fv A7 antibody retains synovial
specificity in vivo. In order to examine the specificity of
scFv A7 targeting in vivo, SCID mice bearing arthritic
synovium (test tissue) and human skin (control tissue)
xenografts were injected intravenously with biotinylated
scFv A7 or biotinylated scFv HEL (as a negative con-
trol). As shown in Figure 3, after in vivo circulation,
biotinylated scFv A7 could be detected in the human
synovial microvasculature by simply adding avidin–
biotin–horseradish peroxidase complex to xenograft sec-
tions. In contrast, scFv A7 reactivity was not observed
with control human skin tissue xenografts. Additionally,
no detectable reactivity was observed with the control
scFv HEL antibody (Figure 3). To confirm the vascular
reactivity of scFv A7 in the positive grafts and to exclude
the possibility that the negative staining observed in
human skin grafts and mouse tongue are not due to an
Figure 3. In vivo targeting of scFv A7 to human arthritic synovial microvasculature. The ability
of scFv A7 to localize to human arthritic synovial microvasculature in vivo was examined by
injecting biotinylated antibody into dual-transplanted SCID mice bearing human arthritic
synovial and human skin xenografts. Immunohistochemistry was used to assess the reactivity of
scFv A7 with the microvasculature in the recovered xenografts. Biotinylated scFv HEL was used
as an antibody negative control. Mouse tongue tissue was used to assess cross-reactivity of scFv
A7 antibody. The presence of microvasculature within human tissue was visualized with
anti-human vWF antibody, while anti-mouse CD31 was used for mouse tissue. Biotinylated scFv
antibodies were detected with avidin–biotin–HRP complex, while anti-vWF and anti-CD31
antibody reactivity was detected using an HRP-labeled antibody. Bar ? 100 ?m. See Figure 1
3762 KAMPERIDIS ET AL
absence of vasculature, all tissues were stained for
human vWF and/or mouse CD31 (Figure 3). No cross-
reactivity with host mouse tissues was observed, as
exemplified by examination of mouse tongue tissue,
where microvasculature was clearly visible (Figure 3).
Taken together, these data further confirm the
synovium-specific reactivity of the scFv A7 antibody and
its capacity to reach its target in vivo.
In vivo targeting of arthritic synovial tissue by
scFv A7. To quantitatively assess antibody tissue speci-
ficity in vivo, we examined the ability of iodinated scFv
A7 to target human synovial tissue xenografts. Iodinated
scFv HEL was used as a nontargeted control antibody,
while human skin was used as control xenograft tissue.
Figure 4 shows the tissue-to-blood ratio of the percent-
age of the injected dose localizing in each tissue at 4
hours and 24 hours. These data demonstrate that 4
hours following injection, 3-fold more radiolabeled scFv
A7 was localized to the human arthritic synovium xeno-
grafts than to the human skin xenografts. Further,
despite an apparent fall in overall activity of scFv A7
in the synovium at 24 hours, significant differential
reactivity was still observed when compared to skin at
this time point. Overall, these data further confirm that
the scFv A7 antibody retains the synovial targeting
specificity of the parental phage clone in vivo.
Single-chain Fv A7 reactivity in normal and
inflamed human tissues. In order to further investigate
scFv A7 binding specificity over and above the original
targeted tissues, we examined the reactivity of this
antibody using an array for a comprehensive range of
normal human tissues. As shown in Figure 5A, scFv A7
did not exhibit reactivity with the various cellular com-
ponents of the tissues represented on this array. In
particular, no detectable reactivity was seen with the
microvasculature of organs such as the adrenal gland,
ovary, heart, ileum, and esophagus, all of which were
shown to be positive for vWF staining with evident
Next we examined scFv A7 reactivity with the
microvasculature of normal human synovial tissue ob-
tained from subjects undergoing joint arthroscopy for
prolonged, unexplained knee pain that did not develop
into arthritic conditions during a 5-year followup survey
(28). The results presented in Figure 5B are represen-
tative of 11 samples and demonstrate that the microvas-
culature found in normal human synovium, as detected
by vWF reactivity, contains a stromal vascular compo-
Figure 4. In vivo targeting of human arthritic synovial tissue with125I-labeled single-chain Fv (scFv) A7 antibody. The ability
of scFv A7 to preferentially target the microvasculature of human arthritic synovial xenografts in vivo was examined by injecting
iodinated scFv A7 (black) into SCID mice bearing dual synovial and skin xenografts. Graft tissues were examined by gamma
counting 4 hours (n ? 5) and 24 hours (n ? 6) after antibody administration. The results were corrected for tissue weight and
background radioactivity in the blood pool and expressed as tissue-to-blood ratios of the percentage of the injected dose.
