From the Department of Biochemistry, University of
Zürich, CH-8057 Zürich, § Division of Oncology,
Department of Internal Medicine, University Hospital,
CH-8044 Zürich, and
Center of Radiopharmaceutical
Science, Paul Scherrer Institute,
CH-5232 Villigen (PSI), Switzerland
Received for publication, December 26, 2000
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ABSTRACT |
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Multimerization of antibody fragments increases
the valency and the molecular weight, both identified as key features
in the design of the optimal targeting molecule. Here, we report the construction of mono-, di-, and tetrameric variants of the
anti-tumor p185HER-2 single chain Fv fragment 4D5 by
fusion of self-associating peptides to the carboxyl terminus. Dimeric
miniantibodies with a synthetic helix-turn-helix domain and tetrameric
ones with the multimerization domain of the human p53 protein were
produced in functional form in the periplasm of Escherichia
coli. We have directly compared these molecules and the
single-chain Fv fragment in the targeting of SK-OV-3 xenografts.
Tetramerization of the 4D5 antibody fragment resulted in increased
serum persistence, significantly reduced off-rate, due to the avidity
effect, both in surface plasmon resonance measurements on purified
p185HER-2 and on SK-OV-3 cells. The
99mtechnetium-tricarbonyl-labeled tetrameric 4D5-p53
miniantibody localized with the highest dose at the tumor and remained
stably bound for at least 72 h. The highest total dose was 4.3%
injected dose/g after 24 h, whereas the highest tumor-to-blood
ratio was found to be 13.5:1 after 48 h, with a total dose of
3.2% injected dose/g. The tetramer shows no higher avidity than the
dimer, presumably since the simultaneous binding to more than two
antigen molecules on the surface of cells is not possible, and the
improvement in performance over the dimer must at least be due in part
to the molecular weight. These results demonstrate that multimerization by self-associating peptides can be used for the development of more
effective targeting molecules for medical diagnostics and therapy.
One of the current visions in the medical application of
recombinant antibody technology (1) is the specific targeting and
delivery of effector agents such as radioisotopes, toxins, or enzymes
to tumors or other disease-related sites in the body. Unfortunately,
recombinant antibody fragments have exhibited poor in vivo
targeting efficiency, probably due to their fast clearance from the
blood circulation resulting in low total dose accumulation (2-4). The
targeting efficacy of antibody fragments can be improved by the use of
multimeric formats of antibody fragments with higher avidity and a
molecular weight slightly above the renal filtration threshold (5-7).
This has first been shown in studies in which Fab and
scFv1 fragments were
multimerized by chemical linkage (8-12). More efficient is the
multimerization of antibody fragments by modification of the
polypeptide sequence itself by recombinant DNA technology and the
subsequent purification of the multimeric protein from the bacterial
host. One strategy is to shorten the flexible peptide linker of scFv
fragments to make it impossible to form monomers (5, 13). The so-called
"diabodies" have been shown to be stable under in vivo
conditions and to enrich efficiently at xenografts (14, 15). Another
strategy is the fusion of the homodimerization domain CH3
to the carboxyl terminus of the polypeptide chain. By using this
approach in combination with a stabilizing intramolecular disulfide
bridge for an anti-carcinoembryonic antigen scFv fragment, very
promising tumor targeting data were reported (7). However, recombinant
molecules that contain additional disulfide bridges are usually
produced at lower yields in heterologous expression systems and are
generally less convenient to handle (16).
Instead of using whole protein domains self-associating peptides can be
used as multimerization modules to form so-called miniantibodies (6).
Here we report on the engineering and production of mono-, di-, and
tetrameric variants of the anti-p185HER-2 scFv fragment 4D5
in the periplasm of Escherichia coli and their in
vivo application for targeting xenografted SK-OV-3 tumors. The
comparison of the various formats is expected to provide insight into
the performance of such multimerized molecules for in vivo targeting purposes and serve as a proof of this concept under in
vivo conditions. We demonstrate that the in vivo
stability of the tetrameric miniantibody is high enough to result in
efficient tumor targeting and that such multimerized antibody fragments can be readily produced in the periplasm of E. coli (17, 18) and be used to deliver radionuclides or other effector molecules for
imaging and therapy.
Mammalian Cell Lines and Recombinant Antigen--
The breast
carcinoma cell line SK-BR-3 (HTB 30, ATCC, Rockville MD) and the
ovarian carcinoma cell line SK-OV-3 (HTB 77, ECACC, Salisbury, Wilts,
UK) were maintained in McCoy's 5A medium (Amimed BioConcept,
Pillschwill, Switzerland), supplemented with 15% bovine serum (Life
Technologies, Inc.). For binding experiments the adherent cell lines
were carefully detached by use of PBS containing 5 mM EDTA.
