Protein Engineering, Design & Selection vol. 19 no. 10 pp. 461–470, 2006
Published online July 25, 2006 doi:10.1093/protein/gzl031
A highly stable polyethylene glycol-conjugated
human single-chain antibody neutralizing
granulocyte-macrophage colony stimulating
factor at low nanomolar concentration
Eva-Maria Krinner1, Julia Hepp1, Patrick Hoffmann1,
Sandra Bruckmaier1, Laetitia Petersen1, Silke Petsch1,
Larissa Parr1, Ioana Schuster1, Susanne Mangold1,
Grit Lorenczewski1, Petra Lutterbüse1, Stefan Buziol1,
Inessa Hochheim1, Jörg Volkland1, Michael Mølhøj1,
Mirnalini Sriskandarajah1, Markus Strasser1,
Christian Itin1, Andreas Wolf1, Amartya Basu2,
Karen Yang2, David Filpula2, Poul Sørensen3,
Peter Kufer1, Patrick Baeuerle1 and Tobias Raum1,4
1
Micromet AG, Staffelseestr. 2, 81477 Munich, Germany, 2Enzon
Pharmaceuticals, 20 Kingsbridge Road, Piscataway, NJ 08854-3969, USA
and 3LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark
4
To whom correspondence should be addressed.
E-mail: [email protected]
E.-M.Krinner and J.Hepp contributed equally to this work
GM-CSF (granulocyte-macrophage colony stimulating
factor) plays a central role in inflammatory processes.
Treatment with antibodies neutralizing murine GM-CSF
showed significant therapeutic effects in mouse models of
inflammatory diseases. We constructed by phage display
technology a human scFv, which could potently neutralize
human GM-CSF. At first, a human VL repertoire was
combined with the VH domain of a parental GM-CSFneutralizing rat antibody. One dominant rat/human scFv
clone was selected, neutralizing human GM-CSF with an
IC50 of 7.3 nM. The human VL of this clone was then combined with a human VH repertoire. The latter preserved the
CDR 3 of the parental rat VH domain to retain binding
specificity. Several human scFvs were selected, which neutralized human GM-CSF at low nanomolar concentrations
(IC50 > 2.6 nM). To increase serum half-life, a branched
40 kDa PEG-polymer was coupled to the most potent GMCSF-neutralizing scFv (3077) via an additional C-terminal
cysteine. PEG conjugation had a negligible effect on the
in vitro neutralizing potential of the scFv, although it caused
a significant drop in binding affinity owing to a reduced
on-rate. It also significantly increased the stability of the
scFv at elevated temperatures. In mouse experiments, the
PEGylated scFv 3077 showed a significantly prolonged
elimination half-life of 59 h as compared with 2 h for the
unconjugated scFv version. PEGylated scFv 3077 is a potential candidate for development of a novel antibody therapy
to treat pro-inflammatory human diseases.
Keywords: GM-CSF/neutralization/PEGylation/phage
display/single chain antibody
Introduction
GM-CSF (granulocyte-macrophage colony stimulating factor)
was originally described as a potent stimulus of granulocyte
and macrophage precursor cells in vitro (Sheridan and Metcalf,
1973). The cytokine is an 23 kDa glycoprotein with a four
a-helical bundle structure (Diederichs et al., 1991). It binds to
a heterodimeric receptor composed of subunits belonging to
the type 1 cytokine receptor family (Shibuya et al., 1991;
Hercus et al., 1994). Further experiments have demonstrated
that GM-CSF also stimulates mature granulocytes and macrophages as well as antigen-presenting dendritic cells (Inaba
et al., 1992) to increase their anti-infective potential. Gene
knockout experiments in mice suggested that the major physiological role of GM-CSF is to maintain or stimulate the
functional activity of mature macrophages and granulocytes
rather than to control hematopoesis (Zhan et al., 1998).
GM-CSF is produced by activated T lymphocytes, endothelial cells, macrophages and stromal cells. In humans,
several chronic inflammatory diseases such as rheumatoid
arthritis (RA), multiple sclerosis (MS) and asthma are associated with systemically or locally increased GM-CSF levels.
One study showed elevated GM-CSF serum levels in 87 RA
patients compared with healthy volunteers (Fiehn et al., 1992).
Synovial fluid from inflamed arthritic joints of RA patients
also showed significantly increased levels of GM-CSF
(Alvaro-Gracia et al., 1991). In MS patients, elevated GMCSF levels were found in the cerebral fluid (Perrella et al.,
1993). Furthermore, GM-CSF plays a key role in respiratory
diseases such as asthma and chronic obstructive pulmonary
disease (COPD) (Balbi et al., 1997; Adkins et al., 1998;
Culpitt et al., 2003). In allergic asthma, GM-CSF produced
by bronchial epithelial cells is involved in enhancing survival
of infiltrated inflammatory cells and, thereby, maintains the
inflammatory response (Esnault and Malter, 2002).
Several animal models have demonstrated a high therapeutic
potential of neutralizing GM-CSF by antibodies. A study in
mice with collagen-induced arthritis (CIA) showed amelioration of the severity and speed of limb deformation by neutralizing murine GM-CSF with antibody 22E9 (Cook et al.,
2001), while administration of the cytokine increased the severity of CIA in DBA/1 mice (Campbell et al., 1997). In mice
with experimental autoimmune encephalo-myelitis (EAE), administration of antibody 22E9 has been shown to not only
prevent the onset of EAE but also to ameliorate established
disease (McQualter et al., 2001). Likewise, therapeutic effects
of GM-CSF neutralization have been observed in various models of lung inflammation and asthma (Bozinovski et al., 2004),
and a psoriasis model (Schon et al., 2000).
The potent anti-inflammatory activity of GM-CSFneutralizing antibodies in a variety of animal models prompted
us to develop a monoclonal antibody fragment with high potential to neutralize human GM-CSF. Because the antibody
fragment would be administered in conditions of enhanced
immune reactivity, we sought to give it the lowest possible
immunogenicity. For high-level production by Escherichia
coli or yeast fermentation, we decided to produce it as an
scFv protein. Lastly, for extension of serum half-life of the
scFv and further reduction of potential immunogenicity, the
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461
E.-M.Krinner et al.
scFv was conjugated with 40 kDa PEG (polyethylene glycol).
Here, we describe the generation and in vitro characteristics
of a PEGylated fully human scFv that neutralizes the
cell proliferation activity of human GM-CSF at low nanomolar
concentrations.
