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Experimental Therapeutics |
Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Switzerland [F. N., D. N.]; Institute of Pathology, Friedrich Schiller University, D-07740 Jena, Germany [H. K.]; and Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, I-16132 Genoa, Italy [L. Z.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A novel approach to cancer therapy based on the antibody-directed targeting of the human coagulation-inducing protein TF2 to tumor vasculature has recently been proposed (10 , 11) . The approach is based on the concept that thrombosis of tumor vessels may stop the supply of nutrients and oxygen to tumor cells, thereby causing their death.
TF is a cell-surface glycoprotein and a major initiator of blood coagulation (12) . At sites of injury, blood comes in contact with the membrane-bound TF, which forms a complex with the serine protease FVIIa present in blood. The resulting complex activates factors IX and X, which leads to thrombin activation and ultimately to blood clotting. tTF consisting of only the extracellular soluble domain (residues 1–219), exhibits an ability to activate the clotting cascade in solution that is five orders of magnitude lower than normal but fully active when retargeted to a membrane surface (10) .
In a first article, Huang et al. (10)
used a
bispecific antibody to target tTF to an artificial marker of
angiogenesis (MHC-II) experimentally induced on tumor vascular
endothelium by grafting in mice neuroblastoma cells that had been
transfected with the IFN-
gene. In that
study, the investigators observed extensive intravascular
thrombosis of the tumor and complete regressions in 38% of the mice
treated (10)
.
In a second study, immunoconjugates were used to target tTF to a naturally occurring marker of tumor vascular endothelium, VCAM-1 (11) . VCAM-1 is expressed by vascular endothelial cells in Hodgkin’s lymphoma and in various solid tumors in mice and humans. It is also expressed in some vessels of thyroid, kidney, and thymus in humans and in heart and lung in mice (11) . In this study, the authors observed selective localization of tTF to VCAM-1-expressing vessels in the tumor, causing thrombosis of those vessels, a 50% reduction of tumor growth rate, but no complete remission. The immunoconjugate also localized to VCAM-1-expressing vessels in the lungs and heart, but did not induce thrombosis at these sites. An immunohistochemical evaluation of a monoclonal anti-PS antibody in the mice showed that the VCAM-1-expressing vessels in the tumor also expressed PS, whereas VCAM-1-expressing vessels in the lungs and heart lacked PS. The authors concluded that PS expression on the luminal aspect of blood vessels is needed to provide the procoagulant surface upon which coagulation complexes can assemble.
The targeted delivery of tTF would be of significant therapeutic relevance if it were directed against a naturally occurring marker of angiogenesis that is expressed in the majority of aggressive solid tumors but undetectable in normal vessels and tissues, and if it mediated the selective thrombosis of tumor blood vessels.
A good quality marker for both tumoral and nontumoral neovasculature is the ED-B domain of fibronectin (a sequence of 91 amino acids identical in mouse, rat, rabbit, dog, and man) that can be inserted into the fibronectin molecule by a mechanism of alternative splicing (13, 14, 15, 16, 17) . B-FN accumulates around neovascular structures in aggressive tumors and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues.
To date, the production of monoclonal antibodies directly recognizing the ED-B domain in B-FN has not been possible using hybridoma technology because of tolerance. However, this problem has been overcome using antibody phage technology (18) with large synthetic antibody repertoires (19 , 20) . Several antibody fragments specific for the ED-B domain of fibronectin have been generated recently. These antibody fragments stain vascular structures in tumor sections and selectively target tumor neovasculature, as shown in tumor-bearing mice using IR fluorescence and radioactive techniques (21 , 22) . Increased binding affinity leads to improved targeting of tumoral angiogenesis, as demonstrated by biodistribution studies performed using the L19 antibody fragment with affinity for the ED-B domain in the pM range and L19 mutants with reduced affinity (23) .
In this study we investigated whether the selective antibody-mediated targeting of tTF to B-FN would result in thrombosis of tumor blood vessels. B-FN is a component of the modified extracellular matrix that surrounds tumor blood vessels, and it was therefore not known whether the delivery of a procoagulant agent at this abluminal site would be capable of mediating the complete and selective intraluminal coagulation of tumor blood vessels.
