Chimeric protein ABRaA-VEGF 121 is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B 16-F 10 melanoma growth

It has been known that VEGF121 isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF121. Expression of the ABRaA-VEGF121 chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF121 preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF121 is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF121 inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF121 with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF121 inhibits growth of B16-F10 murine melanoma tumors.

Among the novel approches to antitumor therapeutic strategy one is based on various engineered chimeric proteins combining toxic agents

2009
A. Smagur and others linked to a carrier (growth factors, immunoglobulins), the latter allowing specific targeting of various molecules involved in the VEGF pathway (Thorpe, 2004;Szala, 2004).The VEGF 121 isoform, which is secreted as a freely difusible non-heparin-binding protein, has been successfully used to deliver toxins to the tumor vascular endothelium.Such chimeric toxins using the VEGF 121 as a targeting domain mediate their cytotoxicity primarily through VEGFR-2 (Arora et al., 1999;Backer et al., 2001;Veenendaal et al., 2002;Liu et al., 2003).
Abrin is a potent plant toxin belonging to the type II family of eukaryotic ribosome-inactivating proteins (Narayanan et al., 2004).Many poisoning features of abrin observed in mammals can be explained by endothelial cell damage leading to increased capillary permeability and vascular leak syndrome (Dickers et al., 2003) as well as apoptotic changes in lymphoid tissues and intestine (Griffiths et al., 1987).Abrin can inhibit tumor growth and prolong survival of mice in several experimental tumor models (Fodstad et al., 1977).Abrin is capable of inducing cell apoptosis by various mechanisms (Shih et al., 2001;Ohba et al., 2004;Qu & Qing, 2004).
Abrin is a heterodimeric protein comprising enzymatic A chain coupled, via a single disulphide linkage, with cell-binding (lectin) B chain (Olsnes & Pihl, 1973;Olsnes et al., 1975;Narayanan et al., 2004).The A chain of abrin-a isoform (ABRaA) is a specific N-glycosidase which causes irreversible inactivation of eukaryotic ribosomes by adenine depurination at position 4324 of 28S rRNA (Endo et al., 1987;Wang et al., 2004).ABRaA is over 10 5 -fold less cytotoxic towards HeLa cells than abrin holotoxin (disulphide-linked A and B chains) (Chow et al., 1999) and, following intravenous injection, does not elicit significant toxic effects in Guinea pig (Hwang et al., 1984).ABRaA has also been used in developing tumor-targeted toxin-antibody therapeutic conjugates (Hwang et al., 1984;Wawrzynczak et al., 1990).Compared with immunotoxins containing other ribosome-inactivating proteins (ricin A, gelonin, momortin), ABRaA immunotoxins show longer serum half-life and, when present in the circulation, retain full cytotoxic activity (Wawrzynczak et al., 1990).Nevertheless, the use of immunotoxins in cancer treatment is limited because of their poor ability to permeate solid tumors (Pastan et al., 2007).Chimeric toxins containing VEGF 121 thus seem to be good candidates for cancer therapeutic agents as they are able to indirectly destroy tumors by selective disruption of tumor vascular endothelium (Veenendaal et al., 2002).
The aim of our study was to generate and purify the ABRaA-VEGF 121 chimeric protein, to test its properties in vitro and to verify the expected anti-tumor properties in treating experimentally induced B16-F10 murine melanoma.

