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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Laboratory of Cell Biology and Laboratory of
Immunopharmacology, Istituto Nazionale per la Ricerca sul Cancro,
Genoa, Italy; Institute of Pathology, Friedrich Schiller University,
Jena, Germany; and Department of Applied Biosciences of the Swiss
Federal Institute of Technology (ETH), Zürich, Switzerland.
Angiogenic processes depend on the precise coordination of
different cell types and a complex exchange of signals, many of which
derive from new specific components of the provisional, angiogenesis-related, extracellular matrix (ECM).
Angiogenesis-associated ECM components thus represent appealing targets
for the selective delivery of therapeutic molecules to newly forming
tumor vessels. Results of a previous study indicated that a high
affinity recombinant antibody (L19) to ED-B, a domain contained in the
angiogenesis-associated isoform of fibronectin (B-FN), selectively and
efficiently targets tumor vessels. The present study shows that a
fusion protein between L19 and interleukin 2 (L19-IL-2) mediates the
selective delivery and concentration of IL-2 to tumor vasculature,
thereby leading to a dramatic enhancement of the therapeutic properties
of the cytokine. By contrast, IL-2 fused to an irrelevant recombinant antibody used as a control fusion protein showed neither accumulation in tumors nor therapeutic efficacy. Tumors in mice treated with L19-IL-2 were significantly smaller compared to those in animals treated with saline, the control fusion protein, or IL-2 alone (P = .003, .003, and .002, respectively). Moreover, no
significant differences in size were observed among the tumors from the
different control groups (using the control fusion protein, a mixture
of IL-2 and L19, or saline alone). Immunohistochemical analysis of tumor infiltrates demonstrated a significantly higher number of T
lymphocytes, natural killer cells, and macrophages, as well as
increased interferon- During tumor progression, the extracellular matrix
(ECM) of the normal tissues in which the tumor grows is remodeled
through 2 different processes: proteolytic degradation and neosynthesis of ECM components by both neoplastic and stromal cells. These processes
generate a "tumoral ECM" that differs quantitatively and
qualitatively from the normal tissue ECM and that apparently gives rise
to a more suitable environment (inductive or instructive) for tumor
progression.1-3 In particular, ECM components modulate vascular cell behavior and angiogenic processes.4,5 This observation is upheld by the recent report that the majority of messenger RNAs (mRNAs) newly expressed by tumoral endothelial cells
encode for ECM proteins.6 Thus, these provisional ECM components that appear during the angiogenic processes represent an
appealing target for the selective delivery of therapeutic molecules to
newly forming blood vessels.7 Furthermore, because new
vessel formation is common to all solid tumors, the
angiogenesis-associated ECM components can be regarded as pan-tumoral
antigens.8-12
One such ECM component is a fibronectin (FN) isoform, B-FN, which
contains an extra FN type III repeat of 91 amino acids, the domain B
(ED-B).13 Because the amino acid sequence of ED-B is
identical in mouse, humans, and other mammals, antibodies to this
domain react equally well with mouse, human, and other species B-FN.
B-FN is detectable only in the stroma of fetal and neoplastic tissues
and around newly forming blood vessels, but not in mature vessels.14,15 Using a radioiodinated human recombinant
single-chain Fv (scFv; L19) to the ED-B domain of FN, we
demonstrated in vivo the possibility of targeting newly forming tumoral
blood vessels, thus laying the groundwork for the selective delivery of
therapeutic agents to neovasculature.16-20 In addition,
Bircher et al21 showed that L19, chemically coupled to a
photosensitizer, selectively accumulates in the newly formed blood
vessels of the rabbit cornea angiogenesis model and, after irradiation
with near infrared light, mediates complete and selective occlusion of
ocular neovasculature. More recently, Nilsson et al22
reported that the immunoconjugate of L19 with the extracellular domain
of tissue factor mediates selective infarction in different types of
murine tumor models.