In this article we show that a fusion protein consisting of the L19
antibody fragment fused to tTF mediates the complete and selective
infarction of three different types of solid tumors (F9, C51, and FE8)
in mice. Injection of 35 µg of the fusion protein led to complete
tumor eradication in 
30% of the treated animals, whereas tTF-fusion
proteins with binding specificity for an irrelevant antigen did not
show any therapeutic effect.
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MATERIALS AND METHODS |
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Cloning, Expression, and Purification of scFv-tTF Fusions.
The scFv(L19)-tTF expression vector (pFN5) was constructed by the
cloning of a synthetic DNA sequence, coding for the human tTF, at the
3'end of the DNA sequence encoding the human scFv(L19) using the
NotI/EcoRI sites of a derivative of vector pDN5
(27)
in which the scFv(D1.3) gene had been
replaced by the scFv(L19) gene. The human tTF DNA sequence
was modified by PCR as follows:
(a) the primer TF-banot (5'-T GAG TCA TTC GCG GCC GCA GGT GGC GGT GGC TCT GGC ACT ACA AAT ACT GTG GCA-3') introduced at the 5'end of the tTF DNA sequence a restriction site for the endonuclease NotI. It also introduced a short linker COOH-terminal of the restriction site consisting of four glycines (GGGG);
(b) the primer TF-fostueco1 (5'-GTC CTT GTA GTC AGG CCT TTC ACG GAA CTC ACC TTT CTC CTG GCC CAT ACA-3'), introduced at the 3'end of the tTF DNA sequence a StuI endonuclease restriction site, and the first four residues of the FLAG tag. It also removed a EcoRI restriction site in the codon for the amino acid 216 in the tTF DNA sequence by a silent mutation;
and (c) the primer TF-fostueco2 (5'-AGA GAA TTC TTA TTA CTT ATC GTC ATC GTC CTT GTA GTC AGG CCT TTC ACG-3') introduced at the 3'end of the product of TF-banot and TF-fostueco1 the rest of the FLAG tag (DYKDDDDK), a EcoRI restriction site, and two stop codons;
The scFv(D1.3)-tTF expression vector was constructed in a similar fashion as described above for scFv(L19)-tTF. In short, the tTF gene was cloned in the NotI/EcoRI sites of vector pDN5 (27) , which already contains the scFv(D1.3) gene.
The vectors were introduced by electroporation in TG1 Escherichia
coli cells. Protein expression and the first affinity purification
step on antigen column were performed as described (22
, 27)
. In the case of scFv(L19)-tTF, eluted fractions were
neutralized with 1 M Tris-HCl (pH 7.4) and loaded
on a M2-resin for affinity purification using the FLAG tag (Sigma, St.
Louis, MO). After serial washes, the protein was eluted with 0.1
M glycine-HCl (pH 3.5) and put directly onto a
Resource S cation exchange column (Amersham Pharmacia Biotech, Uppsala,
Sweden). The monomeric peak fraction was collected and desalted with a
preparative gelfiltration column PD-10 (Amersham Pharmacia Biotech).
Typical yields of tripurified therapeutic fusion protein were 0.2
mg/liter culture, mainly attributable to the low capacity and limited
life of the 
-Flag M2 resin. Protein yields after the first affinity
chromatography step were 1–2 mg/liter.
In the case of scFv(D1.3)-tTF, fractions eluted from a lysozyme column were neutralized with 1 M Tris-HCl (pH 7.4) and loaded on a Sigma M2-resin for affinity purification using the FLAG tag (Sigma). After serial washes, the protein was eluted with 0.1 M Glycine-HCl (pH 3.5) and the pH adjusted to 8.0. The sample was loaded onto a Resource Q anion exchange column (Amersham Pharmacia Biotech). The monomeric peak fraction was collected and desalted with a preparative gelfiltration column PD-10.
Fusion proteins were analyzed under denaturing conditions on SDS-PAGE and in native conditions were analyzed by fast protein liquid chromatography gel filtration on a Superdex S-75 column (Amersham Pharmacia Biotech).
Measurement of Immunoreactivity and Tissue Factor Activity.
The immunoreactivity of the scFv-tTF fusion protein was analyzed by
ELISA immunoassay, by BIAcore, and by affinity chromatography on
antigen column, as described (21, 22, 23)
.