Materials and methods
Sources of recombinant human GM-CSF
Escherichia coli-derived: The hGM-CSF DNA fragment was
subcloned from the pORF-hGM-CSF (Novagen, USA) into
pET22b(+) (Novagen, USA). Expression of the human
(h)GM-CSF was performed in E.coli BL21(DE3) periplasm
according to standard methods. Purification of hGM-CSF
was done by metal affinity chromatography (IMAC) using a
Qiagen Ni-NTA Superflow column, followed by gel filtration
on a Superdex 200 Prep Grade column (Pharmacia, Sweden).
Yeast-derived: Leukine Liquid (Sargramostim) was
obtained from Berlex, USA.
Biotinylation of hGM-CSF
Biotinylation of E.coli-derived hGM-CSF was done in
phosphate-buffered saline (PBS)/5% DMSO (Sigma,
Germany) with a 5-fold molar excess of EZ-Link Sulfo
NHS-LC-LC Biotin (Pierce, USA) for 1 h at room temperature.
Biotinylated hGM-CSF was purified by anion exchange
chromatography according to standard protocols.
Fluorescein labeling of hGM-CSF
Conjugation of E.coli-derived hGM-CSF with FluoresceinNHS (Fluka, Switzerland) was performed in borate buffer
(0.05 M boric acid, 0.1 M NaCl, pH 8.5, 17.5% DMSO)
with a 5-fold molar excess of Fluorescein-NHS for 1 h
at room temperature. The conjugate was purified via gel filtration on Sephadex G25 medium (Amersham Biosciences,
Germany).
Cloning of parental scFv
A rat hybridoma cell line was identified, which produced
a monoclonal antibody neutralizing human GM-CSF. Its VL
and VH gene fragments were amplified as described in Raum
et al. (2001) and subcloned as functionally arranged VH(G4-S)3-VL scFv into the pBluescript derived periplasmic
expression vector pMicHis (Micromet AG, Germany).
Lymphocyte cDNA preparation for human VL and
VH repertoires
PBMCs were isolated by Ficoll density centrifugation from
five healthy donors. IgD+ B-cells were separated from PBMC
using magnetic beads (CELLectionTM Pan Mouse IgG Kit,
Dynal, Germany) coated with mouse anti-human IgD antibody
(PharMingen, Germany). Subsequently total RNA was
prepared from IgD+ B-cells or PBMCs (RNeasy Midi Kit,
Qiagen, Germany) and cDNA was synthesized by random
hexamer priming (Roche, Germany).
PCR amplification of variable regions
For PCR amplification of the Vk regions from IgD+ B-cell
cDNA, each primer of the 50 -huVk primer set (described in
Raum et al., 2001) was combined with the following 30 -huVk
primers: 30 -hu-Vk-J1-SpeI-BsiWI 50 -GACGACACTAGTTGCAGCCACCGTACGTTTGATTTCCACCTTGGTCC-30 ,
462
30 -hu-Vk-J2/4-SpeI-BsiWI 50 -GACGACACTAGTTGCAGCCACCGTACGTTTGATCTCCASCTTGGTCC-30 , 30 -hu-VkJ3-SpeI-BsiW 50 -GACGACACTAGTTGCAGCCA CCGTACGTTTGATATCCACGTTGGTCC-30 and 30 -hu-Vk-J5-SpeIBsiWI 50 -GACGACACT AGTTGCAGCCACCGTACGTTTAATCTCCAGTCGTGTCC-30 . Vk fragments were purified
from an agarose gel and pooled according to their germline
distributions, which were defined through sub-group-specific
primers. VH fragments were PCR-amplified from cDNA of
PBMC combining each primer from the 50 -huVH primer set
(described in Raum et al., 2001) with the following 30 -primers:
30 -huVH-J1-BstEII 50 -CTGAGGAGACGGTG ACC-30 and 30 huVH-J3-BstEII 50 -CTGAAGAGACGGTGACC-30 . The fragments were gel purified and pooled at a J1:J3 ratio of 3:1.
Starting from this pool, a CDR3-truncated intermediate was
generated by PCR amplification using the primers from the
50 -huVH primer set in combination with primers annealing in
the FR3 region of the 30 -huVH-FR3 primer set. In a third PCR
reaction, the parent VH CDR3 and the human FR4 region (JH3)
were added to these fragments by PCR.
Construction of scFv libraries
VL-selection: The Vk pools were cloned via SacI/SpeI into
the phagemid vector pMic5BHis (Micromet) containing the
sequence of the parental rat VH. Transformation of the
ligation products into E.coli Xl-1 blue was accomplished by
electroporation (2.5 kV, 0.2 cm gap cuvette, 25 mF, 200 O.
Determination of the library size and culture conditions after
electroporation was performed as described (Burton et al.,
1991). The bacterial culture was infected with 1 · 1012 pfu
of helper phage VCSM13 resulting in secretion of M13 phage
displaying scFv protein as a translational fusion to coat protein
III on its surface.
VH-selection: The pooled VH fragments were digested
with XhoI and BstEII. These digested fragments were
then ligated into phagemid vector pMic5BHis, which had
been previously restricted with the same two enzymes. Vector
pMic5BHis contained a sequence encoding a human VL (VL
5–306) isolated in the VL-selection to form, together with the
VH of the parental rat antibody, a hGM-CSF-specific scFv
fragment. VL 5–306 was inserted in vector pMic5BHis via
the restriction enzymes SacI and SpeI. Transformation and
rescue with VCSM13 helper phage was carried out as
described above.
Phage display selection
The phage library was harvested from the culture supernatant
according to Burton et al. (1991). For selection 1–10 · 1011
phage particles were incubated with biotinylated hGM-CSF for
1 h in a total volume of 0.5 ml PBS/0.5% BSA. Then 6 · 107
Streptavidin-coated magnetic beads (Dynabeads M-280
Streptavidin, Dynal, Germany) were added for another 0.5 h.
After repeating up to 10 washes with PBS/0.1% BSA, phages
were eluted by HCl-glycine, pH 2.2. After neutralization with
2 M Tris, pH 12, the eluate was used for infection of a 2 ml
culture of E.coli Xl-1 blue (OD600 = 1). Elution was repeated
with HCl-glycine, pH 1 for selection of human VL. In the
human VH selection, the second elution step was performed
by resuspending the selection beads in 100 ml E.coli Xl-1 blue
(OD600 = 1). After incubation for 15 min, beads were removed
and the culture was added to the E.coli culture infected with the
first eluate. Infected E.coli Xl-1 blue cultures were selected for
Neutralizing human anti-human GM-CSF antibody fragment
carbenicillin resistance and were subsequently infected with
VCSM13 helper phage to start the next selection round.