MATERIALS AND METHODS
Human VEGF 121 gene synthesis and construction of expression vector encoding ABRaA-VEGF 121 .Human VEGF 121 coding sequence without a signal sequence was optimized with the help of Prot-2-DNA software (Gustafsson et al., 2004) in order to eliminate rare codons and to achieve effective translation in Escherichia coli.Synthetic VEGF 121 gene was constructed from oligonucleotides (BioTez Berlin-Buch GmbH, Berlin, Germany) (Table 1) as described by Mitrus et al. (2005), with minor changes.The long oligonucleotides #1-#9 yield a VEGF 121 coding sequence with BamHI and HindIII restriction sites and five additional adenines.The oligonucleotides #2-#9 were phosphorylated and oligonucleotide #1 was then added to the reaction mixture and ligated in the presence of the short oligonucleotides #1-2 to #8-9, which should have assured the correct order of linking of the long oligonucleotides #1-#9.The VEGF 121 cDNA was amplified by PCR using Taq DNA Polymerase (Fermentas Inc., Hanover, MD, USA), primer #1 and reverse primer (Table 1) and cloned into pET41a(+) (Novagen, San Diego, CA, USA) using BamHI and HindIII restriction sites, which generated the pET41/VEGF 121 construct.
Codon 121, gat (Asp) in the original ABRaA sequence (GenBank accession number AY458627), was replaced with a synonymous codon (gac) in order to remove the BamHI restriction site.A sequence encoding the G 4 S linker was added to the 3' end.The two terminal amino acids of the linker are encoded by the ggatcc sequence, which is also a BamHI restriction site serving to ligate VEGF 121 .A 43-bp fragment of pET41a(+) plasmid, spanning from BglII site to the enterokinase-recognized site (ER), was added at the 5' end of the ABRaA coding sequence, and this coding cDNA sequence (ER-ABRaA-G 4 S) was synthesized by GenScript Corporation (Piscataway, NJ, USA).The sequence was subsequently cloned, using BglII and BamHI restriction sites, into the GST-His-S•Tag-EK reading frame of the pET41/ VEGF 121 vector, resulting in the formation of pET41/ ABRaA-VEGF 121 +Tag construct.In order to remove additional coding sequences of plasmid origin (GST-His-S•Tag-EK), the ABRaA amino end (223 bp) up to the NdeI restriction site was amplified by PCR (Pfx Platinum Polymerase, Invitrogen, Carlsbad, CA, USA) using L(AV) and R(AV) starters (Table 1) and cloned into the pET41/ABRaA-VEGF 121 +Tag plasmid using NdeI restriction site present in the plasmid and in ABRaA (construct: pET41/ABRaA-VEGF 121 , without tag).Next, the XbaI/XhoI fragment from pET41/ Expression, isolation and purification of recombinant ABRaA-VEGF 121 protein.The E. coli BL21(DE3) strain (Novagen, San Diego, CA, USA) transformed with pET32/ABRaA-VEGF 121 plasmid was grown in LB broth (BD Bioscience, Franklin Lakes, NJ, USA) with 100 μg/ml ampicillin (Sigma, St. Louis, MO, USA) to OD 600 = 0.8-1.0.Protein expression was induced using IPTG (Fluka, Buchs, Switzerland) at a final concentration of 0.5 mM and cells were further grown for additional 3 h at 37°C.Bacterial pellets were harvested by centrifugation (45 000 × g, 30 min, 4°C) and stored at -20°C.
Endotoxin content in ABRaA-VEGF 121 solutions was measured using the LAL QLC 1000 kit (Cambrex, East Rutherford, NJ, USA).Removal of endotoxin (to < 0.0025 EU/1 μg protein) was achieved through affinity chromatography with EndoTrap Red kit components (Profos AG, Regensburg, Germany).Before further use the samples were filter-sterilized and stored at 4°C.
The purified protein was analyzed by 10% SDS/PAGE under reducing and non-reducing conditions and stained by Coomassie blue.Western blots were performed using nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany).Detection of ABRaA-VEGF 121 was achieved with a mouse monoclonal antibody against human VEGF (R&D Systems, Minneapolis, MN, USA), horse anti-mouse IgG biotin conjugate (Vector Laboratories, Burlingame, CA, USA) and streptavidin-biotinylated horseradish peroxidase complex (Amersham Biosciences, Piscataway, NJ, USA).To perform peroxidase reaction and protein visualization 3,3'-diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO, USA) was used as a substrate.
