ARTICLE

Specific Occlusion of Murine and Human Tumor Vasculature by VCAM-1–Targeted Recombinant Fusion Proteins

Ariane Dienst, Andrea Grunow, Maike Unruh, Berit Rabausch, Jacques E. Nör, Jochen W. U. Fries, Claudia Gottstein

Affiliations of authors: Department of Internal Medicine I, University Hospital Cologne, Cologne, Germany (AD, AG, MU, BR, CG); Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan, Ann Arbor (JEN); Department of Pathology, University Hospital Cologne, Cologne, Germany (JWUF)

Correspondence to: Claudia Gottstein, MD, University Hospital Cologne, Department of Internal Medicine I, Experimental Oncology and Vascular Biology, LFI E4 R707, Joseph-Stelzmann-Str. 9, 50924 Cologne, Germany (e-mail: claudia.gottstein{at}uni-koeln.de).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background: The tumor vasculature is increasingly recognized as a target for cancer therapy. We developed and evaluated recombinant fusion proteins targeting the coagulation-inducing protein soluble tissue factor (sTF) to the luminal tumor endothelial antigen vascular cell adhesion molecule 1 (VCAM-1, CD106). Methods: We generated fusion proteins consisting of sTF fused to antibody fragments directed against mouse or human VCAM-1 and characterized them in vitro by flow cytometry, surface plasmon resonance, and two-stage coagulation assays. Their therapeutic effects were tested in three human xenograft tumor models: L540rec Hodgkin lymphoma, Colo677 small-cell lung carcinoma, and Colo677/HDMEC small-cell lung carcinoma with human vasculature. Toxicity was analyzed by histologic examination of organs and determination of laboratory blood parameters. Results: The fusion proteins bound VCAM-1 with nanomolar affinities and had the same coagulation activity as an sTF standard. Xenograft tumor–bearing mice treated with fusion protein (FP) alone or in combination with lipopolysaccharide (FP/L) or doxorubicin (FP/D) exhibited tumor-selective necrosis (L540rec tumors: 74% tumor necrosis [95% confidence interval {CI} = 55% to 93%] with FP/L versus 13% tumor necrosi1s [95% CI = 4% to 22%] with vehicle; Colo677 tumors: 26% [95% CI = 16% to 36%] with FP versus 8% [95% CI = 2% to 14%] with vehicle); tumor growth delay (Colo677/HDMEC: mean tumor weights after 3 days = 42 mg in FP-treated mice versus 71 mg in vehicle-treated mice, difference = 29 mg, 95% CI = 8 to 100, Mann–Whitney P = .008); and some tumor regressions (one of seven FP-treated Colo677 tumor–bearing mice and two of seven FP/D-treated mice). The fusion protein was well tolerated. Conclusions: Recombinant tissue factor–based fusion proteins directed against an intraluminal tumor endothelial cell marker induce tumor-selective intravascular coagulation, tumor tissue necrosis, and tumor growth delay.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The tumor vasculature is increasingly recognized as a target for cancer therapy (14). Targeting the tumor vasculature provides an approach to circumvent the mechanical barriers that impede homogeneous distribution of drugs within the tumor tissue (5,6). Other advantages of targeting the tumor vasculature rather than the tumor cells themselves include a potentiation effect, because one blood vessel nourishes hundreds of tumor cells, and a reduced probability that drug resistance will develop, because endothelial cells have a lower mutation rate than cancer cells (7). Therapeutic vascular-specific approaches that either inhibit neoangiogenesis in tumors or damage the blood vessels supplying the tumor tissue have been tested previously in clinical trials(810). Damage to endothelial cells results in the induction of the coagulation cascade, causing intratumoral vessel occlusion and subsequent tumor necrosis (11). In clinical studies, vascular occluding agents have antitumor effects in locoregional settings. For example, tumor necrosis factor {alpha} (TNF-{alpha})–based isolated limb perfusions of localized soft tissue sarcomas or melanomas resulted in remission rates of 90%–100% in some studies [reviewed in Eggermont et al. (12)]. The antitumor effect was caused by vascular occlusion and subsequent necrosis (13,14).