No trypsin was used to avoid enzymatic cleavage of cell surface
receptors. The purified recombinant antigen p185HER-2-ECD
was a kind gift of Dr. Paul Carter (Genentech Inc., CA).
Cloning of Constructs--
The scFv fragment of the human
anti-p185HER-2 antibody 4D5 was constructed (19) from the
Fab fragment (20) and had been used in several studies before (21-23).
The 4D5-dhlx miniantibody construct was obtained by ligation of
the EcoRI/HindIII fragment of the vector
pKM30-dhlx-his (24), containing the dhlx sequence and the His tag, into
the expression vector pIG6, containing the scFv fragment 4D5 (25). The
nucleotide sequence for the p53 tetramerization domain was isolated as
an EcoRI/AscI fragment from the plasmid pACK9-9p53,2 which contains
the p53 tetramerization domain (26). The construct 4D5-p53 was then
obtained by substituting the dhlx domain in the 4D5-dhlx construct by
the EcoRI/AscI fragment containing the p53 tetramerization domain.
Periplasmic Expression and Purification--
All constructs were
expressed in the periplasm of E. coli using the expression
vector pIG6 under the control of a lac promoter (25). For
large scale expression the constructs were transformed in the E. coli strain SB536 (27) and grown overnight. Twenty-five ml were
then used for inoculation of 1 liter of 2YT medium containing 1%
glucose and ampicillin (50 µg/ml) in a 5-liter baffled shake flask.
The culture was grown at room temperature, and expression was induced
with isopropyl-
Briefly, the monomeric scFv fragment 4D5 was purified as described
earlier (22). For purification of the miniantibodies 4D5-dhlx and
4D5-p53, the pellet of a 1-liter expression culture was resuspended in
50 ml of buffer containing 100 mM Tris (pH 7.0), 150 mM NaCl, 2 mM MgCl2, 0.01% Tween
20, DNase (1 mg/100 ml), RNase (1 mg/100 ml). The cell suspension was
lysed in two cycles with a French pressure cell press (SLS Instruments
Inc., Urbana, IL), and to clear the lysate, it was centrifuged for 30 min in an SS-34-rotor at 48,000 × g at 4 °C. The
protein was purified by a combination of immobilized metal ion affinity
chromatography and ion exchange chromatography. Immobilized metal ion
affinity chromatography purification of the 4D5-dhlx and 4D5-p53 was
performed with a Ni2+-iminodiacetic acid column on a
BioCAD-system (PE Perseptive Biosystems). After loading of the lysate,
the column was washed with 20 mM Tris (pH 7.0), 150 mM NaCl, 0.01% Tween 20 until the absorption reached the
base line. The column was then washed with 20 mM Tris (pH
7.0), 1 M NaCl, 0.01% Tween 20 for 150 column volumes,
followed by a further washing step with 80 mM imidazole, 20 mM Tris (pH 7.0), 150 mM NaCl for 40 column
volumes. Proteins were then eluted with 500 mM imidazole
(pH 7.0), 150 mM NaCl, and samples were collected in a
fraction collector in tubes containing 100 mM Tris (pH
7.0), 8 mM EDTA for immediate 2-fold dilution. The
collected sample was loaded on a HQ-Sepharose column (HQ 4.6/100)
equilibrated with 2-fold diluted PBS. Impurities bound to the column,
and the flow-through, containing the desired miniantibody, was
collected and then dialyzed against PBS overnight at 4 °C, before it
was concentrated to about 200-300 µg/ml by ultracentrifugation using Centricon micro-concentrators (Amersham Pharmacia Biotech).
Analytical Gel Filtration--
Analytical gel filtration with
non-labeled antibody fragments was performed on a SMART system
(Amersham Pharmacia Biotech) using a Superose-12 column (PC3.2)
equilibrated with degassed PBS containing 0.005% Tween 20. Thirty µl
of the antibody fragments were injected at a concentration of 250 µg/ml. Analytical gel chromatography with radiolabeled antibody
fragments was performed on a HiLoad system (Amersham Pharmacia
Biotech). Either a Superose-12 (HR 10/30) column (Amersham Pharmacia
Biotech) or a Superdex-200 (HR 10/30) column (Amersham Pharmacia
Biotech), equilibrated in PBS containing 0.5% BSA, was used. For
calibration of the Superose-12 column alcohol dehydrogenase
(Mr 150,000), bovine serum albumin (Mr 66,000) and carbonic anhydrase
(Mr 29,000) were used. The Superdex-200 column
was calibrated with thyroglobulin (Mr 669,000), apoferritin (Mr 443,000), His Tag-specific 99mTc Labeling of Antibody
Fragments--
99mTc labeling was carried out essentially
as described before (23). To label the various constructs (200 µg/ml)
they were mixed with the same volume of freshly prepared
99mTc-tricarbonyl (pH 6.8, 30 mCi to 1 Ci/ml) and incubated
for 1 h at 37 °C. The reaction was stopped by removing the free
99mTc using a Biospin-6 column (Bio-Rad) equilibrated with
PBS containing 0.005% Tween 20. The eluted fractions were quantitated
for incorporated radioactivity by gamma-scintillation counting.