Neutralization of ABRaA-VEGF 121 cytotoxicity by monoclonal antibody.Subconfluent PAE/KDR cells were trypsinized and plated (3 × 10 3 cells/well) using 96-well plates and allowed to attach for 24 h.The growth medium was then replaced by a medium containing different concentrations of ABRaA-VEGF 121 , previously preincubated (37°C, 2 h) with 2 μg/ml mouse monoclonal antibodies neutralizing the bioactivity of human VEGF (R&D Systems, Minneapolis, MN, USA).As a control, ABRaA-VEGF 121 preincubated without a monoclonal antibody was used.After 72 h, the effect of ABRaA-VEGF 121 on cell viability was determined using the MTT assay, as described above.
In vitro cell-free inhibition of protein biosynthesis by ABRaA-VEGF 121 .In vitro protein translation was performed using a cell-free rabbit reticulocyte lysate-based system (TNT  T7 Quick Coupled Transcription/Translation System, Promega, Madison, WI, USA) and the non-radioactive luciferase control reaction protocol provided with this system.The reaction mix contained 1 μg/reaction of Luciferase T7 Control plasmid and different concentrations of ABRaA-VEGF 121 protein.The translation lev-el (bioluminescence emission) at different timepoints was measured and expressed as luciferase activity using the Luciferase Assay Substrate kit (Promega, Madison, WI, USA) and LUMAT LB9501 luminometer (Berthold Technologies GmbH, Bad Wildbad, Germany).
TUNEL assay.Cells were seeded in gelatin-coated 8-well Lab-Tek™ II Chamber Slide plates (NUNC, Rochester, NY, USA).After 24 h either ABRaA-VEGF 121 in PBS (final concentration 420 ng/ml) or PBS (pH 7.4) was added to the media.The cell cultures were incubated for an additional 24 h and then washed briefly with ice-cold PBS.Cells were fixed for 60 min at room temp.with 4% formaldehyde, rinsed with PBS, permeabilized for 2 min on ice with 0.1% Triton X-100 in 0.1% sodium citrate, and finally washed twice with PBS.Samples were then incubated for 60 min at 37 o C with a TUNEL reaction mixture (In Situ Cell Death Detection Kit, TMR red) (Roche Diagnostics, Basel, Switzerland).To ensure positive control, cells were fixed, permeabilized and treated with DNase I (30 U/ml).TUNEL-positive cells were visualized (excitation λ = 540 ± 25 nm, emission max.λ = 580 nm) by fluorescence microscopy (Nicon Eclipse 80 equipped with Lucia v. 4.8 software) and photographed (magn.400×).
Quantitation of cell death by annexin V and propidium iodide double staining.PAE/KDR cells were seeded (5 × 10 4 cells/well) in a 6-well plate (NUNC, Rochester, NY, USA) and 48 h after seeding exposed for 16 h to different concentrations of ABRaA-VEGF 121 .Adherent cells harvested by mild trypsinization were pooled together with detached cells and washed with ice-cold PBS.Cells were stained with annexin V (AV) and propidium iodide (PI) using the Annexin V-FITC Apoptosis Detection Kit (BD Bioscience, San Diego, CA, USA) according to the manufacturer's protocol.Cells were counted using a BD FACSCanto flow cytometr equipped with BD FACSDiva software (BD Bioscience, San Diego, CA, USA).
Histological analysis.Histological specimens were prepared from tumors or other tissues collected from C57BL/6 mice that had been injected with the ABRaA-VEGF 121 chimeric protein (1 mg/kg body mass) either intravenously or intratumorally.The protein was administered either once on the 6th day following inoculation with cancer cells, or four times (every other day) beginning from the 6th day from inoculation.Mice were sacrificed at 48 h following final injection of the tested protein, tumor material and various tissues were collected and formaldehyde-fixed.Paraffin-embedded sections (6 μmthick) were hematoxylin-and eosin-stained (H&E).Specimens were observed under a light microscope (Nicon Eclipse 80 equipped with Lucia v. 4.8 software).
ABRaA-VEGF 121 in therapy of B16-F10 melanoma-bearing mice.C57BL/6 mice (six-to eight-week-old females) with their left dorsal side shaved (five animals per experimental group) were inoculated subcutaneously with B16-F10 cells (2 × 10 5 cells per animal).Starting on the 6th day after inoculation, when tumors reached about 50 mm 3 , the mice were injected ABRaA-VEGF 121 protein, either via the tail vein or intratumorally.Injection of ABRaA-VEGF 121 was repeated (either 1 mg/kg body mass or 0.25 mg/kg body mass) four times, on every other day.Tumor volume was calculated from the formula: Tumor Volume = (Width) 2 × Length × 0.52.Permission for animal studies was obtained from the local Ethics Commission (Medical University of Silesia, Katowice, Poland).