These promising findings on the targeted accumulation of therapeutic
agents prompted us to investigate the efficacy in tumor therapy of
fusion proteins composed of L19 and cytokines. Interleukin 2 (IL-2)
plays an essential role in the activation phases of both specific and
natural immune responses.23 Although IL-2 has no direct
cytotoxic effect on cancer cells, it can induce tumor regression through its ability to stimulate a potent cell-mediated immune response
in vivo.24 However, its systemic therapeutic efficacy is
limited by its rapid clearance and, at high doses, by a severe toxicity
related mainly to vascular leak syndrome.25 To overcome systemic toxicity, but, at the same time, to selectively deliver therapeutically efficacious concentrations of IL-2 to neoplastic tissues, a number of preclinical experiments on murine tumor models have been carried out using fusion proteins composed of IL-2 and antibodies with different specificities.26-35 Although
these experiments clearly proved the concept that the targeted delivery
of IL-2 enhanced its therapeutic efficacy, the experiments were carried out using artificially overexpressed tumor cell surface antigens, and
often the antigens were of human origin, thus making it difficult to
evaluate both the accumulation in normal mouse organs and the real
therapeutic efficacy of such fusion proteins.
Here we show that a fusion protein between IL-2 and L19, a human
antibody that reacts equally well with a mouse and human antigen that
is naturally expressed on blood vessels during angiogenic processes,
mediates the selective delivery and concentration of IL-2 on tumor
vasculature, thus leading to a dramatic enhancement of the therapeutic
efficacy of the cytokine.
Construction of L19-IL-2 and D1.3-IL-2 fusion proteins
Selection of clones expressing the L19-IL-2 and D1.3-IL-2 fusion
proteins and their purification and characterization
Animals and cell lines Athymic nude mice (8-week-old nude/nude CD1 mice, females) were obtained from Harlan Italy (Correzzana, Milan), 129 (clone SvHsd) strain mice (8-10-week-old mice, female) were obtained from Harlan UK (Oxon, United Kingdom). Mouse embryonal teratocarcinoma (F9), mouse T cells (CTLL-2), and mouse myeloma cells (P3U.1) were purchased from ATCC; N592, human small cell lung cancer (SCLC) cell line,36 was kindly provided by Dr J. D. Minna (National Cancer Institute and Naval Hospital, Bethesda, MD); C51, a mouse colon adenocarcinoma cell line derived from BALB/c, was kindly provided by Dr M. P. Colombo, Milan, Italy.38Biodistribution of L19-IL-2 fusion protein Purified L19-IL-2 was radiolabeled with iodine 125 using the Iodogen method39 (Pierce, Rockford, IL). The immunoreactive radiolabeled L19-IL-2 was more than 90%. Nude mice with subcutaneously implanted F9 murine teratocarcinoma16 were injected intravenously with about 10 µg (4 µCi; 0.148 MBq) protein in 100 µL saline solution. Three animals were used for each time point. Mice were killed at 3, 6, and 24 hours after injection. The organs were weighed and the radioactivity was counted. All organs and tumors were placed in fixative for histologic analysis and microautoradiography. Targeting results of representative organs are expressed as percent of the injected dose per gram of tissue (%ID/g).In vivo treatment of tumor-bearing mice with L19-IL-2 and D1.3-IL-2 fusion proteins and histologic analysis Treatment with purified L19-IL-2 fusion protein was performed in groups of 6 mice each injected subcutaneously with 20 × 106 N592 or with 106 C51 or with 3 × 106 F9 cells. Twenty-four hours or 72 hours after N592, F9, and C51 cell injection, 12 µg L19-IL-2 fusion protein (corresponding to 72 000 IU) was injected into the tail vein of each animal until day 10 (Figure 3A). Similar groups of animals (6/group) were injected with a control fusion protein (D1.3-IL-2) or with a mixture of L19 (8 µg) and recombinant human IL-2 (4 µg, corresponding to 72 000 IU; proleukin, 18 × 106 UI; Chiron, Emeryville, CA) and