Although this particular treatment was too toxic for systemic application, it raised the possibility that molecules could be created that would specifically and effectively induce coagulation in the tumor vasculature when applied systemically (i.e., intravenously). Such a strategy takes advantage of the fact that coagulation can be induced directly, rather than subsequent to damaging endothelial cells, for example by treatment with conjugates containing soluble tissue factor (sTF) (15). Tissue factor, a member of the cytokine receptor family group 2, is the key initiator of the extrinsic coagulation cascade. It is a transmembrane glycoprotein that contains 263 residues; the extracellular domain (sTF) comprises the first 219 amino acids. Although untargeted sTF can induce selective tumor vasculature occlusion under certain circumstances (16), the coagulation-inducing activity of sTF is 10 000-fold less than that of full-length TF; therefore, the replacement of the transmembrane portion with a ligand that binds specifically to endothelial cells should result in specific coagulation induction and ideally increase sTF's ability to induce coagulation. Initiation of coagulation in the tumor vasculature with soluble tissue factor targeted via a bispecific antibody to an artificially induced endothelial cell antigen, the major histocompatibility complex (MHC) class II antigen I-Ad, had dramatic antitumor effects (15).

Ran et al. (17) used a different targeted form of sTF as an antitumor agent. They biochemically conjugated sTF to an antibody directed against vascular cell adhesion molecule 1 (VCAM-1), a luminal tumor-associated endothelial cell antigen that is also known as CD106. VCAM-1 is a type I transmembrane glycoprotein that belongs to the immunoglobulin G (IgG) superfamily. Normal blood vessels generally do not express VCAM-1, although expression or shedding of VCAM-1 has been described for several pathologic conditions (18). In malignant tumors, VCAM-1 is also expressed by the tumor cells and/or on the tumor vasculature (1933). The biochemical conjugate of sTF and anti-VCAM-1 antibody created by Ran et al. (17) selectively occluded tumor vessels and induced necrosis within the tumor tissue. Although the conjugate also bound to endothelial cells in lung and heart, no necrosis was observed in these organs. The finding that tumor vasculature was more sensitive to the conjugates than normal vasculature suggests that the tumor vasculature has an increased procoagulant status—i.e., it supports coagulation better than normal vasculature. Another biochemical conjugate has been developed (34) that targets sTF to prostate-specific membrane antigen, a marker of tumor endothelial cells in humans (35). However, the construct had to be precomplexed with factor VIIa (i.e., activated coagulation factor VII) to be effective in vivo.

Recombinant single-chain fusion proteins are preferable to biochemical conjugates for clinical use because they are exactly defined substances, they can be produced in automated fermenter systems, and they can be engineered precisely. We have developed recombinant fusion proteins that target luminal tumor endothelial cell markers and deliver sTF to these sites (36). Similar sTF-based fusion proteins, but directed against extravascular markers, such as epithelial glycoprotein 2, fibroblast activating protein, the fibronectin ED-B domain, and DNA have been developed by several groups (3740); fusion proteins that target sTF to fibronectin-ED-B domain or to DNA were reported to have antitumor activity in vivo (39,40). By contrast, fusion proteins containing sTF coupled to Arg-Gly-Asp (RGD)-containing peptides or proteins did not cause tumor necrosis or tumor growth delay (40,41).

In this study we developed and evaluated recombinant fusion proteins that use monoclonal antibodies to target sTF to mouse or human VCAM-1. We examined the binding and coagulation activities of these proteins in vitro by flow cytometry, by surface plasmon resonance, and in two-stage coagulation assays. We also tested their antitumor effects when administered systemically to xenograft mouse models for human Hodgkin lymphoma and small-cell lung carcinoma and to a mouse model for small-cell lung carcinoma that contained human tumor vasculature.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell Lines, Antibodies, and Reagents

Hybridoma cell lines used for cloning of single-chain variable fragment (scFv) antibodies were MK271 (anti–murine CD106 [VCAM-1]; American Type Culture Collection [ATCC], Manassas, VA) and 1G11B1 (anti–human CD106 [VCAM1], supplied within a limited material transfer agreement from Chemicon, Temecula, CA). Endothelial cell lines were human umbilical vein endothelial cells (HUVECs; Promocell, Heidelberg, Germany), human dermal microvascular endothelial cells (HDMECs; Clonetics, San Diego, CA), murine endothelial cells 2F2B (ATCC), and murine endothelial cells bEnd3 (a kind gift of Dr. B. Engelhardt, Max-Planck-Institute, Bad Nauheim, Germany; present address: University of Bern, britta.engelhardt{at}tki.unibe.ch). Tumor cell lines were L540rec, a subline of the L540 cell line (42) with increased metastatic potential, Colo677 small-cell lung carcinoma (DSMZ, Braunschweig, Germany), H358 human non–small-cell lung carcinoma (ATCC), and F9 murine teratocarcinoma (ECACC, Salisbury, U.K.). All cell lines were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum, except for HDMEC (EGM-MV medium from Collaborative Biomedical Products, Cambridge, MA) and HUVEC (EGM2 medium from Cambrex BioScience, Walkersville, MD). HUVEC cells were used for experiments at passages 2–15.