Expression and purification of soluble scFv
The pool of fragments obtained after four and five rounds
of panning were subcloned into pMicFlag/His plasmid
(pBluescript derivative for periplasmic expression of antibody
fragments, Micromet). Expression of multiple different clones
was performed in E.coli TG-1 in 96-well format. One hundred
microlitre LB/0.1% glucose were inoculated with 10 ml of
an overnight culture of single clones and grown for 4 h at
37 C. After addition of IPTG to a final concentration of
1 mM, the culture was grown at 30 C for another 18–20 h.
Per well, 40 ml of BEL-buffer (400 mM boric acid, 320 mM
NaCl, 4 mM EDTA pH 8.0 + 2.5 mg/ml lysozyme) was added
and shaken at room temperature for 1 h. Cellular debris was
eliminated by centrifugation and supernatants were tested by
ELISA. For small-scale expression of soluble scFv in E.coli,
preparation of periplasmic extracts was performed as described
in Raum et al. (2001). ScFv was collected for further examination by ELISA. Alternatively, one-step purification of the
scFv was performed in NiNTA spin columns (Qiagen,
Germany) according to the manufacturer’s instructions. For
large-scale production, scFv was either expressed in E.coli
BL21(DE3) from pMicFlag/His or in E.coli BL-21 AI from
pBAD peri-Kan (a derivative of pBAD; Invitrogen, Germany)
for periplasmic expression of antibody fragments. Single
colonies were grown in selective medium to an OD600 =
0.8. Then, IPTG (pMicFlag/His) or L-arabinose (pBAD
peri-Kan) was added to a final concentration of 1 mM or
0.8% (w/v), respectively, and the cultures were grown overnight at 30 C. Following harvest, bacteria were resuspended in
100 ml PBS and periplasmic preparation was performed as
described in Raum et al. (2001). For purification of scFv
produced on a large-scale as described above, a two-step
purification process of IMAC (Ni-NTA Superflow, Qiagen,
Germany) followed by gel filtration (HiLoadTM 16/60 Superdex 75 Prep Grade column, Pharmacia, Sweden) was applied.
Gel filtration resulted in clearly distinguishable peaks signifying monomer and dimer fractions.
DNA sequencing
DNA sequencing was performed at SequiServe (Germany).
Antibody sequences were analyzed according to V-base (http://
vbase.mrc-cpe.cam.ac.uk).
ELISA
Detection of scFv fragments from periplasmic extracts: One
mg/ml hGM-CSF was immobilized on microtiter plates
(Maxisorb, Nunc, Germany). Wells were blocked with PBS/
3% BSA. After incubation of periplasmic extracts for 1 h at
room temperature, bound scFv was detected by an antiFLAG M2 antibody (Sigma, Germany) followed by a PODconjugated goat-anti-mouse IgG antibody (Dianova,
Germany). The ELISA was developed with ABTS substrate
(Roche, Germany) and measured at 405 nm.
Quantification of scFv 3077 or scFv 3077-PEG40 plasma
concentrations: One mg/ml hGM-CSF was immobilized on
microtiter plates (Maxisorb, Nunc, Germany). After blocking
the wells with PBS/3% BSA, samples were added and incubated for 1 h at room temperature. Samples were prediluted
to appropriate concentrations in PBS/50% mouse plasma.
Anti-3077 scFv goat immunoserum was used for detection
of bound scFv, followed by a POD-conjugated rat anti-goat
antibody (Dianova, Germany). The ELISA was developed with
TMB substrate (Sigma, Germany) and measured at 450 nm.
Quantification of scFv 3077 and scFv-PEG40 was done by
means of a standard curve for scFv 3077 and scFv-PEG40,
respectively; calculation of the scFv concentrations was performed from optical readings within the linear range.
TF-1 proliferation inhibition assay
TF-1 cells (DSMZ ACC 334) were cultivated in RPMI 1640/
10% FCS in the presence of 2.5 ng/ml hGM-CSF. For the
inhibition assay TF-1 cells were washed twice with 1· PBS
and seeded at a density of 0.9 · 104 cells/well in RPMI 1640
(containing 10% FCS, but lacking hGM-CSF) in a 96-well flat
bottom microtest plate. A final concentration of 0.3 ng/ml
hGM-CSF was added to stimulate TF-1 cell proliferation. A
dilution series of scFv with final concentrations ranging from
0.4 to 4000 nM was added to inhibit proliferation. After
incubation for 72 h at 37 C in 5% CO2, the proliferative status
was determined by adding WST-1 reagent (Roche, Germany).
Viable cells were quantitated by measuring the absorbance at
450 nm. The data were analyzed and fitted for half-maximal
inhibition of proliferation (IC50) using the GraphPadPrism4
software.
FACS competition assay
Ten mg/ml parent antibody or small-scale purified scFv
was incubated with 0.4 mg/ml hGM-CSF-FITC conjugate in
PBS. The protein samples were left to equilibrate at 25 C for
1 h prior to addition of a suspension of TF-1 cells. TF-1 cells
were starved from hGM-CSF overnight, washed twice with
PBS/1% FCS/0.05% NaN3 and resuspended in 100 ml of
pre-equilibrated protein sample containing the hGM-CSFFITC and scFv. After 1 h at 4 C, cells were analyzed
by flow cytometry on a FACScalibur (Becton Dickinson,
Germany).
Surface plasmon resonance
Association and dissociation rate constants were determined by
surface plasmon resonance on the BIAcore 2000 (Biacore
AB, Switzerland). The surface of the CM5 sensor chip was
activated with NHS/EDC. The hGM-CSF was coupled by injection of 10 mg/ml hGM-CSF in 0.01 M sodium-acetate, pH
4.7. Approximately 800 response units were immobilized.
Binding experiments were performed by injecting serially diluted samples at a flow rate of 5 ml/min and HBS-EP (0.01 M
HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005 % surfactant P20) as running buffer at 25 C. Dissociation was monitored for 100 s. The association and dissociation rates (ka [1/
Ms] and kd [1/s], respectively) were calculated using the BIAevaluation software (3.2 RC1) with a 1:1 Langmuir binding
equation and the equilibrium dissociation constant KD [M]
was calculated from kd/ka. Standard deviations were calculated
from at least three independent fits in dilution experiments.