with saline for the same number of days. At the end of treatment, animals were killed, and tumors, organs, and tissues were weighed and placed in fixative for histologic analysis. Tissue samples were cut within the largest diameter, the total and tumor areas were determined by computer aid, and the percentage of the tumor tissue was calculated.Immunohistochemistry and image analysis For qualitative and quantitative immunohistochemical analysis, 4-µm cryostat sections of the frozen tissue samples were fixed in ice-cold acetone for 15 minutes (the antibody PK136 was applied to native sections). The following primary antibodies were used: rat antimouse CD8a/Ly2 (clone 53-6.7); rat antimouse CD4/L3T4 (clone H129.19); rat antimouse CD11b/Mac-1 chain (clone M1/70); rat
antimouse CD19 (clone 1D3); rat antimouse interferon (IFN)- (clone
R4-6A2) (all from BD Pharmingen, San Diego, CA); rat antimouse CD31
(clone MEC 13.3) kindly supplied by A. Mantovani (Mario Negri Institute, Milan, Italy)43; rat antimouse CD45R/B220
(clone RA3-6B2) (Southern Biotechnology Associates, Birmingham, AL); and biotinylated mouse antimouse CD161b/c/NK1.1 (clone PK136) (Serotec,
Oxford, United Kingdom). The sections were incubated with primary
antibodies overnight at 4°C. Biotinylated mouse antirat IgG2b and
IgG1/2a (BD Pharmingen) were used as secondary antibodies for detection
of unlabeled primary antibodies followed by the alkaline
phosphatase-conjugated ABC system StreptABComplex/AP (Dako, Denmark).
Naphtol-AS-biphosphate (Sigma) and new fuchsin (Merck, Darmstadt,
Germany) were used as substrate and developer, respectively. To
inhibit endogenous tissue enzyme activity, the developing solution was
supplemented with 0.25 mM levamisole (Sigma). As negative control, the
primary antibody was replaced by nonimmune serum. Sections were then
counterstained with hematoxylin and mounted permanently. Quantitative
analysis of inflammatory cells, IFN-, and microvessel density (MVD)
was performed by computer-aided image analysis using the image
processing and analysis system Quantimet 600 and the Qwin software
(Leica, Wetzlar, Germany). A red color threshold was
interactively set by the examiner for each antibody to select the
red-stained immunopositive area. All pixels in the image that met the
defined threshold were transferred into a binary image and analyzed.
For quantitative evaluation of inflammatory cells and [IFN]-, in
each tumor specimen the immunostained area was measured in 5 randomly
selected measurement areas (total measured area 1.35 mm2)
of the tumor periphery to avoid the influence of necrosis. The results
were expressed as percentage of the whole measurement area. For each
animal group the mean value ± SE of all measurements was calculated.
The MVD was assessed according to Weidner at al.40 Intratumoral MVD was evaluated by light microscopic scanning at low power of tumoral areas containing the most capillaries and small venules (so-called neovascular hot spots). The individual microvessel counts were made on a 200 × field in the neovascular hot spots, while a total tumor area of 0.3375 mm2 was evaluated. The objects suspected by the computer-aided image analysis system as microvessels were controlled on the computer monitor before counting. The microvessel counts are given as the average of marked objects per standard area of tumor, which corresponds to one scanned counting field with a size of 0.0675 mm2.41 For each animal group, the mean values and SEs of all measurements were calculated. Microautoradiography analysis and statistical analysis Tumor and organ specimens were processed for microautoradiography to assess the pattern of 125I L19-IL-2 fusion protein distribution within the tumors or organs as described by Tarli et al.16 The statistical significance of different findings between experimental groups of animals was determined using the nonparametric Mann-Whitney test. Findings were regarded as significant if 2-tailed P < .01.