The enzymatic activity of the scFv-tTF fusion protein was analyzed using the Spectrozyme FXa assay (American Diagnostica, Pfungstadt, Germany) as described by Ruf et al. (12) .
Tumor Mouse Models.
Tumor-bearing mice were obtained by s.c. injection of
106 tumor cells of C51 murine colon
adenocarcinoma, FE8 ras-transformed rat fibroblasts, or F9
murine teratocarcinoma in female BALB/c nude mice (Labortierkunde der
Universität Zürich, Zürich, Switzerland). Mice were
monitored daily, and tumor volume was measured with a caliper using the
following formula: volume, = length x width2
x 
/6. Experiments were
performed in agreement with Swiss regulations and under a project
license, "Tumor Targeting," issued to D. N. by the Kantonales
Veterinäramt des Kantons Zürich (Bewillgung 53/97). In
addition to the experiments with nude mice, a set of experiments with
F9 and C51 tumors was also performed with immunocompetent mice,
yielding results comparable with the ones obtained with nude mice (data
not shown). According to our project license, mice had to be euthanized
when tumors became too large, if animals lost >20% of body weight, or
if they showed signs of pain during the therapeutic experiments.
Biodistribution Experiments.
The in vivo targeting performance was evaluated by
biodistribution analysis as described by Tarli et al.
(22)
. Briefly, purified scFv(L19)-tTF fusion protein was
radioiodinated and injected into nude mice with s.c. implanted F9
murine teratocarcinoma. Mice were sacrificed at 24 h after
injection. The organs were weighed and the radioactivity counted.
Targeting results of representative organs are expressed as a
percentage of the injected dose per gram of tissue (%ID/g).
Treatment of Mice with scFv-tTF Fusion Proteins.
Cohorts of mice with tumors of volume 200–300
mm3 (n 
4) were
injected with 14 µg ofscFv-tTF fusion protein in 200 µl of saline
or injected with saline only. The injection was repeated after 48 and
96 h. Mice with tumors of volume 1500 mm3
were injected with a single dose of 20 µg of scFv-tTF fusion protein
in 200 µl of saline.
In a dose-escalation investigation, mice with FE8 tumors of volume
300–500 mm3 were injected with 9, 15, or 35
µg of scFv-tTF fusion protein in 200 µl of saline or injected with
saline only. The injection was repeated after 72 and 144 h. In all
experiments, mice were monitored by tumor volume, weight, and
photographic documentation.
Histology.
To assess the toxicity of treatment by histological analysis, organs
(lung, liver, spleen, intestine, kidney, heart, and brain) were
collected 24 h after injection of 20 µg of scFv-TF or saline,
fixed in 4% buffered paraformaldehyde and embedded in paraffin.
Four-µm sections were cut, stained with H&E, and analyzed. At least
one slide was available per 0.5 cm of diameter of the sample.
Mice were sacrificed at different time points after injection of 20 µg of scFv(L19)-tTF (1 h, 4 h, 6 h, 12 h, and 24 h). The tumors were excised, fixed in 4% buffered paraformaldehyde and embedded in paraffin. Sections were cut and stained with H&E. At least one slide was available per 0.5 cm of tumor diameter. Thrombosis of intratumoral vessels was defined according the following criteria: total or incomplete occlusion by closely packed erythrocytes with blurred outline.
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RESULTS |
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, the tTF was appended at the COOH terminus of the scFv
antibody fragments in order not to impair their immunoreactivity.
Modeling studies based on the known three-dimensional structure of the
TF/Factor VII complex (29)
suggested that modification of
tTF at its NH2-terminal extremity does not
interfere with complex formation and the consequent activation of the
blood-clotting cascade. A FLAG tag (30)
was appended at
the COOH-terminal extremity of scFv-tTF fusions proteins to facilitate
immunodetection and to allow for affinity purification using the
anti-FLAG M2 monoclonal antibody, immobilized on a resin.
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. Both
purified fusion proteins were monomeric as assessed by gel filtration. To check that both moieties of the fusion proteins were functional, immunoreactivity was measured both by affinity chromatography on antigen column (22) and by BIAcore (31 , 32) . Tissue factor activity was measured as described (12) , detecting the cleavage of the FXa fluorogenic peptide mediated by the scFv-tTF/Factor VIIa complex. A half-maximal activity was observed at 45 nM protein concentration, in line with previously published values obtained with tTF (12) .