Site-specific PEGylation
Two subcloning steps were performed to provide an scFv 3077
variant that displayed a C-terminal cysteine suitable for sitespecific PEGylation (Yang et al., 2003). The resulting fragment was ligated into a pBAD peri-Kan vector for periplasmic
expression. Expression of the 3077_His6_Cys construct in
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E.-M.Krinner et al.
E.coli BL21-AI was carried out as described above. PEGylation was performed as in Yang et al. (2003). In brief, the
3077_His6_Cys construct was purified from periplasmic
extracts by a two-step process of IMAC (Ni-NTA Superflow,
Qiagen, Germany) followed by gel filtration (HiLoadTM 16/60
Superdex 75 Prep Grade column, Pharmacia, Sweden). After
IMAC purification and a reducing step with Dithiothreitol
(DTT), gel filtration was carried out. Immediately prior to
PEGylation, a PD10 SEC column (Amersham Biosciences,
Germany) was used to remove the DTT used in the gel filtration step. Protein-containing fractions were determined with a
Bradford assay (Bio-Rad, Germany) and pooled. mPEG2 Maleimide of 40 kDa molecular weight (Nektar Therapeutics,
USA) was added at a 10-fold molar excess over scFv polypeptide. Conjugation was performed for 2 h at room temperature in the dark. Separation of the PEGylated scFv from free
PEG and scFv molecules was performed by cation exchange
chromatography (ResourceS, CV = 1 ml), using a linear NaCl
gradient for elution. The running buffer contained 20 mM
citrate pH 4 and the gradient elution buffer was 1 M NaCl
and 20 mM citrate, pH 4. PEGylated scFv eluted from the
column at 140 mM NaCl. Protein concentration was monitored via absorption of the respective fractions at a wavelength
of 280 nm. Protein containing fractions were pooled, dialyzed
against PBS and analyzed for purity and PEGylation efficiency
using SDS–PAGE.
Determination of thermal stability
PEGylated and non-PEGylated scFv 3077 were diluted to a
final concentration of 25 mg/ml (1 mM). Aliquots (30 ml) were
heated in a water bath at temperatures varying from 40 to
100 C. The actual temperature was monitored in a reference
vial. After 5 min incubation the protein was snap-cooled on ice.
To test the neutralizing activity of a respective sample, a TF-1
proliferation-inhibition assay was performed in duplicate. TF-1
cells (0.9 · 104 per well) were seeded in a 96-well microtest
plate and 10 ml of the heated protein solution was added. A
final concentration of 0.3 ng/ml hGM-CSF was used to stimulate the proliferation of cells. After incubation at 37 C,
5% CO2 for 72 h, the proliferative activity of the TF-1 cells
was quantified by addition of WST-1 and by measurement of
the absorbance at 450 nm. As a control for neutralization, the
untreated scFv 3077 and the untreated PEGylated scFv 3077
were included in the proliferation experiments. Data were normalized defining the maximal neutralizing effect obtained with
the respective untreated scFvs as 0% proliferation and the
minimal neutralizing effect of scFv heated at 100 C as
100% proliferation of TF1 cells. The data were fitted using
the GraphPadPrism4 software.
Guanidinium hydrochloride treatment
scFv 3077 was mixed with the denaturant to a final
protein concentration of 5 mg/ml and guanidinium hydrochloride (GdnHCl) concentrations varying from 0 to 5 M. After
overnight equilibration at 10 C, the fluorescence emission
spectra from 320 to 370 nm were measured at an excitation
wavelength of 280 nm. Maximum emission wavelength was
determined for each spectrum. Data from two experiments
were included, normalized to the fraction of unfolded scFv
protein and analyzed in accordance with Pace (1990).
464
Pharmacokinetics of scFv 3077 and scFv 3077-PEG40
C57BL/6 mice (female, 9–10 weeks old, three animals per
group) were injected intravenously with 1.5 mg per kg of
scFv 3077 or scFv 3077-PEG40, respectively. Different groups
were alternatingly bled at 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8 and
24 h after scFv 3077 injection and after 0.08, 0.25, 0.5, 1, 3, 8,
24, 48, 72, 96 and 168 h after scFv 3077-PEG40 injection.
Serum concentrations of scFv 3077 and scFv 3077-PEG40
were quantified by ELISA.
Pharmocokinetic calculations were performed by the
pharmacokinetic software package WinNonlin Professional
4.1 (Pharsight Corporation, Mountain View, CA, 2003).
Parameters were determined by non-compartmental analysis
based on the model for intravenous bolus injection. The distribution half-life (T1/2-alpha) was calculated using a log-linear
regression of the first three to four sample time points whereas
the terminal elimination half-life (T1/2-beta) was calculated by
the last three to four sample time points with detectable scFv
concentration.
Results
Isolation of parental VH and VL-fragments and expression
of the parental scFv
Total RNA was isolated from a rat hybridoma cell line (MT-G02C) producing an antibody neutralizing hGM-CSF. This RNA
was transcribed into cDNA, and variable heavy (VH) and
light chain (VL) gene segments were amplified by PCR, and
sequenced. Gene segments for VH and VL domains were
re-amplified with primers introducing suitable restriction
sites followed by sub-cloning into a vector for periplasmic
production in E.coli. Periplasmic extracts from four transformants were tested for the presence of functional scFv that
would bind to hGM-CSF in ELISA. However, none of the
clones showed a positive signal. By producing the scFv in
E.coli cytoplasm as inclusion bodies, small amounts of functional scFv could be obtained after a refolding procedure (data
not shown), demonstrating that the scFv was expressed but
not produced in a functional form in E.coli periplasm or
cytoplasm.
Selection of a human light chain
To overcome the lack of functional expression in E.coli and
to convert the parental rat scFv into a human equivalent of
reduced immunogenicity, a two-step phage display guided
selection strategy was applied as schematically outlined in
Figure 1.
In a first step, the parental heavy chain was sub-cloned into a
phage display vector and combined with a human kappa light
chain repertoire from B cells of five healthy donors. Each
plasmid in the library contained the parental rat VH combined
with a distinct human VL gene. After expression on phage, a
pool of scFvs was generated in which rat VH and human VL are
joined via a (Gly4Ser)3 flexible linker. The resulting library
consisted of 3 · 108 independent clones. Five rounds of phage
display selection were carried out using soluble biotinylated
hGM-CSF as antigen. To select for strong binders, the stringency was increased during selection by decreasing antigen
concentration from 100 nM (rounds 1–3) to 10 nM (round 4),
and finally to 1 nM (round 5).