Antibodies used in this study were as follows: MK271 (rat anti–murine VCAM-1, purified from hybridoma supernatant by affinity chromatography), 1G11B1 (mouse anti–human VCAM-1, Chemicon), rabbit anti–human Fc (Dianova, Hamburg, Germany), anti-FLAG antibody M2 (Sigma, St. Louis, MO), polyclonal sheep anti–human tissue factor (Haemochrom, Essen Germany), 124 (anti–human bcl-2, Dako Cytomation, Hamburg, Germany), V9 (anti–human vimentin, Dako Cytomation), CI:A3-1 (anti–mouse macrophage marker F4/80, Serotec, Oxford, U.K.), KG7/30 (anti–human-von Willebrand Factor [vWF], BMA Biomedicals, Augst, Switzerland), polyclonal rabbit anti–human fibrin(ogen) antibody (No. 0080, Dako Cytomation). Isotype-matched control antibodies were designated isotype controls and were obtained from Becton Dickinson Pharmingen (San Diego, CA).

Unless otherwise specified, tissue culture reagents came from Invitrogen, molecular biology reagents from New England Biolabs (Beverly, MA), and chemical reagents from Merck/VWR (Darmstadt, Germany) or Sigma.

Expression of VCAM-1 on Tumor Vasculature

Expression of VCAM-1 on human or murine tumor endothelial cells was determined by immunohistochemistry. Immunostaining was performed on cryostatic sections of primary human tumors or human xenograft tumors derived from mice. Primary human Hodgkin lymphoma sections were obtained from the tumor bank of the Department of Oncology and Hematology at the University Hospital Cologne, which contains patient samples that are obtained within clinical studies upon written informed consent. The murine xenograft tumors were from groups of three to five mice that had been injected subcutaneously with 107 tumor cells (L540 rec Hodgkin lymphoma, Colo677 small-cell lung carcinoma, H358 non–small-cell lung carcinoma, or F9 teratocarcinoma). Tumors were measured in three perpendicular directions, a, b, and c, where a, b, and c represent the three perpendicular diameters, and volumes were calculated according to the formula V = {pi}/6 x a x b x c. When tumors reached a size of 150–400 µL, mice were killed by ether overdose and tumors were snap-frozen in liquid nitrogen and stored at –80 °C until further use. Two mice with L540rec Hodgkin lymphoma were injected with 500 ng of lipopolysaccharide (LPS; from Escherichia coli, serotype O55:B5; Sigma) 72 hours before being killed to investigate VCAM-1 induction by LPS.

Cryostatic sections were thawed, fixed in acetone, and incubated with th e primary antibodies MK271 or 1G11B1 (directed against mouse and human VCAM-1, respectively) at 10 µg/mL in phosphate-buffered saline (pH 7.4), containing 0.1% Tween 20 (PBS-T). Next, they were incubated with rabbit anti–rat IgG-peroxidase or rat anti–mouse IgG-peroxidase secondary antibodies (Dianova) at 4 µg/mL in PBS-T containing 10% fetal calf serum, and developed with AEC in carbazole buffer (3-amino-9-ethylcarbazole at 2.4 mg/mL in dimethylformamide diluted 1 : 10 in 25 mM sodium acetate [pH 5.2] plus 0.15% H2O2).