Determination of the Immunoreactive Fraction--
The
immunoreactive fraction of the antibody constructs on cells was
determined as described by Lindmo et al. (28) and/or by gel
filtration analysis. For the determination on cells, duplicate samples
with increasing numbers of cells (0.5-10 × 106
cells/ml) were incubated in suspension with constant amounts (1-5 ng)
of radiolabeled antibody fragments for 1 h at 4 °C on a shaker.
Nonspecific binding was determined on control samples of cells,
preincubated with a 100-fold excess of unlabeled antibody fragments in
PBS containing 0.5% BSA for 1 h at 4 °C. Cells were washed
three times with PBS containing 0.5% BSA, and the bound radioactivity
in the cell pellets was determined by gamma-scintillation counting
(28).
Alternatively, the immunoreactivity of the antibodies was determined by
a gel filtration shift assay. After radiolabeling the constructs were
separated from free 99mTc-tricarbonyl by gel filtration on
a Superdex-200 (HR 10/30) column, equilibrated with PBS containing
0.5% BSA, on a HiLoad system (Amersham Pharmacia Biotech). The
fraction containing the radiolabeled antibody fragment was identified
by gamma-scintillation counting of the eluted fractions, and 10-20 ng
of the radiolabeled antibody fragment were then incubated with a
100-fold molar excess of recombinant antigen p185HER-2-ECD
for 1 h at room temperature. After the binding equilibrium had
been established, the sample was analyzed again on a second Superdex-200 (HR 10/30) column, and the eluted radioactivity in the
collected fractions was monitored by gamma-scintillation counting and
compared with the elution profile of the radiolabeled control antibody
fragment not incubated with p185HER-2-ECD antigen.
Immunoreactivity was then estimated from the percentage of
radioactivity eluting at higher molecular weight, indicating the
formation of complexes of antigen and active radiolabeled antibody fragment.
Evaluation of Thermal and Serum Stability--
The in
vitro thermal and serum stability of the
anti-p185HER-2-antibody fragments was estimated in a gel
filtration assay. After incubation at nanomolar concentration in human
serum at 37 °C for 20 h radiolabeled antibody fragments were
analyzed on a Superdex-200 (HR 10/30) column equilibrated in PBS
containing 0.5% BSA. The elution profile of the radioactivity was then
compared with the elution profile of the radiolabeled control antibody
fragment diluted in PBS (containing 0.5% BSA) and stored at 4 °C.
The loss of the peak with the desired molecular weight over time was examined.
Comparison of the Dissociation Kinetics of the
Miniantibodies--
A comparison of the dissociation kinetics of the
various constructs was carried out by surface plasmon resonance by
using a BIAcore instrument (Amersham Pharmacia Biotech), as well as on
p185HER-2-overexpressing SK-OV-3 cells. For the surface
plasmon resonance measurements a CM5-Sepharose Chip (Amersham Pharmacia
Biotech) was coated with 3000 RU p185HER-2-ECD antigen by
amine chemistry. Antibody fragments were injected in a volume of 30 µl on the coated surface and for estimation of nonspecific binding on
an uncoated reference surface. The antibody concentration was chosen
such that the surface was not saturated, and binding was about 50% of
the maximally reachable RU values. The sensorgrams were obtained at a
flow rate of 30 µl/min, and dissociation was followed for 6000 s. Data were evaluated with the BIAevaluation (3.0) software (Amersham
Pharmacia Biotech).
Alternatively, dissociation of the miniantibodies was studied on cells
overexpressing the p185HER-2 antigen. Duplicate samples of
the radiolabeled 4D5 constructs were incubated with 0.5 × 106 SK-OV-3 cells suspended in 100 µl of PBS containing
0.5% BSA for 1 h at 4 °C. The cells were washed three times
with PBS containing 0.5% BSA to remove initially unbound radioactivity
and were then incubated at 37 °C on a shaker to start dissociation.
Antibodies were used at non-saturating concentrations in which about
50% of maximum binding was reached (50% Bmax).