Construction of cDNA encoding ABRaA-VEGF 121
In order to assure more efficient expression of the chimeric protein in bacterial system, the codons for ABRaA and human VEGF 121 -encoding sequences which are rarely used in E. coli host were replaced with more frequent ones (codon optimization).The sequence encoding human VEGF 121 (GenBank accession No. AF214570), without the signal sequence, was codon-optimized using Prot2DNA software v. 1.0 and an E. coli codon usage table.The designed sequence (GenBank accession No. EF424789) was synthesized de novo from oligonucleotides (Table 1).The ABRaA synthetic coding sequence, together with a short G 4 S spacer, was linked to the cDNA encoding human VEGF 121 in the pET41a(+) vector.cDNA encoding the ABRaA-VEGF 121 chimeric protein (GenBank accession No. EF424790) was cloned into pET32b(+) vector.The pET32/ABRaA-VEGF 121 plasmid was sequenced to verify the coding sequence of ABRaA-VEGF 121 .

Expression in E. coli and purification of ABRaA-VEGF 121
The recombinant ABRaA-VEGF 121 protein was expressed in E. coli BL21(DE3) strain and isolated from bacterial inclusion bodies which were solubilized in 2 M urea buffer.Protein refolding was achieved by eliminating urea by gradual dialysis in the presence of oxidized and reduced glutathione.Under reducing conditions the purified protein migrates in SDS/polyacrylamide gel as a single about 42-kDa band whereas under non-reducing condi-tions two bands are present: an about 84-kDa one (homodimer) and an about 42-kDa one (monomer) (Fig. 1A).The experimental ABRaA-VEGF 121 mass agrees well with the theoretical molecular mass.The monomer/dimer ratio observed for SDS/PAGE was about 1 : 1. ABRaA-VEGF 121 shows immunoreactivity towards mouse monoclonal anti-human VEGF antibody (Fig. 1B).

Neutralization of VEGF 121 domain abrogates ABRaA-VEGF 121 toxicity towards PAE/KDR cells
Preincubation of the ABRaA-VEGF 121 chimeric protein with a monoclonal antibody neutralizes biological activity of human VEGF almost totally abrogates its toxicity towards PAE/KDR cells (Fig. 2B).These data show that the VEGF 121 domain of ABRaA-VEGF 121 is essential for recognition of VEG-FR-2 (KDR) receptor-expressing cells (PAE/KDR).

ABRaA-VEGF 121 inhibits protein biosynthesis in vitro
Abrin A chain is known as a "ribosome-inactivating protein" that inhibits protein translation.The ability of the chimeric ABRaA-VEGF 121 protein to inhibit protein translation was confirmed with a rabbit reticulocyte cell-free system (Promega).Translation was observed to begin 15 min after the start of control reaction.A dose-dependent inhibition of protein translation by ABRaA-VEGF 121 was found in the 4.2-21.0ng/ml protein concentration range (Fig. 3A).These data demonstrate that protein translation is indeed inhibited by the chimeric protein.

ABRaA-VEGF 121 induces death of PAE/KDR cells in vitro
A 24-hour incubation with 0.42 μg/ml ABRaA-VEGF 121 brought positive TUNEL assay results for PAE/KDR cells but not for PAE/hFlt1 or parental PAE cells (Fig. 3B) or untreated cell lines.This indicates that ABRaA-VEGF 121 specifically induces death in cells displaying VEGFR-2 (KDR) receptors.Additionally, the treated PAE/KDR cells exhibit some morphological changes typical of abrin-caused apoptotic cell death, such as cell rounding, shrinking and detachment from the bottom of culture plate (not shown).A similar observation for abrin-treated HeLa cells was made by Qu and Qing (2004).
The apoptotic death assay based on Annexin V (AV) and propidium iodide (PI) staining followed by FACS analysis showed a dose-dependent apoptotic effect of ABRaA-VEGF 121 on PAE/KDR cells.FACS analysis with annexin V/PI staining of control cells revealed a large subpopulation of viable cells (marked as AV − /PI − ) with some staining seen also for early apoptotic (AV + /PI − ), late apoptotic and/or necrotic (AV + /PI + ) as well as dead cells (AV − /PI + ).On the other hand, a 16-hour exposure to increasing ABRaA-VEGF 121 concentrations (0.21, 0.42, 0.84 μg/ml) decreased the number of live cells, increased the subpopulation of early apoptotic cells and caused no significant changes in the late apoptotic/necrotic or dead cell subpopulations (Fig. 3C).