Generation of Anti-CD106-scFv-soluble Tissue Factor ({alpha}-CD106-sTF) Fusion Proteins and Control Proteins

The DNA coding for the variable regions of the anti-CD106 (VCAM-1) monoclonal antibodies MK271 (directed against murine CD106) and 1G11B1 (directed against human CD106) were cloned from the corresponding hybridoma cell lines, and DNA coding for the extracellular domain of human tissue factor was cloned from J82 cells (ATCC) as described (36). Variable antibody regions were cloned into the cloning site between HindIII and XbaI of the expression plasmid pswc4 (36), which contains the sTF coding sequence downstream of the cloning site. The resulting fusion proteins, {alpha}-mCD106-sTF and {alpha}-hCD106-sTF, were assembled in the order 5'-FLAG–6xHis–anti-CD106(scFv)–sTF-3', (where FLAG = epitope for detection and purification, 6 x His = six histidines for affinity purification, scFv = single-chain variable fragment, and sTF = soluble tissue factor). The DNA segments encoding the scFvs alone were subcloned into the vector pswc5 (36), and the DNA encoding sTF had been previously subcloned into pswc7 (36). The control proteins, i.e., scFv and sTF, also contained a FLAG tag and a 6xHis tag at the 5' end. All proteins were expressed in E. coli and purified by affinity chromatography and gel filtration as described (36). Proteins were stored at 0 °C for short-term storage (several weeks) or at –20 °C for long-term storage.

Biochemical Characterization of Fusion Proteins

Purified fusion proteins and control proteins were subjected to gel electrophoresis on 4%–15% gradient SDS–polyacrylamide gels. Proteins were visualized by Coomassie Blue staining. Total protein concentrations were determined by scanning UV spectrophotometry of the protein solution (absorption at 278 nm), and specific protein concentrations were calculated as 1/100 x (% purity) x total protein concentration. The identity of the proteins was confirmed by western blot analysis. For western blotting, proteins were separated by gel electrophoresis (as described above) and then transferred via semidry capillary heat transfer to nitrocellulose membranes. Nonspecific binding was blocked by incubating the membranes for 1 hour at room temperature with PBS-T containing 1% membrane-blocking reagent (Amersham Biosciences, Piscataway, NJ), and the membranes were then incubated with sheep anti–human tissue factor antibody or murine anti-FLAG antibody M2 at a concentration of 1 µg/mL in PBS-T. Secondary antibodies were rabbit anti–sheep IgG-peroxidase (Pierce, Rockford, IL) and rat anti–mouse IgG-peroxidase (Dianova) at 0.2 µg/mL. Signals were detected by chemoluminescence using the SuperSignal West Pico kit (Pierce).

Concentrations of endotoxin LPS, a bacterial contaminant, in purified fusion proteins and controls were measured by astandard limulus amebocyte lysate (LAL) assay (Biowhittaker, Walkersville, MD) according to the manufacturer's instructions.

Flow Cytometry Analysis of Fusion Proteins

Murine endothelial cells 2F2B and bEnd3 and HUVECs were stimulated with TNF-{alpha} at 500 U/mL for 4 hours at 37 °C. Cells were detached with 0.25% trypsin, and 2 x 105 cells were incubated with the {alpha}-mCD106-sTF or {alpha}-hCD106-sTF fusion proteins at concentrations ranging from 1 nM to 1 µM in 100 µL of PBS containing 0.2% bovine serum albumin (BSA) and 0.2% NaN3 for 30 minutes on ice. Positive controls were anti-mouse VCAM-1 or anti–human VCAM-1 IgG antibodies, and negative controls were isotype-matched control antibodies or buffer. Cells were then washed and incubated with a sheep anti–human tissue factor antibody at 10 µg/mL for 30 minutes on ice, washed, and incubated with donkey anti–sheep IgG coupled to fluorescein isothiocyanate (Serotec) for 30 minutes on ice. Binding of fusion proteins or control proteins to the endothelial cells was detected with a flow cytometer (Becton Dickinson, San Jose, CA), and MFI50 values (i.e., the concentration at which 50% of the maximum mean fluorescence intensity is reached) were determined.

Enzyme-linked Immunosorbent Assay of{alpha}-hCD106-sTF

Each well of a 96-well microtiter plate was coated with human VCAM-1 (R & D Systems, Minneapolis, MN) at 1 µg/mL in coating buffer (35 mM Na2CO3, 15 mM NaHCO3) at 4 °C overnight, blocked with PBS-T containing 1% BSA for 1 hour at 37 °C, and incubated with {alpha}-hCD106-sTF that had been diluted in PBS-T at concentrations ranging from 10 nM to 60 nM for 1 hour at room temperature on a shaker. Negative controls were sTF or PBS-T. Wells were washed with PBS-T, and binding of fusion protein to VCAM-1 was detected by adding sheep anti–human tissue factor antibody at 1 µg/mL to each well. After 1 hour at room temperature, the wells were again washed, and rabbit anti–sheep-peroxidase (Pierce) at 1 µg/mL was added. After 1 hour at room temperature, the plates were washed and developed in 0.4 mg/mL O-phenylenediamine in 55 mM sodium citrate, 100 mM Na2HPO4, and 0.01% H2O2, and absorption at 405 nm (absorption maximum for substrate) minus absorption at 650 nm (background absorption) was determined on an enzyme-linked immunosorbent assay (ELISA) reader.