After 0, 5, 10, 15, 30, 60, 120, and 180 min, samples were taken and
immediately washed three times with PBS containing 0.5% BSA to remove
dissociated antibody fragments. When the last sample was taken, all
samples were measured for the remaining radioactivity in a
gamma-scintillation counter. For an estimation of nonspecifically bound
radioactivity control samples were preincubated with a 100-fold excess
of unlabeled antibody constructs for 1 h at 4 °C.
Determination of the Functional Affinity on SK-OV-3 Cells by
Radioimmunoassay (RIA)--
The functional affinity of the
99mTc-labeled anti-p185HER-2 antibody fragments
on SK-OV-3 cells was determined by RIA. SK-OV-3-cells (0.5 × 106) were incubated with increasing amounts of the
radiolabeled antibody fragments (50 pM to 30 nM) for 1 h at 4 °C, washed three times with PBS
containing 0.5% BSA to remove unbound radioactivity, and measured for
bound radioactivity in a gamma-scintillation counter. To correct for
nonspecific binding, control samples were preincubated with 100-fold
excess of unlabeled antibody fragments for 1 h at 4 °C. All
measurements were performed in duplicate. The corrected radioactivity
was plotted against the scFv fragment concentration, and the functional
affinity was calculated from the fit of the data, assuming a simple 1:1
model with the approximate function y = ymax· × /(KD + x), where x is the concentration of radioligand
(corrected for activity); y is the radioactivity attributable to specific binding; and ymax is
its plateau value.
Blood Clearance and Biodistribution--
Blood clearance studies
were performed in 6-8-week-old female Balb/c mice. Biodistribution
analysis was carried out in athymic CD1 nu/nu mice (Charles River,
Germany). For blood clearance studies each mouse received intravenous
injections of 5-10 µg of radiolabeled antibody fragments (90-130
µCi/mouse). Mice were sacrificed after 7.5, 15, 30, 60, 120, and 180 min, and blood, liver, and kidney samples were taken and measured for
radioactivity in a gamma-scintillation counter. The percentage of the
injected dose/g tissue (% ID/g) was calculated for each time point.
t1/2
The serum stability of the multimeric miniantibodies in the circulation
of Balb/c mice was examined by gel filtration of serum samples after
administration of the radiolabeled constructs. Each mouse received
intravenous injection of 100 µl of PBS containing 2-5 µg of
radiolabeled antibody fragments. After 30 min mice were sacrificed, and
blood samples were taken and centrifuged for 5 min at maximum speed in
an Eppendorf lab centrifuge at room temperature. Then 150 µl of the
serum was analyzed by gel filtration in PBS containing 0.5% BSA using
a Superose-12 (HR10/30) column on a HiLoad system (Amersham Pharmacia
Biotech). The eluted fractions were monitored in a gamma-scintillation
counter for serum radioactivity. All data were normalized to the same
amount of injected radioactivity (10 × 106 cpm).
Tumor localization studies of the 99mTc-labeled 4D5
antibody fragments were performed in nude mice xenografted with SK-OV-3
tumors. Tumors were raised subcutaneously at the lateral flanks by
injections of 107 SK-OV-3 carcinoma cells in a total volume
of 100 µl. Ten days after tumor inoculation, when tumors reached a
size of 20-50 mm3, each mouse received intravenous
injections of 10-15 µg of radiolabeled miniantibody (1-2
mCi/mouse). The anti-fluorescein binding scFv fragment FITC-E2 (29) was
used as a nonspecific control antibody. Mice were killed at 15 and 30 min and 1, 4, 24, 48, and 72 h after injection, and organs were
removed, and radioactivity was measured in a gamma-scintillation
counter. The areas under the curve (AUC) values were calculated with
the GraphPad Software version 3.0 (GraphPad Software, San Diego CA).
Construction, Periplasmic Expression, and Purification of
Multimeric Miniantibodies--
The synthetic helix-turn-helix peptide
dhlx (30) and the tetramerization peptide of the human tumor suppressor
protein p53 were previously found to mediate the spontaneous di- and
tetramerization of fused antibody fragments in the periplasm of
E. coli to produce the so-called miniantibodies (6, 26, 31).
We used these multimerization devices for the construction of di- and
tetravalent miniantibodies of the anti-tumor anti-p185HER-2
antibody fragment 4D5 (19-22) (Fig. 1),
expressed them in the periplasm of E. coli, and were able to
purify them to greater than 95% purity (Fig.
2A). For the unmodified scFv
fragment 4D5, we routinely obtained 1-2 mg/liter E. coli
culture, whereas for the 4D5-dhlx and 4D5-p53 constructs 500 and ~250
µg/liter were obtained, respectively. Upon concentration by
ultrafiltration, concentrations of 2-3 mg/ml could be obtained for the
scFv 4D5, 400-500 µg/ml for the 4D5-dhlx miniantibody, and about
200-250 µg/ml for the 4D5-p53 tetrameric miniantibody.