ABRaA-VEGF 121 induces necrosis in B16-F10 tumors
Mice harboring B16-F10 tumors were injected (intravenously or intratumorally) with ABRaA-VEGF 121 protein (1 mg/kg body mass/injection), either once or four times (every other day), starting from the 6th day after inoculation of mice with cancer cells.Forty-eight hours following the final injection mice were sacrificed; tumor material and other tissues were excised and hematoxylin and eosin-stained.Tumor tissue sections revealed tumor necrosis even after a single dose of intravenously administered chimeric protein (Fig. 4A and B).Similar effects were observed following ABRaA-VEGF 121 intratumoral injection (not shown).In contrast, no necrosis was found in the case of sections obtained   Female C57BL/6 mice (n = 3) were inoculated intradermally with B16-F10 cells as described in Materials and Methods.Six days after inoculation, when tumors reached approx.50 mm 3 , ABRaA-VEGF 121 (1 mg/kg) was injected into the tail vein.Control mice obtained PBS only.Animals were sacrificed 48 h later, tumor material was excised, fixed and paraffin-embedded.Sections were H&E-stained.Tumor sections from mice that received ABRaA-VEGF 121 reveal extensive areas of eliminated neoplastic cells and extravasated red blood cells (A and B).
No such damage to the vasculature is seen in sections from untreated control tumors (C and D).
from control tumors (Fig. 4C and D) or normal tissues such as liver, spleen, kidneys and lungs, even after four repetitive injections (total of 4 mg/kg body mass, not shown).

ABRaA-VEGF 121 inhibits B16-F10 tumor growth in mice
ABRaA-VEGF 121 (1 mg/kg body mass/injection) inhibited B16-F10 primary tumor growth after intratumoral (Fig. 5A) or intravenous (Fig. 5B) injection.A stronger inhibitory effect was obtained with the intratumoral mode of administration.The therapeutic effect, at the dose of 0.25 mg/kg of body mass/ injection, was visible only in the case of intratumoral ABRaA-VEGF 121 injection.Treated mice showed no noticeable weight loss (not shown).These data suggest that the inhibitory effect of ABRaA-VEGF 121 on tumor growth depends on both the dose and mode of administration.

DISCUSSION
This report concern design, purification and properties of ABRaA-VEGF 121 , a novel chimeric protein.We constructed a cDNA encoding a fusion protein containing the VEGF 121 domain linked to the Achain of abrin-a (ABRaA).The latter is a plant toxin which irreversibly inactivates eukaryotic ribosomes (Endo et al., 1987;Wang et al., 2004).To ensure efficient expression of chimeric ABRaA-VEGF 121 protein in a bacterial host, codons rarely used in E. coli and naturally occurring in the ABRaA and human VEGF 121 sequences were replaced (codon optimization) with the frequently used ones (Wang et al., 2004;Gustafsson et al., 2004).
When the VEGF biological activity was blocked with a specific monoclonal antibody, it strongly abrogated the cytotoxicity of ABRaA-VEGF 121 towards PAE/KDR cells (Fig. 2B).Such result indicates that the VEGF 121 domain of ABRaA-VEGF 121 is responsible for specific interactions with cells presenting VEGFR-2 and thus mediates toxicity of the chimeric protein.
Since the mode of action of ABRaA is based on irreversible inactivation of eukaryotic ribosomes (Endo et al., 1987;Wang et al., 2004), we investigated whether such activity would also be displayed by ABRaA-VEGF 121 chimeric protein.Using a cellfree system we found a concentration-dependent inhibition of in vitro protein translation by ABRaA- Female C57BL/6 mice were inoculated intradermally with B16-F10 cells as described in Materials and Methods.Six days after inoculation, when tumors reached about 50 mm 3 , ABRaA-VEGF 121 was administered intratumorally (A) or intravenously (B).A total of four injections was given (every other day, see arrows) using either 0.25 mg/ kg or 1 mg/kg per injection.Control mice received PBS only.The higher dose of ABRaA-VEGF 121 inhibited tumor growth irrespective of administration route.For mice receiving 1 mg/kg per injection statistically significant differences in tumour volumes (vs.control) were seen from day 8 of the experiment (for intratumoral administration) or from day 13 (for intravenous administration) (P < 0.05; Mann-Whitney U-test).One of two independent experiments is shown.Data points are mean ± S.D. (n = 5).
VEGF 121 (Fig. 3A).We also found that ABRaA-VEGF 121 induces death of PAE/KDR cells expressing VEGFR-2 receptors (TUNEL test, Fig. 3B).Treatment of PAE/KDR cells with increasing concentrations of ABRaA-VEGF 121 resulted in a dose-dependent distribution shift from a population of live cells to that of early apoptotic cells (Fig. 3C).
Histological staining of sections from B16-F10 tumors (Fig. 4A, C) excised 48 h after a single intravenous administration of ABRaA-VEGF 121 (1 mg/kg body mass) documented necrotic tumor areas with extravasated red blood cells.Treatment of animals with ABRaA-VEGF 121 caused neither a weight loss nor destruction of normal tissues at least as seen for liver, spleen, kidneys and lungs, even after four repetitive administrations (total of 4 mg/kg body mass, not shown).
We also show that ABRaA-VEGF 121 inhibits growth of B16-F10 murine melanoma tumors, following intratumoral or intravenous administration (Fig. 5A and B).The therapeutic effect is dose-and mode of injection-dependent.The best results were obtained following intratumoral administration of the fusion protein, presumably because of its accumulation within the tumor.Because our in vitro experiments demonstrate that B16-F10 cells are less sensitive to ABRaA-VEGF 121 (IC 50 ≈ 84 μg/ml) than other cell lines (HUVEC (16.8 μg/ml), PAE (27.3 μg/ml), PAE/KDR (0.067 μg/ml), BAEC (29 μg/ ml), HECa10 (29.5 μg/ml)), it is quite possible that the antitumor effect of the fusion protein results mainly from the disruption of an established tumor vascular network.However, we cannot exclude the possibility of ABRaA-VEGF 121 directly eliminating B16-F10 cells, especially after intratumoral administration.
The best therapeutic effects were observed a few days after administration of the chimeric protein (between day 15th and 21st).At that time, over 90% of tumor mass was necrotized (not shown).Despite that, cancer cells surviving the ABRaA-VEGF 121 therapy and still present at the periphery of destroyed tumors caused of their subsequent regrowth.The appearance of necrosis in tumors has been well described for numerous drugs known as Vascular Disrupting Agents (Thorpe, 2004;Szala, 2004).