For the preparation of scFv-IL-2 fusion proteins, we used the human scFv L19 and, for the preparation of the "control" fusion protein endowed with an irrelevant specificity, we used the scFv D1.342 that reacts with hen egg lysozyme but not with mouse lysozyme. As shown in Figure 1A, the cDNA
corresponding to the open reading frame of mature IL-2 was appended to
the 3' of the cDNA of the scFv by a linker of 45 bp. Chimeric cDNAs
were cloned into the vector pcDNA3.1 and used to transfect mouse
myeloma cells (P3U1). The 2 fusion proteins were purified from the
conditioned media of the transfected cells by affinity chromatography
columns. Figure 1B shows an SDS-PAGE of the 2 purified proteins.
L19-IL-2 was tested for immunoreactivity of the scFv by
immunohistochemistry and ELISA, and both fusion proteins were evaluated
for biologic activity of IL-2 by CTLL-2 proliferation
assay.36 Equimolar amounts of IL-2 and of the fusion
proteins L19-IL-2 and D1.3-IL-2 showed identical IL-2 activity.
Equimolar amounts of L19-IL-2 and of scFv L19 showed identical
immunoreactivity (data not shown).
Fusion proteins were radioiodinated and intravenously injected in
tumor-bearing animals for biodistribution analysis. Figure 2A shows the tumor-blood ratio of the
percentage of injected dose per gram using the 2 radiolabeled proteins.
Twenty-four hours after intravenous injection of radiolabeled
L19-IL-2, this ratio was 33, whereas using D1.3-IL-2 it was less than
1 (Figure 2A). Using L19-IL-2, the ratio between the percentage of
injected dose per gram of tumor and of the other organs, 24 hours after
injection, was higher than 10. By contrast, no specific tumor
accumulation was seen using the control fusion protein. Intratumoral
accumulation of the radiolabeled proteins was analyzed by
microautoradiography. As shown in Figure 2B, L19-IL-2 selectively
accumulated around tumoral vasculature, similarly to L19
alone.16 No accumulation was detectable using D1.3-IL-2.
To test whether the targeted delivery of IL-2 to tumor vasculature was
able to inhibit tumor growth, we treated groups of syngeneic mice
grafted with F9 murine teratocarcinoma with the fusion proteins
L19-IL-2 or D1.3-IL-2 or saline alone. Figure 2A shows the treatment
schedule used. Table 1 reports the
typical results obtained treating tumor-bearing mice for 6 days with
daily intravenous doses of 72 000 U IL-2 per mouse as either L19-IL-2 or D1.3-IL-2. Figure 3A shows the tumors
removed from the 3 groups of mice at the end of the treatment. The
weights of tumors from animals treated with L19-IL-2 were compared to
those from animals treated with D1.3-IL-2 using the Mann-Whitney
statistical test and were found to be highly significantly different
(P = .003, Table 1).
Tumors were also analyzed histologically and immunohistochemically, and
the results are summarized in Table 1. The major observations were: (1)
a higher lymphocyte infiltration in tumors from animals treated with
L19-IL-2 with respect to controls (about 70 times higher with respect
to animals treated with D1.3-IL-2 and 100 times higher with respect to
animals treated with saline as shown in Figure 3B); (2) the amount of
connective tissue and necrosis in tumors from animals treated with
L19-IL-2 was nearly 70% of the tumor mass, but only 18% and 11% in
tumors from animals treated with D1.3-IL-2 and saline, respectively.
Similar results were obtained using different tumor models such as C51
murine adenocarcinoma and N592 human SCLC (Table
2). No significant differences were
observed in tumors grafted in syngeneic or athymic nude mice,
suggesting that the antitumor response may occur in the absence of T
cells.
We also tested the ability of L19-IL-2 to inhibit the growth of
well-established tumors. Six days after grafting F9 teratocarcinoma into syngeneic mice, tumors reached a volume of nearly 0.4 to 0.5 cm3. Three groups of these animals were then treated for 6 days with daily intravenous injection of 250 000 U IL-2 as L19-IL-2,
unconjugated IL-2/L19 mixture, or saline alone. The results, depicted
in Figure 4, show that although L19-IL-2
induced a regression of about 50% of the tumor mass, the mixture of
unconjugated IL-2/L19 did not significantly inhibit tumor growth
compared to treatment with saline alone. In the group of animals
treated with L19-IL-2, the tumor masses remained stable until 5 days
after interruption of treatment; thereafter, tumors resumed growth, but
at a reduced rate compared to controls (Figure 4).