Neutralizing human anti-human GM-CSF antibody fragment
Fig. 1. Strategy of guided selection and modification leading from a rat
monoclonal antibody to a PEGylated fully human scFv.
Phages selected after four and five rounds were expressed
as scFvs in the periplasm of E.coli. The scFv molecules
were affinity-purified by spin columns, followed by testing in
ELISA for specific binding to hGM-CSF. Human GM-CSF
used for phage selection had been produced in prokaryotes
and, therefore, lacked N-glycosylation, an attribute that,
however, allowed for more effective biotinylation. To ignore
binders that specifically recognized the non-glycosylated form
of the cytokine, a glycosylated form of hGM-CSF produced
in yeast was used for screening of scFvs in ELISA. A total
of five clones showed strong binding to the antigen (4–301,
4–306, 5–301, 5–306, 5–310) (Figure 2A). All three binders
identified from the fifth selection round (5–301, 5–306, 5–310)
and clone 4–306 were identical in sequence. These clones are
in the following collectively referred to as clone 5–306. Clone
4–301 had a different sequence but showed homology to clone
5–306 at the amino acid level. Both sequences were derived
from the V kappa 1 germline subfamily gene Vk1-O12.
GM-CSF neutralizing activity of half-human scFvs
Cytokine binding is necessary but not sufficient for neutralizing GM-CSF. ELISA-positive clones 5–306 and 4–301
were, therefore, tested for the ability to inhibit the binding of
hGM-CSF to its high-affinity receptor expressed on TF-1 cells.
Binding of fluorescently-labeled hGM-CSF-FITC to TF-1 cells
was detected by flow cytometry. Purified scFvs from clones 5–
306 and 4–301 were pre-incubated with hGM-CSF-FITC and
the mixture used for staining of TF-1 cells. ScFv from the
dominant clone 5–306 as well as the parental rat antibody,
inhibited binding of FITC-labeled GM-CSF, while the scFv
from clone 4–301 still allowed binding of labeled GM-CSF to
TF-1 cells (Figure 2B and C). Clone 5–306 was used for further
analysis and guided selection.
An assay was developed that monitored the effect of scFvs
on neutralizing the proliferating activity of hGM-CSF on
cell line TF-1. ScFv from clone 5–306 was purified from
Fig. 2. Characterization of rat/human scFvs selected by phage display. (A)
Analysis for hGM-CSF binding in ELISA. Crude periplasmic extracts from
20 selected clones were tested. Higher absorption signals indicate stronger
binding to hGM-CSF. The DNA sequences of the five labeled clones were
determined. Identical sequences are indicated by asterisks (*). (B) FACS
analysis of scFvs for inhibition of labeled GM-CSF binding to TF-1 cells.
ScFv 4-301 and 5-306 were incubated with 0.4 mg/ml FITC-labeled hGMCSF and used to stain TF-1 cells that had been starved from hGM-CSF
overnight. Filled graphs in each plot indicate the signal of hGM-CSF FITC on
TF-1 cells in the absence of antibodies; empty graphs show signals in the
presence of antibodies. A bar graph of the respective mean fluorescence
intensities of peak signals from FACS histograms is depicted in (C). Lower
MFI values indicate inhibition of cell binding by labeled hGM-CSF.
periplasmic preparations by IMAC. Monomeric and dimeric
isoforms were separated by gel filtration and both tested for
bioactivity. Proliferation of TF-1 cells was induced by hGMCSF in the presence of a serial dilution of scFv and viable cells
quantitated after 72 h by addition of WST-1. ScFv 5-306
inhibited proliferation of GM-CSF-stimulated TF-1 in a
dose-dependent form. Monomeric and the dimeric variants
were similarly active. IC50 values of 7.3 nM and 3.5 nM
were determined for monomeric and dimeric scFvs, respectively (Figure 3).
Selection of a human heavy chain
For the final generation of a hGM-CSF-neutralizing scFv, the
parental rat VH domain in clone 5–306 was complemented by a
human VH repertoire that preserved the 10 amino acid-long
CDR3 from the parental VH domain. The resulting phage
library was then used for phage selection with soluble biotinylated hGM-CSF as antigen. Combination of the pre-selected
VL 5–306 with the human VH repertoire resulted in an antibody
phage library of 1.6 · 108 independent clones. Antigen concentration was reduced for selection from 100 nM in the first
round to 10 nM for the following three rounds. Highly enriched
465
E.-M.Krinner et al.
Fig. 3. Effect of rat/human scFv 5-306 monomer and dimer on GM-CSF
induced proliferation of TF-1 cells. Purified monomeric (filled squares) and
dimeric (filled triangle) scFv 5-306 were analyzed in dilution series for
inhibition of GM-CSF-induced TF-1 cell proliferation in a 72 h assay. Bars
give standard deviations from duplicate experiments.
phages were harvested and VH/VL fragments were sub-cloned
into a vector for periplasmic expression in E.coli. ScFv proteins from 160 clones were prepared and tested for binding to
immobilized hGM-CSF in ELISA. Over 80% of scFvs showed
strong antigen binding. Sequence analysis of the 13 strongest
binders led to the identification of four different sequences.
ScFv proteins expressed by representative clones encoding
these four sequences (clones nos 3035, 3039, 3077 and
3080) gave strong binding signals to hGM-CSF in ELISA
(Figure 4A). All VH sequences were derived from the
human germline sequence VH-1 1-O2.
GM-CSF neutralizing activity of human scFvs
ScFvs from all four clones were produced and monomeric
fractions analyzed for their neutralizing potential in the TF-1
cell proliferation assay. All proteins inhibited hGM-CSFdependent cell proliferation in a 72 h assay (Figure 4B).
IC50 values were 130 nM for scFv 3039, 19.1 nM for scFv
3080, 3.2 nM for scFv 3025 and 2.6 nM for scFv 3077.
Repetitions of the assay showed considerable inter-assay variance of IC50 values, but the relative potency of the molecules
remained constant.
Binding constants of scFvs 3035, 3039, 3077 and 3080 were
determined by surface plasmon resonance analysis using
immobilized hGM-CSF. As shown in Table I, dissociation
constants (KD) ranged from 106 to 109 M. With one exception, binding affinities were in line with the neutralizing
activity of scFvs in the TF-1 cell proliferation assay. Although
scFvs from clones 3035 and 3077 showed a very similar
neutralizing efficacy in the TF-1 assay, the KD of scFv 3077
(KD = 1.1 · 109 M) was seven times lower than for scFv 3035
(KD = 9 · 109 M) due to a high association rate (ka = 1.7 · 106
M s1). The better kinetic properties of 3077 in this low
nanomolar affinity range may have been concealed in the neutralization assay because of the long incubation period of 72 h.