Iodinated scFv HEL (gray) was used as a negative control. Results are expressed as the mean ? SEM. ?? ? P ? 0.0079;
? ? P ? 0.0513 versus skin graft injected with125I-labeled scFv A7, by unpaired 2-tailed t-test.
TARGETED THERAPY OF HUMAN ARTHRITIS3763
nent as detected by ?-smooth muscle actin reactivity. In
contrast, scFv A7 showed no reactivity with the micro-
vasculature found in these synovium samples.
Finally, in order to establish whether the reactiv-
ity of scFv A7 is specific to the microvasculature of
arthritic synovium or a common feature of neovasculo-
genesis related to the presence of inflammation, we
examined scFv A7 staining in tissue samples from pa-
tients with Crohn’s disease (n ? 7) and psoriasis (n ? 5),
where the presence of microvasculature was detected
using anti-human vWF. The results presented in Figure
6 demonstrate that scFv A7 exhibits no detectable
reactivity with the microvasculature found in tissues
from patients with either Crohn’s disease or psoriasis.
Thus, these results demonstrate that the target epitope
for scFv A7 is absent from normal human tissues and
microvasculature and is not expressed in the neovascu-
logenesis seen in inflammatory conditions. Taken to-
gether, these results further support the conclusion that
scFv A7 is specific for the microvasculature found in
Over the past decade, the therapy of RA has been
transformed through the application of recombinant
Figure 5. Immunohistochemistry of scFv A7 in normal human tissues. A, Assessment of reactivity of scFv A7 antibody with normal human tissues
using a paraffin-embedded whole-body survey tissue microarray. Bound biotinylated scFv A7 antibody was detected using avidin–HRP conjugate.
The presence of blood vessels in tissue samples was visualized with anti-human vWF antibody and an HRP-labeled secondary antibody.
Representative areas in selected samples at top (dashed circles) are presented at higher magnification at bottom. Bar ? 100 ?m. B, Reactivity of
scFv A7 with normal human synovial tissue. Sequential sections show blood vessels that stain with vWF and ?-actin. No reactivity is observed using
scFv A7 antibody. Images shown are representative of 11 independent samples examined. Arrows indicate the position of microvessels. Bar ? 50
?m. See Figure 1 for definitions.
Figure 6. Reactivity of scFv A7 antibody with the microvasculature of
inflammatory tissues. The reactivity of scFv A7 with normal colon,
colon from patients with Crohn’s disease, normal skin, and psoriatic
skin was assessed. The presence of microvasculature was visualized
using anti-human vWF antibody. Biotinylated scFv A7 was detected
with avidin–biotin–HRP complex, while anti-vWF antibody reactivity
was detected using an HRP-labeled secondary antibody. Images shown
are representative of the 5 normal colon samples, 7 samples of colon
from patients with Crohn’s disease, 5 psoriatic skin samples, and 3
normal skin samples examined. Bar ? 100 ?m. See Figure 1 for
3764KAMPERIDIS ET AL
antibodies targeting inflammatory cytokines (24). How-
ever, despite the obvious impact of these agents, high-
magnitude responses and treatment-free remission re-
main elusive goals (29,30). Further, many patients
remain nonresponders or partial responders (14), and
within the responder cohort a loss of efficacy can be seen
over time, as can specific adverse effects (31,32).