Analysis of Multimer Formation--
The occurrence of
multimerization of the anti-p185HER-2 antibody fragments
was demonstrated by gel filtration analysis of the purified proteins on
a Superose-12 column (Fig. 2B). The unmodified scFv fragment
4D5 eluted at a retention volume of 1.56 ml as expected for a monomeric
species. The 4D5-dhlx construct eluted at a volume of 1.43 ml, which
corresponds to a calculated Mr of about 60,000 consistent with a dimer. The p53-multimerized species eluted at 1.32 ml, which corresponds to a calculated Mr of
about 130,000 consistent with a tetramer. In no cases were higher
molecular weight aggregates detected, and the elution of single
symmetric peaks indicated the homogeneity of the protein preparations.
Efficiency of the His Tag-specific 99mTc
Labeling--
All 4D5 constructs could be labeled with
99mTc-tricarbonyl, which forms an extremely stable complex
with clusters of histidine residues (23). No precipitate was observed
in the reaction mixture. For the monomeric 4D5 about 70% of the free
99mTcCO (which is present in molar trace amounts) was
incorporated when the protein was used at 1 mg/ml. For the dimeric
4D5-dhlx usually 30% incorporation was obtained with an initial
protein concentration of 500 µg/ml. To compare the data between the
proteins of this study, all antibody fragments were used at
concentrations of 250 µg/ml, which was the highest concentration
achievable for the 4D5-p53 construct, and the routinely obtained
incorporation yields of 99mTc-tricarbonyl were between 10 and 20% of the radionuclide (100-200 mCi/ml).
Immunoreactivity of the Multimers after Radiolabeling--
To
ensure the conservation of the binding activity of the multimers
after radiolabeling, we measured the immunoreactivity of the
constructs. In binding assays on SK-OV-3 cells we determined the
immunoreactive fractions to be about 80-90% for the monomeric 4D5
scFv, above 95% for the 4D5-dhlx dimer, and 80% for the 4D5-p53 tetramer (Table I).
In a gel filtration shift assay (Table I), we found for scFv 4D5 and
4D5-dhlx that about 95% of the labeled molecules formed antigen
complexes, whereas for 4D5-p53 about 55-60% of the radioactivity eluted earlier from the column than in the control experiment without
antigen, indicating antigen complex formation. As the elution profiles
comprised species of different stoichiometry, they were too complex to
determine reliably the number of reactive binding sites (data not shown).
Thermal and Serum Stability of Radiolabeled Multimers--
A
sufficient stability of the antibody in serum and at high temperature
is essential for tumor targeting (22). For this reason we compared the
elution profile of 99mTc-labeled 4D5 constructs incubated
in human serum at 37 °C and stored at 4 °C. For the unmodified
4D5 scFv fragment after serum incubation for 20 h around 50-60%
of the protein still eluted at the volume expected for its respective
molecular weight (13.5 ml), for the 4D5-dhlx construct 60% (12.2 ml),
and for the 4D5-p53 90% (11.5 ml). These data indicate that the
presence of the interaction domains does not increase aggregation and
that the miniantibodies are at least as stable at 37 °C as the
parent scFv, and possibly more.
Comparison of the Dissociation Kinetics of the
Miniantibodies--
To demonstrate the differences in the binding
behavior due to the multimerization, we tested the dissociation
kinetics of the various 4D5 constructs from their receptor both in
surface plasmon resonance (BIAcore) experiments with purified
p185HER-2 and on p185HER-2 overexpressing
SK-OV-3 cells (Fig. 3).
For surface plasmon resonance (BIAcore) measurements, we coated the
chip with high antigen densities to allow multivalent binding, which is
not restricted by the distance between antigen molecules, but used high
flow rates to minimize rebinding of dissociated molecules. Injected
concentrations of the 4D5 constructs and the duration of the injection
were chosen as to create a situation at the beginning of the off-rate
measurements in which the binding surface was only saturated up to 50%
of the maximum RU value, independently determined for each construct.
The dissociation was followed for 100 min and revealed a slower
dissociation for the 4D5-dhlx miniantibody (>92% after 100 min still
bound to the receptor) and the 4D5-p53 miniantibody (85%) in
comparison to the monomeric 4D5 scFv (58%). However, the observed
dissociation for the dimeric 4D5 and the tetrameric 4D5 was similar and
even slightly slower for the dimeric species.
In measurements performed on p185HER-2-overexpressing
SK-OV-3 cells, we found the unmodified 4D5 scFv to dissociate rapidly
(65% bound after 100 min and 49% after 180 min), while bound 4D5-dhlx (76 and 63% after 100 and 180 min, respectively) and 4D5-p53 (73 and
60% after 100 and 180 min, respectively) dissociated more slowly.