The ability of scFv(L19)-tTF to selectively localize on tumor blood
vessels was examined in mice bearing a s.c. grafted F9 murine
teratocarcinoma by quantitative biodistribution analysis using a
radioiodinated protein sample (22)
. Fig. 2
shows the results of this analysis expressed in terms of the percentage
of injected dose per gram of tissue (%ID/g) 24 h after i.v.
injection. At this time point, the %ID/g (tumor) was 16.8, with a
tumor:blood ratio of 17:1, which is comparable with the ratio published
for radiolabeled scFv(L19) (22)
. Tumor:blood ratios >2
can be observed as early as 3 h after injection. Considering that
L19 localizes to tumoral blood vessels, which represent only a small
percentage of the weight of F9 tumors (22)
, the
results of Fig. 2
confirm that scFv(L19)-tTF is able to accumulate at a
high density around vascular structures.
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Mice bearing small F9, C51, or FE8 tumors (<500 mg) were injected at
different time points with three doses of 14 µg scFv-tTF (or saline).
A few hours after the first injection, all tumors turned black
only with the L19 fusion protein (Fig. 3a)
, but not with the saline or with scFv(D1.3)-tTF (Fig. 3b)
, suggesting that scFv(L19)-tTF did mediate a selective
intraluminal blood coagulation in tumor blood vessels, as confirmed by
histochemical analysis (Fig. 4)
.
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). However, animals were not cured. In the groups of
negative control mice, there was no statistically significant
difference between mice injected with saline or with the scFv(D1.3)-tTF
fusion protein, confirming that the targeting of tTF to B-FN was
necessary for the antitumor effect.
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shows a plot of tumor volume
versus time for the three different tumor models after a
single i.v. injection of D1.3-tTF or the L19-tTF fusion protein. Within
1–2 h after injection of scFv(L19)-tTF [but not scFv(D1.3)-tTF],
tumors turned black. At 2 days after injection, most of the tumor had
been converted into a crusty mass. However, a portion of the tumor
typically survived at the border with the peritoneal cavity and would
eventually grow back and kill the animal (Fig. 6a)
. A histochemical analysis of tumors revealed that, at the
dose used, only a small fraction of vessels close to the peritoneal
cavity was occluded (Fig. 6b)
in spite of the fact that all
tumor vessels were ED-B-positive. By contrast, blood vessels in other
parts of the tumors were completely thrombosed.
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Fig. 7
shows FE8 tumor volumes, plotted versus time, of mice
injected three times with saline, 35 µg of scFv(D1.3)-tTF, or
escalating doses of scFv(L19)-tTF. Even at these high doses,
scFv(D1.3)tTF did not show any significant difference relative to the
saline control. In contrast, scFv(L19)-tTF showed a therapeutic effect,
which improved with an increase in the administered dose. At the
highest dose tested (35 µg), 30% of the mice exhibited a complete
tumor regression. In these conditions, mice showed a transient loss of
10% body weight, indicating the onset of some toxicity. Mice
regained weight after tumor eradication. Fig. 8
shows pictures of the mice responding to treatment.
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50% of intratumoral blood vessels were completely
occluded just 1 h after injection. Four h after injection,
80% of the tumor vessels were thrombosed. The number of occluded
intratumoral vessels did not change 24 h after the injection, and
no additional thrombosis or thrombolysis could be observed. |
DISCUSSION |
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The observation of complete remissions of tumor-bearing mice treated with scFv(L19)-tTF described in this article may support an alternative view on the requirements of an effective fusion protein for the complete and selective thrombosis of tumoral vessels. B-FN is an abundant component of the modified extracellular matrix of tumors, which accumulates in the abluminal side of tumor blood vessels. ScFv(L19)-tTF displays an extremely high accumulation on neovasculature a few hours after injection, as measured by quantitative biodistribution analysis using radioiodinated fusion proteins or by ex vivo immunofluorescence analysis of tumor sections from mice injected with fluorescently labeled scFv(L19) (data not shown; Ref. 21 ). It is plausible that the fenestration and leakiness of tumor blood vessels allows the extravasation of Factor VIIa, which could then bind to the tTF anchored at high density on B-FN via the fusion protein. Conversion of Factor X into Factor Xa in the perivascular space immediately around the blood vessels would facilitate the diffusion of Factor Xa in the blood stream with consequent continuation of the blood clotting cascade. Alternatively, fibrin deposition could start in the perivascular space, and would propagate back into the luminal aspects of tumor blood vessels.