The development of new therapeutics for RA,
with the ability to elicit greater clinical responses and
acceptable safety profiles, remains an unmet need. To-
ward this aim, we have used a human synovial xenograft
model established in our laboratory (19,20) to carry out
in vivo phage display selection of scFv antibodies with
specificity for the human synovial microvasculature. In
this model, synovial grafts implanted subcutaneously
into SCID mice remain viable and continue to express
human tissue–specific markers (17,20). Using this ap-
proach we have isolated and characterized scFv A7, a
novel human scFv antibody that efficiently and prefer-
entially targets the synovial microvasculature in RA, and
we have demonstrated that this antibody specifically
recognizes perivascular cells in this tissue. Abnormalities
of vascular morphology and angiogenesis in arthritic
synovium have been previously described at the macro-
scopic, histologic, and molecular levels (11,33). It is well
established that blood vessels of inflammatory tissue
lack the tight endothelial monolayer essential for normal
barrier function, resulting in increased endothelial per-
meability (leakiness) and extravasation of immune cells
to the extracellular space (34,35). In this context, given
that our in vivo phage screening strategy is against
human synovial grafts vascularized by permeable vessels,
the selection of an antibody that recognizes a stromal
vascular antigen is not surprising.
Our results demonstrate that the reactivity of
scFv A7 is specific to the microvasculature of arthritic
synovium, since the antibody does not exhibit reactivity
with the microvasculature or other cellular components
of normal human tissue from a spectrum of organs.
Further, expression of the scFv A7 epitope is not a
general feature of neovasculogenesis, since we detected
no binding to the microvasculature of the tissue from
patients with Crohn’s disease or psoriasis. These results
indicate that the expression of the epitope for scFv A7 is
likely to be tissue specific and restricted to the micro-
vasculature found in arthritic synovium rather than a
feature of the microvasculature seen in neoangiogenesis
or vasculogenesis in inflammatory diseases. The specific
reactivity of scFv A7 suggests that the target molecule
for scFv A7 may have potential as a biomarker in
arthritis and may also have applications as an immuno-
therapeutic target in the development of new strategies
for therapy of this condition.
Angiogenesis is an important and possibly a
primary event in the pathogenesis of the chronic inflam-
matory process of RA (36). Hence, targeting angiogen-
esis could play a part in a polypharmacy intervention
strategy for the treatment of arthritic disease (9,37–39).
In the course of angiogenesis, the associated tissue
remodeling leads to the expression and/or exposure of
molecules on endothelial and perivascular cells, which
are inaccessible, much lower in abundance, or undetect-
able in healthy adult tissues. For example, the oncofetal
extra domain B (ED-B) of fibronectin represents one of
the best characterized markers of angiogenesis (37) and
is abundantly expressed in many diseases including RA
(38,40). Moreover, antibody-mediated targeted delivery
of proinflammatory cytokines using L19, the human
antibody specific for ED-B (41), can result in a signifi-
cant increase in the therapeutic index of biopharmaceu-
ticals in animal models of cancer (42,43) and has re-
cently been evaluated for interleukin-2 (IL-2) and TNF?
delivery in phase I and II clinical trials (39). Further, L19
together with F8, an antibody specific for the ED-A of
fibronectin, have been used to deliver IL-10 to inhibit
the progression of collagen-induced arthritis (38,44).
Most recently, 3 recombinant human antibodies specific
for matrix metalloproteinases 1, 2, and 3 have been
developed and are currently being evaluated for
antibody-based pharmacodelivery applications in arthri-
Taken together, these findings clearly demon-
strate the utility of perivascular and stromal targeting for
the development of ligand-based strategies for treat-
ment of arthritic disease. In this context, we have shown
that in vivo, scFv A7 can target the microvasculature of
arthritic synovium efficiently and is preferentially re-
tained on the target tissue for at least 24 hours after
systemic administration. These data provide functional
evidence for the potential use of scFv A7 as an agent to
target therapeutics to the arthritic joint.