Again, the dissociation rates of 4D5-dhlx and the 4D5-p53 construct
were very similar.
RIA Measurements of Functional Affinities on SK-OV-3 Cells--
To
complement the dissociation data, we also determined the equilibrium
binding of the various constructs to
p185HER-2-overexpressing SK-OV-3 cells (Table
II). An increase in functional affinity
for the di- and tetramerized miniantibody was found in this cell line
compared with the monomeric scFv 4D5. No significant difference in
functional affinity (avidity) was found between di- and tetramer,
however.
Blood Clearance and Biodistribution--
The use of multimeric
miniantibodies is expected to result in an increase in serum
persistence. Therefore, we measured blood serum levels in clearance
studies, and indeed we observed longer serum half-lives with increasing
degree of multimerization (Fig. 4). The
analysis of the obtained curves (Fig. 4A) yielded for the
monomeric 4D5 a t1/2
The gel filtration analysis of serum samples taken from the circulation
30 min after injection (Fig. 4B) shows that the various species are stable with respect to their multimerization, as no other
peaks were observed, and they are cleared with different rates from the
circulation, since the levels dropped at different rates. No peaks
originating from dissociated multimers were observed, probably because
such species would not accumulate, as they are rapidly cleared.
To analyze the tumor targeting potential of the various
constructs, we performed biodistribution studies in xenografted nude mice (Table III). The monomeric scFv 4D5
enriched to 1.1% ID/g in SK-OV-3 tumors with a tumor-to-blood ratio of
6.5. This is consistent with results of earlier targeting experiments
with this scFv fragment (23). After 48 h, 0.98% ID/g and a
tumor-to-blood ratio of 13.5 were found. The dimeric miniantibody
4D5-dhlx accumulated at the tumor site with 1.47% ID/g after 24 h
with a tumor-to-blood ratio of 7, and it followed the same kinetics as
the monomeric scFv 4D5. Dimerization of the scFv 4D5 by the dhlx domain
and formation of a dimeric miniantibody thus did not lead to a
significant improvement (Table III). In contrast, an improved
selectivity and an increased total dose enrichment was obtained with
the tetrameric 4D5-p53 construct. After 24 h 4.32% ID/g was
accumulated at the tumor site with a tumor-to-blood ratio of 3.4, and
the antibody fragment remained stably bound, since after 48 h
3.24% ID/g tissue was still present at the tumor with a tumor-to-blood
ratio of 13.5. Regarding the residence time at the tumor the tetrameric 4D5-p53 construct was the most efficient with a total calculated AUC
value of 10,270 after 48 h and 13,400 after 72 h. For the monomeric 4D5 an AUC value of 3285 was calculated after 48 h, and
for the dimeric 4D5-dhlx values of 3573 (48 h) and of 4380 (72 h) were
calculated.
Two aspects of multimerization are of importance for efficient
tumor targeting as follows: (i) multimerization leads to higher functional affinity by increasing the number of binding sites, and (ii)
the molecular weight is automatically increased by the presence of
multiple copies of the binding domains. This higher molecular weight
extends the serum persistence of molecules in the circulation, because
they are not filtrated into the kidney glomeruli (4, 9, 32). For
molecules that are too large to pass this filtration barrier, the blood
pool remains at a high concentration level over time, and thus there is
a higher chance for these molecules to bind to their target antigens.
On the other hand, there is an inverse correlation between the
molecular weight of these molecules and their ability to penetrate into
the tumor tissue. To overcome this drawback a compromise has to be
found in the design of the targeting molecules with respect to the
molecular weight. Because of the conflicting nature of these
requirements the optimal molecular design can only be determined experimentally.
In the present study we report the production and the in
vitro and in vivo properties of mono-, di-, and
tetramerized anti-p185HER-2 scFv fragments in the format of
miniantibodies. Multimerization was achieved by the use of
self-associating peptides (31), which lead to spontaneous assembly of
the fused antibody fragments directly in the periplasm of E. coli (Fig. 1). The multimeric antibody fragments could be
expressed and purified in good yields from E. coli as native
proteins without refolding (Fig. 2A). The degree of
multimerization was checked by gel chromatography analysis, and the
presence of the expected mono-, di-, and tetrameric species was
confirmed (Fig. 2B). The anti-p185HER-2
(anti-c-erbB2) 4D5 scFv fragment was chosen for this study,
because it was reported to be of high affinity (20), above average
equilibrium thermodynamic (21) and thermal stability (22), and could be purified in high yields from the periplasm of E. coli (19,
33). Furthermore, we have shown that the 4D5 scFv fragment could be labeled by His tag-specific 99mTc labeling to high specific
activities and that it sufficiently localizes to SK-OV-3 xenografts in
nude mice (23). Our expectation was that this antibody fragment confers
favorable biophysical properties to the miniantibodies and that the
effect of multimerization by the self-associating peptides on the
overall integrity of the miniantibody and its tumor targeting
properties could be studied without limitation by the antibody fragment
used. Experimental analysis indeed showed that the thermal and serum
stability, which are important prerequisites for efficient targeting,
were retained during multimerization and not lost in the miniantibody formats.