Using a panel of antibodies, we studied and compared the inflammatory
cells infiltrating the tumors from the groups of animals treated with
the fusion protein L19-IL-2, an equimolar mixture of IL-2 and L19, or
saline. We also studied the amount of IFN-
We also studied immunohistochemically the MVD in nonnecrotic, nonfibrotic areas of tumor sections from different groups of animals using a monoclonal antibody specific for mouse CD31.43 We observed only a slight reduction of MVD in the tumor tissues of mice treated with L19-IL-2 compared to the tumors from animals of the control groups (Table 3).
The targeted delivery of cytokines to solid tumors using antibody-cytokine fusion proteins represents a promising strategy in cancer immunotherapy. This approach achieves the purpose of selectively distributing cytokine concentrations sufficient to elicit a significant antitumor activity in primary and disseminated tumors, while limiting systemic toxicity. We chose to selectively deliver therapeutic agents to tumor ECM components, which are generally more abundant and stable with respect to tumor-associated cell surface antigens.7 Here we report the preclinical therapeutic evaluation of the fusion protein L19-IL-2, which binds to the provisional tumor ECM component B-FN, a marker of angiogenesis. We show that L19-IL-2, injected intravenously in tumor-bearing mice, accumulates selectively around tumor blood vessels, as does the scFv L19 alone.16 Using murine tumor models, we found that L19-IL-2 inhibits tumor
growth more effectively than either the control fusion protein D1.3-IL-2 or an equimolar mixture of IL-2 and L19. In all 3 experimental animal models we studied, most of the tumor mass (70%) in
animals treated with L19-IL-2 was composed of connective and necrotic tissue. In addition, a dramatic increase in lymphocyte infiltration and
IFN- Moreover, because endothelial cells migrate along the fibers of the provisional ECM, and because IL-2 is known to be cytotoxic to endothelial cells,25 the possibility to selectively deposit IL-2, and thereby convey cytotoxic properties to this ECM, should allow the cytokine to more effectively elicit its cytotoxic action on migrating endothelial cells. To assess this possibility, we measured the MVD in neovascular of tumors, and found only a slight decrease in tumoral MVD in animals treated with L19-IL-2 compared to animals receiving a mixture of IL-2 and L19 or saline alone. Nevertheless, considering that necrotic and fibrotic areas represented more than 70% of the total mass of the tumors from animals treated with L19-IL-2, we cannot rule out a priori that the direct cytotoxic effect of IL-2 on endothelial cells could have contributed to the generation of the observed fibrosis and necrosis. Further experiments are needed to validate or disprove this concept. Furthermore, the targeted delivery of therapeutic agents to the subendothelial ECM could also help to overcome one of the major problems hindering cancer therapy, namely, the poor delivery of drugs owing to the interstitial hypertension that is characteristic of solid tumors.45,46 The L19-IL-2 fusion protein reported here serves this purpose, because it binds avidly to B-FN, which is a component of the subendothelial matrix of blood vessels during angiogenic processes. We have set up an assay to quantitatively determine B-FN in tissues from murine experimental tumor models and from fragments of human tumors. Our results indicate that in the murine teratocarcinoma F9 there are about 300 µg B-FN/g fresh tissue. This figure is similar to the levels of B-FN found in human tumors; in fact, we found from 125 to 320 µg B-FN/g fresh tissue in different specimens of human lung cancer, and from 100 to 400 µg B-FN/g in specimens of human glioblastoma, whereas in normal lung and brain tissues B-FN was undetectable (L.Z., unpublished results, manuscript in preparation). In conclusion, we demonstrate that the targeted delivery of IL-2 to provisional, angiogenesis-related ECM components is feasible and therapeutically effective. Furthermore, because B-FN is a naturally occurring angiogenesis marker identical in mouse and in humans, and because the amount of ED-B in human tumors is similar to that expressed in murine tumor models, the scFv L19 appears to be a very powerful and fitting reagent to be used as a means to deliver a variety of effector molecules to newly forming blood vessels, thus providing a new approach to therapy for solid tumors.