Based on highest affinity and biological activity, scFv 3077
was selected for further analysis and modification.
Thermodynamic stability of scFv 3077
The selected scFv 3077 was investigated for its thermodynamic stability using GdnHCl equilibrium denaturation. The
GndHCl-induced unfolding of the protein was measured by the
change in emission maximum at an excitation wavelength of
280 nm after equilibration with varying concentrations of the
466
Fig. 4. Characterization of fully human scFvs obtained from completed
guided selection by phage display. (A) Analysis of scFvs for hGM-CSF
binding in ELISA. Crude periplasmic extracts were tested. Higher absorption
signals indicate stronger binding to hGM-CSF. Phosphate-buffered saline
(PBS) and rat/human scFv 5-306 were included as controls. (B) Analysis of
purified monomeric human scFvs 3077 (open square), 3080 (closed triangle),
3039 (inverted triangle) and 3035 (closed circle) for neutralization of hGMCSF in the TF-1 cell proliferation assay. Error bars show standard deviations
from duplicate experiments.
Table I. Binding kinetics of the human anti-GM-CSF scFvs
ka (1/Ms)
3035
3039
3077
3080
1.6
5.7
1.7
1.0
6
6
6
6
1.1
0.4
1.1
0.5
kd (1/s)
·
·
·
·
105
103
106
105
1.5
9.7
1.9
3.5
6
6
6
6
0.4
0.1
0.2
0.2
·
·
·
·
103
103
103
103
KD (M)
IC50 (nM)
9 · 109
1.7 · 106
1.1 · 109
3.5 · 108
3.2
130.5
2.6
19.1
denaturing reagent. Figure 5 shows a co-operative two-step
unfolding behavior of the scFv with a first transition and midpoint of unfolding at 2.05 M GdnHCl and a second transition
and midpoint of unfolding at 4.23 M GdnHCl. This reflects a
high thermodynamic stability of scFv 3077.
PEGylation of scFv 3077
Owing to their small size, scFv proteins are rapidly cleared
from the circulation through the kidneys. Pharmacokinetic
studies have demonstrated that the circulating half-life of scFvs
and of other small proteins and chemicals can be substantially
prolonged by conjugation with PEG (Lee et al., 1999). Owing
to its favorable binding properties and stability, scFv from
clone 3077 was chosen for PEGylation. To this end, a variant
of scFv 3077 was constructed containing a C-terminal sulfhydryl group for site-directed conjugation by a maleimide-PEG
polymer (Yang et al., 2003). The coding sequence of scFv
3077 was amplified with primers introducing histidine and
Neutralizing human anti-human GM-CSF antibody fragment
Fig. 5. Normalized denaturation of scFv 3077 by guanidinium hydrochloride.
The unfolding transitions were measured by the change in the emission
maximum as a function of denaturant concentration at an excitation
wavelength of 280 nm.
cysteine codons at the C-terminus and restriction sites for
ligation into a new expression vector.
Production of scFv 3077 yielded 2.8 mg monomeric scFv
from a 5 l shaking flask culture. The proportion of monomer to
dimer was approximately 62% to 38%. In small-scale experiments, we observed a reduction of monomeric scFv portion
bearing a C-terminal cysteine and, therefore, aimed to produce
larger quantities of monomeric scFv 3077_His_Cys. For
improved expression, the scFv gene was subcloned into a
prokaryotic expression vector based on pBAD containing a
signal peptide coding region for efficient transport of the protein into the periplasm and produced in a 2 l fed batch fermenter run. The E.coli pellet from 750 ml culture volume was
used for periplasmic extraction and purification. We obtained
5.2 mg monomeric scFv 3077_His_Cys at a proportion of 42%
monomer and 58% dimer. The resulting monomeric scFv
3077_His_Cys was then exposed to reducing conditions by
addition of DTT for dissociation of C-terminally linked
scFv dimers and to generate reactive C-terminal sulfhydryl
groups. Reducing conditions were maintained during separation of monomeric scFv by gel filtration. DTT was removed
right before conjugation of branched 40 kDa PEG-maleimide.
These experimental conditions allowed selective conjugation
of the 40 kDa PEG-Mal to the free C-terminal cysteine residue
by formation of a thioether bond between maleimide and sulfhydryl groups.
Figure 6 shows that PEGylation of scFv 3077_His_Cys
was very effective and yielded a homogeneous product
with an apparent molecular size of 100 kDa. In the elution
profile of PEGylated scFv 3077_His_Cys from a cation
exchange chromatography column, the signal for residual nonPEGylated scFv was at the limit of detection (Figure 6A).
Analysis of column fractions A9 and A10 by SDS–PAGE
(Figure 6B, lane 4) showed that cation exchange chromatography completely removed non-PEGylated scFv left
after coupling reaction (compare with lanes 2 and 3). NonPEGylated, maleimide-activated scFv 3077_His_Cys had a
molecular size of 31 kDa (Figure 6B, lane 1), which increased
to 100 kDa upon reaction with a 40 kDa PEG moiety.
Under the present reaction conditions, more than 90% of
the scFv was conjugated with PEG, as can be estimated
from Figure 6A and B.
Affinity and activity of naked and PEGylated scFv
Purified PEGylated scFv 3077_His_Cys (3077-PEG40) was
compared with scFv 3077 for kinetic parameters and the
Fig. 6. Purification and analysis of PEGylated scFv 3077. (A) Purification of
PEGylated scFv 3077 by cation exchange chromatography. A linear salt
gradient shown by the dashed line was used for elution. The solid line shows
the elution profile recorded at an absorption of 280 nm. (B) SDS–PAGE
analysis. Lane 1, monomeric non-PEGylated scFv 3077 after blocking the
reduced C-terminal cysteine residue with ethylmaleimide; lane 2, scFv 3077
after the PEGylation reaction in conjugation buffer; lane 3, PEGylated scFv
3077 dialyzed against running buffer before cation exchange chromatography;
lane 4, PEGylated scFv from pooled cation exchange chromatography
fractions A9 to 10. Molecular size markers are in the right lane and the sizes
in kDa are indicated.