Real-time Binding Analysis of {alpha}-mCD106-sTF to Murine VCAM-1

A real-time binding analysis of {alpha}-mCD106-sTF was carried out using surface plasmon resonance on a Biacore 2000 (Biacore, Uppsala, Sweden). To this end, a rabbit anti–human Fc antibody was immobilized on a CM5 sensor chip (Biacore) by amine coupling at 5000 response units (RU) using the amine coupling kit (Biacore) according to the manufacturer's instructions. A chimeric protein of murine VCAM-1 and human Fc fragment (R & D Systems) was captured at 400 RU, and the {alpha}-mCD106-sTF fusion protein was injected at concentrations ranging from 0.5 µg/mL to 20 µg/mL (8 nM to 330 nM) at a flow speed of 30 µL/minute. The flow cells were regenerated with glycine buffer (10 mM glycine, pH 1.5). A blank flow cell (without immobilized ligand) served as a negative control. Binding curves were analyzed with BiaEvaluation software 3.1 (Biacore). Curves were fitted by use of the Langmuir 1 : 1 binding model. The association rate, dissociation rate, and the dissociation constant were determined by global fitting of binding curves from five different concentrations. Some samples were also analyzed at higher flow rates, yielding the same results; thus, there was no indication for mass transport limitation of the binding.

Cell-free Coagulation Assay of {alpha}-CD106-sTF Fusion Proteins

In vitro coagulation activity of the fusion proteins was tested in a cell-free two-stage coagulation assay that measured factor X activation. For this assay, 5 µM phosphatidylserine and 15 µM phosphatidylcholine (Sigma) in calcium buffer (50 mM Tris–HCl [pH 8.1], 150 mM NaCl, BSA at 2 mg/mL, 5 mM CaCl2) were mixed with Factor VIIa (Haemochrom) at 10 nM in a total volume of 100 µL, and 10 µL of {alpha}-CD106-sTF fusion proteins were added at final concentrations ranging from 1 nM to 50 nM. sTF was used as standard and buffer as a negative control. Samples were incubated for 5 minutes at 37 °C, and 50 µL of factor X (Sigma) was added to a final concentration of 30 nM. After 5 minutes at room temperature, 100 µL of the chromogenic substrate S2765 (Haemochrom) was added at pH 8.0 to a final concentration of 0.3 mM, and absorption at 405 nm (absorption maximum for substrate) minus adsorption at 650 nm (background) was measured after 3 minutes.

Coagulation Induction by Fusion Proteins on the Surface of Endothelial Cells

Murine endothelial (2F2B) cells or HUVECs were added to 48-well tissue culture plates at densities of 5 x 104 cells per well and 1 x 104 cells per well, respectively, and allowed to adhere overnight. Cells were then incubated with {alpha}-mCD106-sTF (2F2B) or {alpha}-hCD106-sTF (HUVEC) at final concentrations ranging from 1 nM to 100 nM diluted in tissue culture medium or with negative control reagents (sTF or tissue culture medium) for 1 hour at room temperature, washed with calcium buffer, and incubated with 100 µL of a coagulation factor mixture containing 10 nM factor VIIa, 50 nM factor IX, 200 nM factor X, and 20 µM phospholipids in calcium buffer. Ninety microliters of the supernatant of each well was transferred to the wells of a new 96-well plate. One hundred microliters of the substrate S2765 (pH 8.0) was added to a final concentration of 0.3 mM, and factor Xa generation was measured as described above. For competition assays, different amounts of anti–mouse VCAM-1 antibody or an IgG of irrelevant specificity (Intraglobin; Biotest Pharma, Dreieich, Germany) were added during the incubation along with the {alpha}-mCD106-sTF fusion protein.