This manuscript is dedicated to Professor Leonardo Santi, founder of the Istituto Nazionale per la Ricerca sul Cancro, Genoa, for his 75th birthday.
Submitted May 17, 2001; accepted October 22, 2001.
Supported in part by the Associazione Italiana per la Ricerca sul Cancro (AIRC), EU BIO4-CT97-2149 Project "Neo-vasculature markers," Italian Health Ministry, Krebforschung Schweiz, and the Bundesamt für Bilding und Wissenschaft and Philogen s.r.l.
Two of the authors (L.Z. and D.N.) are consultants of and hold shares in Philogen (La Lizza 7, Siena, Italy), a new biotechnology company dedicated to the development of antibody-based anti-angiogenesis compounds.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Luciano Zardi, Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10 16132 Genoa, Italy; e-mail: lzardi{at}tin.it.
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  E. Ventura, F. Sassi, S. Fossati, A. Parodi, W. Blalock, E. Balza, P. Castellani, L. Borsi, B. Carnemolla, and L. Zardi Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins J. Biol. Chem., September 25, 2009; 284(39): 26646 - 26654. [Abstract] [Full Text] [PDF]
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 C. Schliemann, A. Palumbo, K. Zuberbuhler, A. Villa, M. Kaspar, E. Trachsel, W. Klapper, H. D. Menssen, and D. Neri Complete eradication of human B-cell lymphoma xenografts using rituximab in combination with the immunocytokine L19-IL2 Blood, March 5, 2009; 113(10): 2275 - 2283. [Abstract] [Full Text] [PDF]
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S. Sauer, P. A. Erba, M. Petrini, A. Menrad, L. Giovannoni, C. Grana, B. Hirsch, L. Zardi, G. Paganelli, G. Mariani, et al. Expression of the oncofetal ED-B-containing fibronectin isoform in hematologic tumors enables ED-B-targeted 131I-L19SIP radioimmunotherapy in Hodgkin lymphoma patients Blood, March 5, 2009; 113(10): 2265 - 2274. [Abstract] [Full Text] [PDF]
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 J. Marlind, M. Kaspar, E. Trachsel, R. Sommavilla, S. Hindle, C. Bacci, L. Giovannoni, and D. Neri Antibody-Mediated Delivery of Interleukin-2 to the Stroma of Breast Cancer Strongly Enhances the Potency of Chemotherapy Clin. Cancer Res., October 15, 2008; 14(20): 6515 - 6524. [Abstract] [Full Text] [PDF]
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K. Wagner, P. Schulz, A. Scholz, B. Wiedenmann, and A. Menrad The Targeted Immunocytokine L19-IL2 Efficiently Inhibits the Growth of Orthotopic Pancreatic Cancer Clin. Cancer Res., August 1, 2008; 14(15): 4951 - 4960. [Abstract] [Full Text] [PDF]
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 M. Kaspar, E. Trachsel, and D. Neri The Antibody-Mediated Targeted Delivery of Interleukin-15 and GM-CSF to the Tumor Neovasculature Inhibits Tumor Growth and Metastasis Cancer Res., May 15, 2007; 67(10): 4940 - 4948. [Abstract] [Full Text] [PDF]
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 T. von Lukowicz, M. Silacci, M. T. Wyss, E. Trachsel, C. Lohmann, A. Buck, T. F. Luscher, D. Neri, and C. M. Matter Human Antibody Against C Domain of Tenascin-C Visualizes Murine Atherosclerotic Plaques Ex Vivo J. Nucl. Med., April 1, 2007; 48(4): 582 - 587. [Abstract] [Full Text] [PDF]
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 V. Castronovo, D. Waltregny, P. Kischel, C. Roesli, G. Elia, J.-N. Rybak, and D. Neri A Chemical Proteomics Approach for the Identification of Accessible Antigens Expressed in Human Kidney Cancer Mol. Cell. Proteomics, November 1, 2006; 5(11): 2083 - 2091. [Abstract] [Full Text] [PDF]