Although vascular targeting research has mainly
focused on tumor angiogenesis, the development of
nononcologic applications has recently gained momen-
tum and is likely to become an important area of
pharmaceutical intervention. Over the last decade, a
spectrum of innovative bispecific antibody formats has
been described, with scFv fragments being extensively
used as fundamental building blocks to develop 13 novel
antibodies that are currently in clinical phase I and II
trials (46), as well as a spectrum of new candidate
antibodies with biologic potency (47, 48). As described
TARGETED THERAPY OF HUMAN ARTHRITIS 3765
here, scFv A7 represents a new building block for the
development of vascular targeting of biopharmaceuti-
cals, capable of selective accumulation at neovascular
sites in RA.
We are grateful to Professor Thomas MacDonald for
providing tissue samples from patients with Crohn’s disease
and Professor Rino Cerio for providing tissue samples of
psoriatic skin. We thank Dr. Vineeth Rajkumar for advice and
assistance with image analysis.
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Pitzalis had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Kamperidis, Kamalati, Ferrari, Jones,
Garrood, Smith, Diez-Posada, Hughes, Finucane, Mather, Nissim,
Acquisition of data. Kamperidis, Kamalati, Ferrari, Jones, Garrood,
Smith, Diez-Posada, Hughes, Finucane, Mather, Nissim.
Analysis and interpretation of data. Kamperidis, Kamalati, Ferrari,
Jones, Garrood, Smith, Diez-Posada, Hughes, Finucane, Mather,
Nissim, George, Pitzalis.
1. Callahan LF. Awareness of the prevalence and impact of arthritis:
the role of health professionals [editorial]. Arthritis Care Res
2. Young A, Koduri G, Batley M, Kulinskaya E, Gough A, Norton S,
et al. Mortality in rheumatoid arthritis: increased in the early
course of disease, in ischaemic heart disease and in pulmonary
fibrosis. Rheumatology (Oxford) 2007;46:350–7.
3. Paleolog EM. Angiogenesis in rheumatoid arthritis. Arthritis Res
4. Choy EH, Panayi GS. Cytokine pathways and joint inflammation
in rheumatoid arthritis. N Engl J Med 2001;344:907–16.
5. Szekanecz Z, Koch AE. Vascular involvement in rheumatic dis-
eases: ‘vascular rheumatology.’ Arthritis Res Ther 2008;10:224.
6. Paleolog EM. The vasculature in rheumatoid arthritis: cause or
consequence? Int J Exp Pathol 2009;90:249–61.
7. Hirohata S, Sakakibara J. Angioneogenesis as a possible elusive
triggering factor in rheumatoid arthritis. Lancet 1999;353:1331.
8. Clavel G, Bessis N, Lemeiter D, Fardellone P, Mejjad O, Menard
JF, et al. Angiogenesis markers (VEGF, soluble receptor of VEGF
and angiopoietin-1) in very early arthritis and their association
with inflammation and joint destruction. Clin Immunol 2007;124:
9. Khong TL, Larsen H, Raatz Y, Paleolog E. Angiogenesis as a
therapeutic target in arthritis: learning the lessons of the colorectal
cancer experience. Angiogenesis 2007;10:243–58.
10. Lainer-Carr D, Brahn E. Angiogenesis inhibition as a therapeutic
approach for inflammatory synovitis. Nat Clin Pract Rheumatol
11. Szekanecz Z, Besenyei T, Paragh G, Koch AE. New insights in
synovial angiogenesis. Joint Bone Spine 2010;77:13–9.
12. Rothe A, Power BE, Hudson PJ. Therapeutic advances in rheu-
matology with the use of recombinant proteins. Nat Clin Pract
13. Kremer JM. Rational use of new and existing disease-modifying
agents in rheumatoid arthritis. Ann Intern Med 2001;134:695–706.