The binding behavior was analyzed on immobilized recombinant
p185HER-2-ECD in BIAcore experiments and on
p185HER-2-overexpressing cells. In both experiments di- and
tetrameric 4D5 miniantibodies showed a reduced dissociation rate
compared with the monomeric 4D5 scFv, and this was undoubtedly due to
the increased avidity of these molecules. Nevertheless, in none of the
experiments 4D5-p53 exhibited a slower dissociation than the 4D5-dhlx
construct. A dissociation of the tetramer into dimers seems to be
unlikely, since it has been demonstrated that the tetramer still exists
at the nanomolar concentrations used (Fig. 3). From the binding
experiments and the RIA-measurements (Table II), we conclude that in
targeting the 4D5 epitope on SK-OV-3 cells not more than two
antigen-binding sites could be simultaneously engaged. However, this
result can certainly not be generalized, as the avidity effect of going
from a dimer to a tetramer will depend on the geometric orientation of
the targeted epitope, its surface density, its accessibility, and the
concentration of the multimer. In a previous study mono-, di-, and
tetrameric anti-LewisY scFv constructs were tested in a
similar experiment, and increased avidity was found with increasing
number of binding sites (26).
In clearance studies we found longer serum persistence with an
increasing degree of multimerization (Fig. 4). Analysis of serum
samples 30 min after intravenous injection showed stable multimerization of all miniantibody constructs. In biodistribution experiments in tumor xenografted nude mice, we monitored the organ and
tumor distribution over a period of 72 h (Table III) and observed that the dimeric 4D5-dhlx construct did not show any significant improvement in tumor localization, compared with the monomeric scFv
4D5, despite its higher avidity. Although the 4D5-dhlx miniantibody cleared more slowly from the circulation than the monomeric scFv 4D5,
its clearance rate was still faster than that of a well localizing iodinated diabody used in a recent study (14). The dissociation constants for the dhlx and p53 module into monomeric constituents are
not known, and we cannot exclude that, at the high dilution in the
serum, the non-covalent dimerization provided by the dhlx domain may
slowly dissociate over time, leading to insufficient serum persistence
and poorer tumor localization. As a consequence, the avidity gain by
dimerization of molecules not yet bound could also be slowly lost under
these conditions. The 4D5-p53 construct, on the other hand, was the
most efficient in terms of tumor localization. Due to its tetrameric
nature this 130-kDa molecule was above the renal threshold and showed
the slowest clearance rate. The optimal time point for 4D5-p53
localization was reached only after 48 h with a 6-fold higher
tumor localization than the dimeric construct and a tumor-to-blood
ratio of 13.5:1 (Table III and Fig. 5).
To our knowledge no 99mTc-biodistribution study for the 4D5
monoclonal antibody was reported so that we cannot directly compare it
to the tetramer. However, the radiometal-labeled DOTA-conjugated
anti-HER2/neu antibody 4D5 accumulated in nude mice with high total
dose in transfected MCF7/HER2 tumor xenografts, but at no time point
were tumor-to-blood ratios better than 2.6 obtained (34).
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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-D-thiogalactopyranoside (Roche Molecular Biochemicals, 1 mM final concentration) for miniantibody
production when it reached an A550 nm of
0.5. Expression was continued for 3-4 h at 24 °C until the culture
reached a final A550 nm of 4-5. The harvested
pellet was stored at
80 °C.
-amylase
(Mr 200,000), and alcohol-dehydrogenase (Mr 150,000).
and t1/2
were obtained
from the analysis of the plot of the % ID/g of the blood values over
time with a biphasic exponential function (GraphPad software).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Construction scheme of multimerization of
scFv 4D5 by self-associating peptides. Shown are the
Xba/HindIII cassettes of the expression vector
pIG6 for production of the monomeric anti-p185HER-2 scFv
(I), the di- (II), and tetrameric
(III) miniantibodies in the bacterial periplasm. By simple
exchange of the EcoRI/AscI module one can easily
switch between the molecular formats.
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Fig. 2.