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 M. Silacci, S. S. Brack, N. Spath, A. Buck, S. Hillinger, S. Arni, W. Weder, L. Zardi, and D. Neri Human monoclonal antibodies to domain C of tenascin-C selectively target solid tumors in vivo Protein Eng. Des. Sel., October 1, 2006; 19(10): 471 - 478. [Abstract] [Full Text] [PDF]
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D. Moosmayer, D. Berndorff, C.-H. Chang, R. M. Sharkey, A. Rother, S. Borkowski, E. A. Rossi, W. J. McBride, T. M. Cardillo, D. M. Goldenberg, et al. Bispecific antibody pretargeting of tumor neovasculature for improved systemic radiotherapy of solid tumors. Clin. Cancer Res., September 15, 2006; 12(18): 5587 - 5595. [Abstract] [Full Text] [PDF]
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S. S. Brack, M. Silacci, M. Birchler, and D. Neri Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clin. Cancer Res., May 15, 2006; 12(10): 3200 - 3208. [Abstract] [Full Text] [PDF]
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E. Balza, L. Mortara, F. Sassi, S. Monteghirfo, B. Carnemolla, P. Castellani, D. Neri, R. S. Accolla, L. Zardi, and L. Borsi Targeted Delivery of Tumor Necrosis Factor-{alpha} to Tumor Vessels Induces a Therapeutic T Cell-Mediated Immune Response that Protects the Host Against Syngeneic Tumors of Different Histologic Origin Clin. Cancer Res., April 15, 2006; 12(8): 2575 - 2582. [Abstract] [Full Text] [PDF]
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D. Berndorff, S. Borkowski, S. Sieger, A. Rother, M. Friebe, F. Viti, C. S. Hilger, J. E. Cyr, and L. M. Dinkelborg Radioimmunotherapy of Solid Tumors by Targeting Extra Domain B Fibronectin: Identification of the Best-Suited Radioimmunoconjugate Clin. Cancer Res., October 1, 2005; 11(19): 7053s - 7063s. [Abstract] [Full Text] [PDF]
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 J. Smith, R. E. Kontermann, J. Embleton, and S. Kumar Antibody phage display technologies with special reference to angiogenesis FASEB J, March 1, 2005; 19(3): 331 - 341. [Abstract] [Full Text] [PDF]
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 P. Carter, L. Smith, and M. Ryan Identification and validation of cell surface antigens for antibody targeting in oncology Endocr. Relat. Cancer, December 1, 2004; 11(4): 659 - 687. [Abstract] [Full Text] [PDF]
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P. E. Thorpe Vascular Targeting Agents as Cancer Therapeutics Clin. Cancer Res., January 15, 2004; 10(2): 415 - 427. [Abstract] [Full Text] [PDF]
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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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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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Top
Abstract
Introduction
) encoding for 15 amino acids
(SSSSG)3 and cloned into the pcDNA3 mammalian expression
vector using the HindIII and BamHI restriction
sites.
is the intron inside
the genomic sequence); and white boxes, the VH or
VL of scFV L19 or D1.3 and IL-2 sequences. (B)
Four percent to 18% SDS-PAGE gradient of scFv L19, L19-IL-2, and
D1.3-IL-2 proteins after purification on ED-B/Sepharose affinity
column. The values reported on the left indicate the molecular masses,
in kilodaltons, of the standards. The fusion proteins L19-IL-2 and
D1.3-IL-2 showed an apparent molecular mass of about 43 and 45 kd,
respectively, in accordance with their expected sizes.