Cell Viability Assay

Potential direct toxic effects of the {alpha}-mCD106-sTF fusion protein on either tumor or endothelial cells were examined by carrying out viability assays with two human tumor cell lines, Colo677 and L540rec, and two murine endothelial cell lines, bEnd3 and 2F2B. For each cell line, optimal culture density was determined in a pilot experiment. Cells—2.5 x 104 Colo677 cells per well, 2 x 104 L540rec cells per well, 3 x 103 2F2B per well, and 3 x 104 bEnd3 cells per well—were added to wells of96-well microtiter plates and incubated with {alpha}–mCD106-sTF fusion protein at concentrations ranging from 10–12 M to10–7 M (in triplicate) at 37 °C in a 5% CO2 atmosphere. After 48 hours, 50 µL of 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (Roche, Mannheim, Germany) at 1 mg/mL in tissue culture medium containing phenazine methosulfate at 1.2 µg/mL (Sigma, St. Louis, MO) was added. Absorption at 405 nm minus absorption at 650 nm (background) was measured after 48 hours. The difference reflects the metabolic activity of the cells, because viable cells reduce the tetrazolium salt to a formazan salt, resulting in a color change that can be measured at the wavelength of 405 nm. Average values for each concentration of fusion protein were calculated as a percentage of those in untreated control cells.

Treatment Studies in Xenograft Mice

All animal experiments were performed in accordance with institutional guidelines and followed the recommendations of the Society of Laboratory Animal Science. Mice were kept in a barrier facility with a hygienic status of specific-pathogen-free (for the human angiogenesis model) or specific- and opportunistic-pathogen-free (SOPF, for all other models). In the SOPF facility, the air is filtered and overpressured, and the mice are housed in a separately air-filtered shelf within microisolator cages. The hygienic status of the animal facility was routinely tested by an external laboratory. Access was restricted, and all incoming mice were obtained from certified suppliers accompanied by appropriate health certificates or from our own breeding colony, which is housed within the same room in isolators and microfilter cages. Mice of different treatment groups were housed in the same room and were handled in the same way by the same people. We used female mice at the age of 4–5 weeks, weighing 20–25g. In one experiment, half of the mice were male, and female and male mice were equally distributed among treatment groups.

Short-term experiments. L540rec Hodgkin lymphoma cells or Colo677 SCLC cells (107) were injected subcutaneously into 30 SCID (severe combined immunodeficiency) mice (from our breeding colony or from Charles River WIGA, Sulzfeld, Germany) or 72 CD1 nude mice (Charles River WIGA), respectively. Tumors developed in 90% of SCID mice injected with L540rec cells and 73% of nude mice injected with Colo677 cells. When subcutaneous tumors became measurable (i.e., after 1–2 weeks), they were measured daily with a caliper in three perpendicular directions. The person measuring the tumor volumes was blinded to the treatment groups, and for each experiment one individual carried out all measurements for the duration of the entire experiment. When tumors reached 150–300 µL in volume, groups of six to 13 mice were treated once intravenously with 20 µg of {alpha}-murine-CD106-sTF or with equimolar amounts of control reagent (10 µg of sTF, 10 µg of scFv, or vehicle, i.e., 0.9% NaCl solution, clinical grade) in 0.4 mL. For SCLC-bearing mice, we added LPS from E. coli (serotype O55:B5; Sigma) to all control reagents to a final concentration of 6 ng (the highest total dose provided to mice with the fusion protein; see "Results"). For lymphoma-bearing mice, we added 500 ng of LPS to the treatment reagent prior to injection (treatment and controls) to increase VCAM-1-expression on tumor vessels, because constitutive VCAM-1 expression was low. Because LPS by itself can influence the coagulation system, the same amount was used in treatment groups and control groups. Mice were observed for clinical signs of toxicity at prespecified times after treatment (5, 10, 15, 30, 60, and 120 minutes and 1, 2, and 3 days). Three days after treatment, mice were killed by ether overdose, and an autopsy was performed. For evaluation of early coagulation changes in fusion protein–treated mice, we also killed fusion protein–treated Colo677 tumor–bearing mice at earlier time points. Citrated blood was obtained from the vena cava, and tumors and organs were removed for histologic evaluation.