A, purity of mono-, di-, and tetrameric
anti-p185HER-2 antibody fragments. SDS-polyacrylamide
gel electrophoresis under reducing and non-reducing conditions shows
the result of the purification of the 4D5 scFv fragment and of the
miniantibodies 4D5-dhlx and 4D5-p53. B, analysis of
multimerization by size exclusion chromatography. Top, gel
filtration analysis of unmodified 4D5 scFv fragment; middle,
4D5-dhlx; and bottom, 4D5-p53 on a Superose-12 column.
Molecular weight standards are as follows: alcohol dehydrogenase
(Mr 150,000); BSA (Mr
66,000); and carbonic anhydrase (CA,
Mr 29,000).
Immunoreactivity of multimerized anti-p185HER-2-antibody
fragments
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Fig. 3.
Increase in avidity by multimerization.
The increase of functional affinity obtained by multimerization was
measured by surface plasmon resonance (BIAcore) on recombinant
p185HER-2-ECD (A) and on living SK-OV-3 cells
overexpressing p185HER-2 (B).
Measurement of functional affinity of 4D5 constructs on SK-OV-3 cells
= 1 min and
t1/2
= 0.34 h, for the 4D5-dhlx a
t1/2
= 2.05 min and
t1/2
= 0.54 h, and for the 4D5-p53 a
t1/2
= 7 min and
t1/2
= 2.18 h.
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Fig. 4.
Influence of multimerization on the clearance
of 4D5 antibody fragments. A, blood clearance was
examined in Balb/c mice and followed over 24 h. B,
serum samples were taken from sacrificed mice 30 min after injection
and analyzed by size exclusion chromatography on a Superose-12 column.
Each mouse received the same amount of antibody fragment, and the
measured radioactivity was normalized to 10 × 106
cpm.
Biodistribution of 99mTc-labeled scFv fragments in athymic mice
bearing SK-OV3 tumor xenografts
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Tumor enrichment of mono-, di-, and
tetrameric anti-p185HER-2 antibody fragments. Each
mouse received intravenously 10-15 µg of 99mTc-labeled
4D5 antibody fragments. Mice (n = 3 per time point)
were sacrificed after 15 and 30 min and 1, 4, 24, 48, and 72 h.
Tumor and blood samples were taken, measured for radioactivity, and the
% ID/g was calculated.
In summary, our results show that miniantibodies multimerized by self-associating peptides in the periplasm of E. coli have the potential to localize efficiently to tumor xenografts in vivo and to remain stably bound to their target antigen. This is the first study investigating the in vivo performance of this approach, and it has several consequences for the further development of these molecules in the future. From the two types of modules tested the tetrameric miniantibody was suitable to obtain significant tumor-to-blood ratios and efficient tumor localization. Nevertheless, it appears that the multimerization modules investigated dissociate over a time course of 72 h. This is not surprising, given the fact that they are held together by non-covalent forces and were highly diluted after injection into the animals, whereas they were at micromolar concentrations during the radiolabeling step, such that the oligomeric molecule was maintained at equilibrium. Further variants of these domains may help to better address this issue, either by evolving high affinity domains (35) and/or by introducing disulfide bonds (36). The final format will not only have to provide stability against dissociation, but should also result in facile production, which at least for the disulfide-bridged molecules is usually less favorable, and some formats even have to be made in eukaryotes (7). For non-covalent diabodies some dissociation would be expected as well over time, but to our knowledge this has not yet been determined.
The advantage of the self-associating peptides is that they allow a
modular engineering approach in which it is possible to switch from one
format to the other depending on the aim of the in vivo
application. The approach is also completely general, as it does not
depend on the details of the heavy chain variable domain/light chain
variable domain interface and can be used for the multimerization of
modules other than antibody fragments as well. Moreover, the
self-associating peptides will allow the production of recombinant
proteins containing additional effector domains with the smallest
possible size in the periplasm of E. coli. Such developments
are potentially of great significance for the development of novel and
more effective targeting strategies in cancer therapy.
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ACKNOWLEDGEMENTS |
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We thank Dr. Paul Carter for the gift of p185HER-2-ECD, Christine de Pasquale for excellent technical assistance, and Drs. Ilse Novak-Hofer and Alain Tissot for helpful discussions.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Schering AG, Experimental Oncology, 13342 Berlin, Germany.
** To whom correspondence should be addressed. Tel.: 41-1-635-5570; Fax: 41-1-635-5712; E-mail: plueckthun@biocfebs.unizh.ch.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011669200
2 P. Pack, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: scFv, single-chain Fv fragment; AUC, area under curve; ECD, extracellular domain; ID/g, injected dose/g; RIA, radioimmunoassay; RU, response units; 99mTc, 99mtechnetium; PBS, phosphate-buffered saline; BSA, bovine serum albumin; DOTA, 1,4,7,10-tetraazacyclodecane-N,N',N'',N'''-tetraacetic acid.
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