14. Taylor PC, Feldmann M. Anti-TNF biologic agents: still the
therapy of choice for rheumatoid arthritis. Nat Rev Rheumatol
15. Khraishi M. Comparative overview of safety of the biologics in
rheumatoid arthritis. J Rheumatol Suppl 2009;82:25–32.
16. Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJ. The
safety and side effects of monoclonal antibodies. Nat Rev Drug
17. Garrood T, Blades M, Haskard DO, Mather S, Pitzalis C. A novel
model for the pre-clinical imaging of inflamed human synovial
vasculature. Rheumatology (Oxford) 2009;48:926–31.
18. Lee L, Buckley C, Blades MC, Panayi G, George AJ, Pitzalis C.
Identification of synovium-specific homing peptides by in vivo
phage display selection. Arthritis Rheum 2002;46:2109–20.
19. George AJ, Lee L, Pitzalis C. Isolating ligands specific for human
vasculature using in vivo phage selection. Trends Biotechnol
20. Wahid S, Blades MC, De Lord D, Brown I, Blake G, Yanni G, et
al. Tumour necrosis factor-alpha (TNF-?) enhances lymphocyte
migration into rheumatoid synovial tissue transplanted into severe
combined immunodeficient (SCID) mice. Clin Exp Immunol
21. De Wildt RM, Mundy CR, Gorick BD, Tomlinson IM. Antibody
arrays for high-throughput screening of antibody-antigen interac-
tions. Nat Biotechnol 2000;18:989–94.
22. Hughes C, Faurholm B, Dell’Accio F, Manzo A, Seed M, Eltawil
N, et al. Human single-chain variable fragment that specifically
targets arthritic cartilage. Arthritis Rheum 2010;62:1007–16.
23. Goletz S, Christensen PA, Kristensen P, Blohm D, Tomlinson I,
Winter G, et al. Selection of large diversities of antiidiotypic
antibody fragments by phage display. J Mol Biol 2002;315:
24. Harrison JL, Williams SC, Winter G, Nissim A. Screening of
phage antibody libraries. Methods Enzymol 1996;267:83–109.
25. Visser GW, Klok RP, Gebbinck JW, ter Linden T, van Dongen
GA, Molthoff CF. Optimal quality131I-monoclonal antibodies on
high-dose labeling in a large reaction volume and temporarily
coating the antibody with IODO-GEN. J Nucl Med 2001;42:
26. Barlow AL, Macleod A, Noppen S, Sanderson J, Guerin CJ.
Colocalization analysis in fluorescence micrographs: verification of
a more accurate calculation of Pearson’s correlation coefficient.
Microsc Microanal 2010;16:710–24.
27. Adler J, Parmryd I. Quantifying colocalization by correlation: the
Pearson correlation coefficient is superior to the Mander’s overlap
coefficient. Cytometry A 2010;77:733–42.
28. Smith MD, Barg E, Weedon H, Papengelis V, Smeets T, Tak PP,
et al. Microarchitecture and protective mechanisms in synovial
tissue from clinically and arthroscopically normal knee joints. Ann
Rheum Dis 2003;62:303–7.
29. Emery P, Fleischmann RM, Moreland LW, Hsia EC, Strusberg I,
Durez P, et al. Golimumab, a human anti–tumor necrosis factor ?
monoclonal antibody, injected subcutaneously every four weeks in
methotrexate-naive patients with active rheumatoid arthritis:
twenty-four–week results of a phase III, multicenter, randomized,
double-blind, placebo-controlled study of golimumab before meth-
otrexate as first-line therapy for early-onset rheumatoid arthritis
[published erratum appears in Arthritis Rheum 2010;62:3005].
Arthritis Rheum 2009;60:2272–83.
30. Keystone EC, Genovese MC, Klareskog L, Hsia EC, Hall ST,
Miranda PC, et al. Golimumab, a human antibody to tumour
necrosis factor ? given by monthly subcutaneous injections, in
3766 KAMPERIDIS ET AL
active rheumatoid arthritis despite methotrexate therapy: the
GO-FORWARD Study. Ann Rheum Dis 2009;68:789–96.