The remissions were observed in extremely aggressive tumor
models, which are not cured by conventional chemotherapy (data not
shown). At the highest dose, complete remissions were observed in 30%
of the mice treated. All tumors showed extensive thrombosis of blood
vessels, but in some cases residual tumor cells grew back. Typically,
these cells would be located in tumor areas in which blood vessel
occlusion was not complete. However, the rapid tumor debulking observed
with our fusion protein may facilitate removal of residual tumor mass
by other means. Furthermore, the clear dose dependence of therapeutic
benefit (Fig. 7)
obtained with scFv(L19)-tTF suggests that the efficacy
of this therapeutic fusion protein would further improve using slightly
higher doses or different administration schedules. In principle, a
better formulation of the fusion protein, combination with other
therapeutic modalities (e.g., chemotherapy), or
coadministration of rapidly clearing heparins may be avenues toward
increasing the dose administered and/or the therapeutic index of
scFv(L19)-tTF. Indeed, glycosylated scFv(L19)-tTF, produced in stably
transfected human embryonic kidney cells, appears to yield a fusion
protein with biological activity similar to its counterpart produced in
E. coli, but less prone to aggregation, as evidenced by
freeze-thaw cycles and chromatographic analysis (F. N., and D. N.;
data not shown).
The ED-B domain sequence is identical from mice to humans, allowing the use of scFv(L19)-tTF in a number of different syngeneic animal models. Whereas our results suggest that scFv(L19)-tTF may offer a benefit to cancer patients in a therapeutic setting, experiments with slow growing tumors and studies in larger animals (preferentially in animal patients with spontaneously disseminated tumors) are required before considering clinical trials. The appropriate formulation of scFv(L19)-tTF, which strictly prevents protein aggregation and/or precipitation, appears to be essential to avoid side effects attributable to the undesired intraluminal blood coagulation in healthy organs and tissues.
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FOOTNOTES |
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1 Financial support from Krebforschung Schweiz and
the Bundesamt für Bilding und Wissenschaft are gratefully
acknowledged. 
2 To whom requests for reprints should be
addressed, at Institute of Pharmaceutical Sciences, Swiss Federal
Institute of Technology Zurich, Building 36 M14, Winterthurerstrasse
190, CH-8057 Zurich, Switzerland. Phone: 41-1-635-60-63; Fax:
41-1-635-68-86; E-mail: neri{at}pharma.ethz.ch
3 The abbreviations used are: TF, tissue factor;
tTF, truncated form of TF; VCAM-1, vascular cell adhesion molecule-1;
PS, phosphatidylserine; B-FN, fibronectin containing ED-B.
Received 7/14/00. Accepted 11/13/00.