Fig. 7. Comparison of hGM-CSF inhibitory activities of scFv 3077 and scFv
3077-PEG40. Purified monomeric scFvs 3077 (closed square) and 3077PEG40 (closed triangle) were side-by-side tested for their capability to inhibit
hGM-CSF-dependent proliferation of TF-1 cells. Error bars show standard
deviations from duplicate experiments.
potential to neutralize the activity of hGM-CSF. For 3077PEG40, an association rate of 2.7 · 104 Ms1 and a dissociation rate of 1.1 · 103s1 were determined by surface plasmon
resonance analysis, resulting in a KD of 4.1 · 108 M. This
corresponded to a 60-fold reduction in the association rate of
3077-PEG40 compared with its non-PEGylated counterpart,
while the dissociation rate remained essentially unaffected
by PEGylation. Overall, PEGylation led to a 36-fold decrease
in affinity from 1.1 · 109 M for scFv 3077 to 4.1 · 108 M for
3077-PEG40.
We next tested the influence of PEGylation on GM-CSF
neutralization in the TF-1 cell proliferation assay. Surprisingly,
3077-PEG40 exhibited the same neutralizing efficacy as nonPEGylated scFv 3077 (Figure 7). Apparently the decrease in
affinity caused by PEGylation did not result in a corresponding
reduction of the biological activity of the molecule in a longterm assay.
Thermal stability of naked and PEGylated scFv 3077
In order to investigate the impact of PEGylation on the scFv
with regard to its thermal stability, purified scFv 3077 and its
PEGylated counterpart were exposed to temperatures varying
from 40 C up to 100 C. After heating the compounds for 5 min
467
E.-M.Krinner et al.
Fig. 8. Comparison of thermal stability of scFv 3077 and scFv 3077-PEG40.
Purified scFvs 3077 (closed square) and 3077-PEG40 (closed triangle) were
tested for their biological activity in a TF-1 proliferation inhibition assay after
exposure to different temperatures. Data were normalized. Error bars show
standard deviations from duplicate experiments. Lower proliferation values
indicate higher biological activity of the respective scFv.
at the respective temperatures, samples were cooled down
on ice and assayed for their capability to neutralize GM-CSF
dependent TF-1 cell proliferation. As shown in Figure 8, a
significant difference in the sensitivity of the respective
PEGylated and non-PEGylated scFvs to increasing temperature was observed. scFv 3077 reached half-maximal temperature inhibition of its activity at 73 C, whereas the PEGylated
scFv 3077 reached half-maximal temperature inhibition at
83 C. Complete loss of neutralizing activity was seen after
incubation at 85 C for scFv 3077 and at 95 C for the PEGylated scFv 3077.
Pharmacokinetic properties of scFv 3077 and scFv
3077-PEG40
C57BL/6 mice were intravenously injected with 1.5 mg/kg
scFv 3077 or scFv 3077-PEG40, respectively, and mice bled at
different time points after injection. ScFv 3077 and scFv 3077PEG40 serum concentrations were quantified by specific ELISAs and serum concentrations versus time profiles generated
(Figure 9). Peak serum concentrations of the two constructs
were detected 5 min after bolus i.v. injection and reached
the lower limit of quantification after 6 h for scFv 3077 and
after 168 h for the scFv3077-PEG40. Elimination rate constants were determined and resulted in distribution half-lives
T1/2-alpha of 0.12 6 0.01 for scFv 3077 and 0.39 6 0.04 for scFv
3077-PEG40, respectively, and terminal elimination half-lives
T1/2-beta of 1.99 6 0.49 h for scFv 3077 and 59.36 6 0.31 for
scFv 3077-PEG40. It was observed that the prolonged half-life
of the PEGylated version correlated with an increased area
under the curve (AUClast) of 156.80 6 9.11 h mg/ml as compared with 4.91 6 0.37 h mg/ml for scFv 3077. Accordingly, a
significantly reduced clearance of 8.52 6 0.38 mg/h kg was
observed for the PEGylated molecule in comparison with
301.22 6 22.47 mg/h kg of the unconjugated scFv.
Discussion
The present work describes the construction and characterization of a PEGylated human scFv neutralizing hGM-CSF. As
a starting point, the scFv of a rat monoclonal antibody neutralizing the biological activity of hGM-CSF was constructed
and produced in the periplasm of E.coli. In this manner, no
468
Fig. 9. Pharmocokinetics of scFv 3077 and scFv 3077-PEG40. C57/BL6
mice were intravenously injected with 1.5 mg/kg scFv 3077 or scFv 3077PEG40. Groups of three mice each were bled at different time points and scFv
serum concentrations were quantified by specific ELISAs. In (A) scFv serum
concentrations determined at time points within the first 25 h after i.v.
administration of scFv 3077 (closed square) or scFv 3077-PEG40 (closed
triangle) are plotted versus time. (B) depicts scFv serum concentrations
determined for time points up to 168 h after administration.
functional scFv could be obtained despite the fact that
the protein, when expressed in inclusion bodies, regained
biological activity after refolding. Deficient activity of scFvs
is frequently observed when these are constructed from VH and
VL domains of monoclonal antibodies originating from hybridoma cells (e.g. Rojas et al., 2004). After replacing the original
rat kappa light chain with a human kappa repertoire and
selecting for hGM-CSF binding using phage display technology, rat/human scFvs could be isolated that bound specifically
to the antigen and could be well expressed in a functional form
in the periplasm of E.coli. This finding demonstrates that
exchange of the light chain can foster functional producibility
of an scFv, which is otherwise not expressed in a soluble form
in the periplasm of prokaryotes. A similar observation was
published very recently. An scFv derived from a monoclonal
antibody binding to N-glycolyl GM3 ganglioside could only be
expressed functionally in the periplasm of E.coli after the
exchange of the original light chain against a light chain
selected from human and murine VL repertoires by phage display (Rojas et al., 2004). Light chain selection can thus be
a general means of stabilizing scFvs while preserving their
binding specificity.
One of the two human light chains allowing functional
expression of an hGM-CSF-specific scFv in E.coli was
Neutralizing human anti-human GM-CSF antibody fragment
dominantly selected. Both human sequences are derived from
the human Vkappa 1 subgroup, in particular the O12 Vkappa
segment. The human Vkappa 1 subgroup is known to be preferentially selected in phage display experiments (e.g. Xu
et al., 2004), probably due to its high intrinsic stability
(Ewert et al., 2003). This potential gain in intrinsic stability
could also be a reason that the selected human kappa light
chains are able to stabilize the original rat VH upon expression
in E.coli. The difference between the original rat VL and the
selected human VLs is also illustrated by the observation that
the two selected human sequences share only 60% identity to
the original rat VL. Analysis of the selected human VLs and the
parental rat VL regarding their CDR structures according to
Al-Lazikani et al. (1997) revealed the same canonical structures in CDR1 and CDR2 indicating a common architecture of
the CDRs. Apparently, owing to epitopic guidance by retaining
the parental VH (‘guided selection’), the selected rat/human
scFvs still showed GM-CSF neutralizing activity in a low
nanomolar range. In most cases, guided selection approaches
maintain the epitopic specificity of the parental antibody
(Jespers et al., 1994; Figini et al., 1998; Raum et al., 2001),
although differences in the fine specificity have been observed
(Ohlin et al., 1996).