31. Van Vollenhoven RF. Treatment of rheumatoid arthritis: state of
the art 2009. Nat Rev Rheumatol 2009;5:531–41.
32. Lipsky PE. Are new agents needed to treat RA? Nat Rev
33. Szekanecz Z, Besenyei T, Szentpetery A, Koch AE. Angiogenesis
and vasculogenesis in rheumatoid arthritis. Curr Opin Rheumatol
34. Middleton J, Americh L, Gayon R, Julien D, Aguilar L, Amalric F,
et al. Endothelial cell phenotypes in the rheumatoid synovium:
activated, angiogenic, apoptotic and leaky. Arthritis Res Ther
35. Pitzalis C, Garrood T. From ubiquitous antigens to joint-specific
inflammation: could local vascular permeability be the missing
link? Trends Immunol 2006;27:299–302.
36. Alam C, Colville-Nash P, Seed M. Modelling angiogenesis in in-
flammation. In: Seed MP, Walsh DA, editors. Angiogenesis
in inflammation: mechanisms and clinical correlates. Basel:
Birkhauser Verlag; 2008. p. 99–148.
37. Rybak JN, Trachsel E, Scheuermann J, Neri D. Ligand-based
vascular targeting of disease. ChemMedChem 2007;2:22–40.
38. Trachsel E, Bootz F, Silacci M, Kaspar M, Kosmehl H, Neri D.
Antibody-mediated delivery of IL-10 inhibits the progression of
established collagen-induced arthritis. Arthritis Res Ther 2007;9:
39. Schliemann C, Neri D. Antibody-based vascular tumor targeting.
Recent Results Cancer Res 2010;180:201–16.
40. Trachsel E, Kaspar M, Bootz F, Detmar M, Neri D. A human mAb
specific to oncofetal fibronectin selectively targets chronic skin
inflammation in vivo. J Invest Dermatol 2007;127:881–6.
41. Pini A, Viti F, Santucci A, Carnemolla B, Zardi L, Neri P, et al.
Design and use of a phage display library: human antibodies with
subnanomolar affinity against a marker of angiogenesis eluted
from a two-dimensional gel. J Biol Chem 1998;273:21769–76.
42. Menrad A, Menssen HD. ED-B fibronectin as a target for
antibody-based cancer treatments. Expert Opin Ther Targets
43. Dela Cruz JS, Huang TH, Penichet ML, Morrison SL. Antibody-
cytokine fusion proteins: innovative weapons in the war against
cancer. Clin Exp Med 2004;4:57–64.
44. Schwager K, Kaspar M, Bootz F, Marcolongo R, Paresce E, Neri
D, et al. Preclinical characterization of DEKAVIL (F8-IL10), a
novel clinical-stage immunocytokine which inhibits the progres-
sion of collagen-induced arthritis. Arthritis Res Ther 2009;11:
45. Pfaffen S, Hemmerle T, Weber M, Neri D. Isolation and charac-
terization of human monoclonal antibodies specific to MMP-1A,
MMP-2 and MMP-3. Exp Cell Res 2009;316:836–47.
46. Chames P, Baty D. Bispecific antibodies for cancer therapy: the
light at the end of the tunnel? MAbs 2009;1:539–47.
47. Michaelson JS, Demarest SJ, Miller B, Amatucci A, Snyder WB,
Wu X, et al. Anti-tumor activity of stability-engineered IgG-like
bispecific antibodies targeting TRAIL-R2 and LT?R. MAbs 2009;
48. Mabry R, Lewis KE, Moore M, McKernan PA, Bukowski TR,
Bontadelli K, et al. Engineering of stable bispecific antibodies
targeting IL-17A and IL-23. Protein Eng Des Sel 2010;23:115–27.
TARGETED THERAPY OF HUMAN ARTHRITIS3767