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L. Borsi, E. Balza, B. Carnemolla, F. Sassi, P. Castellani, A. Berndt, H. Kosmehl, A. Biro, A. Siri, P. Orecchia, et al. Selective targeted delivery of TNF{alpha} to tumor blood vessels Blood, December 15, 2003; 102(13): 4384 - 4392. [Abstract] [Full Text] [PDF]
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 S. Christian, J. Pilch, M. E. Akerman, K. Porkka, P. Laakkonen, and E. Ruoslahti Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels J. Cell Biol., November 24, 2003; 163(4): 871 - 878. [Abstract] [Full Text] [PDF]
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J. S. Palumbo, K. E. Talmage, H. Liu, C. M. La Jeunesse, D. P. Witte, and J. L. Degen Plasminogen supports tumor growth through a fibrinogen-dependent mechanism linked to vascular patency Blood, October 15, 2003; 102(8): 2819 - 2827. [Abstract] [Full Text] [PDF]
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P. Hu, J. Yan, J. Sharifi, T. Bai, L. A. Khawli, and A. L. Epstein Comparison of Three Different Targeted Tissue Factor Fusion Proteins for Inducing Tumor Vessel Thrombosis Cancer Res., August 15, 2003; 63(16): 5046 - 5053. [Abstract] [Full Text] [PDF]
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C. Halin, V. Gafner, M. E. Villani, L. Borsi, A. Berndt, H. Kosmehl, L. Zardi, and D. Neri Synergistic Therapeutic Effects of a Tumor Targeting Antibody Fragment, Fused to Interleukin 12 and to Tumor Necrosis Factor {alpha} Cancer Res., June 15, 2003; 63(12): 3202 - 3210. [Abstract] [Full Text] [PDF]
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M. Santimaria, G. Moscatelli, G. L. Viale, L. Giovannoni, G. Neri, F. Viti, A. Leprini, L. Borsi, P. Castellani, L. Zardi, et al. Immunoscintigraphic Detection of the ED-B Domain of Fibronectin, a Marker of Angiogenesis, in Patients with Cancer Clin. Cancer Res., February 1, 2003; 9(2): 571 - 579. [Abstract] [Full Text] [PDF]
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 P. Castellani, L. Borsi, B. Carnemolla, A. Biro, A. Dorcaratto, G. L. Viale, D. Neri, and L. Zardi Differentiation between High- and Low-Grade Astrocytoma Using a Human Recombinant Antibody to the Extra Domain-B of Fibronectin Am. J. Pathol., November 1, 2002; 161(5): 1695 - 1700. [Abstract] [Full Text] [PDF]
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C. Liu, H. Huang, F. Donate, C. Dickinson, R. Santucci, A. El-Sheikh, R. Vessella, and T. S. Edgington Prostate-specific Membrane Antigen Directed Selective Thrombotic Infarction of Tumors Cancer Res., October 1, 2002; 62(19): 5470 - 5475. [Abstract] [Full Text] [PDF]
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T. Fukuda, N. Yoshida, Y. Kataoka, R.-i. Manabe, Y. Mizuno-Horikawa, M. Sato, K. Kuriyama, N. Yasui, and K. Sekiguchi Mice Lacking the EDB Segment of Fibronectin Develop Normally but Exhibit Reduced Cell Growth and Fibronectin Matrix Assembly in Vitro Cancer Res., October 1, 2002; 62(19): 5603 - 5610. [Abstract] [Full Text] [PDF]
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D. C. Blakey, F. R. Westwood, M. Walker, G. D. Hughes, P. D. Davis, S. E. Ashton, and A. J. Ryan Antitumor Activity of the Novel Vascular Targeting Agent ZD6126 in a Panel of Tumor Models Clin. Cancer Res., June 1, 2002; 8(6): 1974 - 1983. [Abstract] [Full Text] [PDF]
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 K. Porkka, P. Laakkonen, J. A. Hoffman, M. Bernasconi, and E. Ruoslahti A fragment of the HMGN2 protein homes to the nuclei of tumor cells and tumor endothelial cells in vivo PNAS, May 28, 2002; 99(11): 7444 - 7449. [Abstract] [Full Text] [PDF]
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D. E. J. G. J. Dolmans, A. Kadambi, J. S. Hill, C. A. Waters, B. C. Robinson, J. P. Walker, D. Fukumura, and R. K. Jain Vascular Accumulation of a Novel Photosensitizer, MV6401, Causes Selective Thrombosis in Tumor Vessels after Photodynamic Therapy Cancer Res., April 1, 2002; 62(7): 2151 - 2156. [Abstract] [Full Text] [PDF]
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B. Carnemolla, L. Borsi, E. Balza, P. Castellani, R. Meazza, A. Berndt, S. Ferrini, H. Kosmehl, D. Neri, and L. Zardi Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix Blood, March 1, 2002; 99(5): 1659 - 1665. [Abstract] [Full Text] [PDF]
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 C. Halin, L. Zardi, and D. Neri Antibody-Based Targeting of Angiogenesis Physiology, August 1, 2001; 16(4): 191 - 194. [Abstract] [Full Text] [PDF]
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 Vet. Pathol., May 1, 2001; 38(3): 355 - 355. [Full Text] [PDF]
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scFv(D1.3)-tTF; •, saline; 
, scFv(L19)-tTF.
, saline; 
, 9 µg
scFv(L19)-tTF; 
, 15 µg scFv(L19)-tTF; 