Since human-derived proteins, especially antibodies and
fragments thereof, are thought to have reduced immunogenic
potential in man compared with non-human proteins (Qu et al.,
2005), the rat/human scFv 5–306 was subjected to a further
round of phage display selection. In this second round, the rat
VH was exchanged with VH domains from a human repertoire.
On the notion that especially the CDR3 of VH is dominant for
antigenic fine specificity (Kabat and Wu, 1991; Collet et al.,
1992; Xu and Davis, 2000; Sidhu et al., 2004), the original VH
CDR3 was maintained in combination with one human FR4
(from JH3) and a diverse human repertoire of FR1-CDR1-FR2CDR2-FR3 derived from peripheral B cells (Rader et al., 1998;
Steinberger et al., 2000). Owing to the generally high sequence
diversity within the CDR3 of VH (Sanz, 1991), the 10 amino
acid CDR3 sequence in our scFv cannot be identified as
species-specific. Consequently, because the CDR3 is
embedded in a human VH domain and combined with a
human VL domain, the complete scFvs may be considered
to be fully human.
Using soluble hGM-CSF as antigen for phage display selection, four human scFvs could be isolated, which specifically
bound to hGM-CSF. All four human scFvs also showed GMCSF-neutralizing activity, indicating that the guided selection
approach preserved the binding specificity of the parental rat
monoclonal antibody. An IC50 range from 2 to 130 nM was
observed for the GM-CSF-neutralizing activity of human
scFvs. IC50 values correlated with binding affinities that
ranged from 1 to 1700 nM, indicating that increased binding
affinity may translate into prolonged retention of the antigen by
the antibody. A 10-fold higher on-rate of scFv 3077 compared
with scFv 3035 could apparently not produce a significant
difference in neutralizing activity under the experimental conditions of the TF-1 neutralization assay, because IC50 values
were comparable (2.6 and 3.2 nM, respectively). The molecule
chosen for further development, scFv 3077, was characterized
and exhibited properties comparable with stability engineered
scFv proteins (Worn and Pluckthun, 1998).
Serum half-life for scFvs has been determined to be on the
order of a few hours in humans (Larson et al., 1997; Fitch et al.,
1999) probably due to renal clearance (Hamilton et al., 2002).
Strategies have been established to prolong the half-life of such
molecules by coupling to serum protein (Smith et al., 2001),
or to polymeric organic compounds of high molecular weight.
It could be demonstrated that PEGylation of scFv molecules
dramatically increases their serum half-life (Lee et al., 1999).
To generate a more long-lived GM-CSF neutralizer, a version
of scFv 3077 was constructed with a C-terminal cysteine for
site-directed and quantitative coupling of 40 kDa PEG. When
compared side-by-side, non-PEGylated scFv 3077 and PEGylated 3077_His_Cys showed identical biological activity with
respect to neutralization of hGM-CSF. This was despite the
fact that PEGylated scFv 3077 suffered a distinct loss in affinity due to a reduced on-rate. Such an effect has previously
been observed (Yang et al., 2003). The reduced on-rate may
be attributable to several circumstances. One is a reduced
diffusion rate of PEGylated scFv 3077, which has double
the size of unconjugated scFv 3077. The other could be steric
hindrance for GM-CSF access and binding by the large hydrated PEG polymer. The equal biological activity of naked
and PEGylated scFv in the TF-1 assay suggests that over longer periods of time only off-rate matters for neutralizing activity. The same may be true for the in vivo situation. The high
biological activity of the molecules in a cell culture system
running for 72 h also suggests that the scFvs are sufficiently
stable and resistant to proteases in fetal calf serum or released
by TF-1 cells.
To further investigate the stabilizing effect of PEG described
for other proteins (Diwan and Park, 2001), scFv 3077 and
its PEGylated version were exposed to temperatures from 40
to 100 C and then tested in an activity-based assay. We
observed a significant increase in thermal stability of the
PEGylated scFv of 10 C. This effect may be due to the
conformational shielding of the scFv by the surrounding
PEG molecule that potentially reduces the ‘breathing’ of the
VH and VL domain and, therefore, stabilizes the respective
scFv. Besides an increase in molecular size, this stabilizing
effect of the PEG may contribute to the prolonged half-life of
PEGylated molecules in vivo by protection against serum
proteases.
C57BL/6 mice i.v. bolus injection experiments were
performed to address the pharmacokinetic profile of the
unconjugated scFv3077 versus its PEGylated counterpart. In
this experiment a 30-fold prolongation in in vivo half-life of the
PEGylated scFv 3077 could be observed, probably due to a
reduced renal clearance. Comparable data were observed
previously using PEGylated scFv fragments in mouse experiments (Yang et al., 2003).
The human scFv 3077 and its PEGylated counterpart 3077PEG40 are able to neutralize the proliferating activity of the
hGM-CSF molecule at low nanomolar levels with off-rates that
predict a durable sequestration of hGM-CSF preventing the
cytokine from binding to immune cells bearing high-affinity
GM-CSF receptor. This long-term sequestration of GM-CSF
may have clinical benefits in inflammatory and autoimmune
diseases comparable with the in vivo effects reported for the
monoclonal anti-mouse GM-CSF antibody 22E9. Owing to its
human origin, scFv 3077 and derivatives are expected to
exhibit very low immunogenicity in patients. This is particularly important when treating pro-inflammatory disease presenting with an enhanced reactivity of immune cells. The
non-PEGylated scFv may be useful in clinical settings
469
E.-M.Krinner et al.
where short-term GM-CSF-neutralizing activity is desirable, or
administration to a compartment where clearance is reduced.
Use of the PEGylated version of scFv 3077 may be more
desirable in clinical settings where long half-life and low immunogenicity may matter.
Acknowledgments
We thank Steve Zeman for his assistance and critical review in the preparation
of this manuscript.
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470
Received February 16, 2006; revised June 1, 2006;
accepted June 12, 2006
Edited by Prof Dr Hennie Hoogenboom