Figure 1 .
Figure 1.Chimeric ABRaA-VEGF 121 protein.Recombinant ABRaA-VEGF 121 was expressed in E. coli BL21(DE3) strain and isolated from inclusion bodies.After refolding, purification on Q-Sepharose and endotoxin removal using EndoTrap red columns, the recombinant protein was analyzed by electrophoresis on SDS/PAGE and either stained with Coomassie blue (A) or identified by Western blotting (B) using mouse anti-human VEGF 121 monoclonal antibodies and diaminobenzidine as color developer.Purified ABRaA-VEGF 121 was electrophoresed in 10% SDS/polyacrylamide gel under nonreducing (lane 1) or reducing (lane 2) conditions, M = molecular mass standard.

Figure
Figure 4. ABRaA-VEGF 121 induces death of tumor cells adjacent to destroyed blood vessels.Female C57BL/6 mice (n = 3) were inoculated intradermally with B16-F10 cells as described in Materials and Methods.Six days after inoculation, when tumors reached approx.50 mm 3 , ABRaA-VEGF 121 (1 mg/kg) was injected into the tail vein.Control mice obtained PBS only.Animals were sacrificed 48 h later, tumor material was excised, fixed and paraffin-embedded.Sections were H&E-stained.Tumor sections from mice that received ABRaA-VEGF 121 reveal extensive areas of eliminated neoplastic cells and extravasated red blood cells (A and B).No such damage to the vasculature is seen in sections from untreated control tumors (C and D).

Figure 5 .
Figure 5. Inhibition of B16-F10 primary tumor growth by ABRaA-VEGF 121 .Female C57BL/6 mice were inoculated intradermally with B16-F10 cells as described in Materials and Methods.Six days after inoculation, when tumors reached about 50 mm 3 , ABRaA-VEGF 121 was administered intratumorally (A) or intravenously (B).A total of four injections was given (every other day, see arrows) using either 0.25 mg/ kg or 1 mg/kg per injection.Control mice received PBS only.The higher dose of ABRaA-VEGF 121 inhibited tumor growth irrespective of administration route.For mice receiving 1 mg/kg per injection statistically significant differences in tumour volumes (vs.control) were seen from day 8 of the experiment (for intratumoral administration) or from day 13 (for intravenous administration) (P < 0.05; Mann-Whitney U-test).One of two independent experiments is shown.Data points are mean ± S.D. (n = 5).