Histologic Analysis and Quantitation of Tumor Cell Necrosis and Analysis of Organs

Histologic analysis and quantitation of tumor cell necrosis were performed as described (16). In brief, several hematoxylin and eosin–stained paraffin sections from each tumor were analyzed by light microscopy. The percentage of necrotic tissue per tissue section was calculated with the aid of densitometry scanning (16).

All major organs (i.e., heart, lung, brain, liver, kidney, spleen, intestine, and pancreas) of every mouse from the short-term treatment experiments were analyzed by light microscopy for thrombosis or necrosis. The number of thrombotic events per tissue section were documented. No tissue necrosis was found in these organs. All evaluations were performed by two independent investigators, one of them being a pathologist and blinded to the treatment groups.

Laboratory parameters of treated mice. General laboratory values and several coagulation parameters were determined. Blood counts included the numbers of leukocytes and platelets and hemoglobin content. Coagulation parameters included soluble fibrin monomers, thrombin–antithrombin complexes (TAT), and antithrombin III (AT III).

Leukocytes and platelets were counted in a Neubauer chamber after diluting the blood 1 : 20 in 3% acetic acid for leukocyte counts or 1% ammonium oxalate for platelet counts and after incubating them for 10 minutes at room temperature on a shaker. Hemoglobin was measured in a spectrophotometer (absorption at 546 nm) after diluting blood 1 : 250 in transformation solution (potassium ferricyanide at 0.2 g/L, sodium cyanide at 0.05 g/L, and sodium bicarbonate at 1 g/L in water) and incubating for 10 minutes at room temperature.

For coagulation parameters, thrombocyte-free plasma was obtained from vena cava blood by sequential centrifugation (10 minutes at 500g followed by 10 minutes at 8000g). Soluble fibrin was analyzed in fresh plasma, and the remainder was stored at –80 °C until further analysis.

Soluble fibrin monomers were detected in plasma by use of the FM test kit (Roche, Mannheim, Germany) according to the manufacturer's instructions, with modifications (plasma was used at 1 : 20 and 1 : 50 dilutions). TAT complexes in 20 µL of plasma were detected with the Enzygnost TAT micro-assay (Dade-Behring, Marburg, Germany) according to the manufacturer's instructions. ATIII levels were determined using the Coamatic antithrombin-assay (Haemochrom) by the manufacturer's instructions. Normal laboratory values for the parameters tested here had been determined previously (data not shown) by analyzing plasma samples from mice matched in mouse strain and tumor type. TNF-{alpha} levels in serum of Colo677 tumor–bearing nude mice were determined with the Quantikine-M kit (R & D Systems) according to the manufacturer's instructions, because we had previously observed measurable TNF-{alpha} levels in this tumor model (data not shown) and wanted to rule out that the presence of TNF-{alpha} would influence the treatment outcome.

Long-term experiments. SCID mice (n = 40) were injected subcutaneously with L540rec cells and nude mice (n = 64) were injected subcutaneously with Colo677 cells as described for the short-term experiments. Tumors developed in 91% of the for L540rec/SCID mice and in 69% of the Colo677/nude mice. Once tumor volumes reached 200–250 µL (as measured by caliper measurement in three dimensions), five to 10 mice per group were treated by intravenous injection with 20 µg of {alpha}-murine-CD106-sTF fusion protein or equimolar amounts of control reagents (10 µg sTF, 10 µg scFv, saline as the vehicle) in a total volume of 0.4 ml on days 0, 4, 8, and 12 (for lymphoma-bearing mice) or on days 0, 4, and 8 (for SCLC-bearing mice). LPS (500 ng) was added to the treatment reagents given to the lymphoma-bearing mice but not the SCLC-bearing mice. In the Colo677 SCLC model, two additional treatment groups were given either combination treatment with doxorubicin, which had good efficacy against Colo677 cells in vitro (data not shown), or doxorubicin alone. These mice were treated by intravenous injection with 8 mg/kg doxorubicin on days 0, 7, and 11, in addition to fusion protein or vehicle treatment.

All mice were observed daily for clinical signs of toxicity, and body weights were measured regularly. Mice were followed up for at least 2 weeks, mice with regressed tumors for 3 weeks. Tumors were measured daily by caliper measurement in three dimensions, and volumes were calculated as described for short-term experiments. At the end of the experiment mice were killed by ether overdose, an autopsy was performed, and tumors were removed and snap-frozen in liquid nitrogen.

SCID mouse model of human tumor angiogenesis. The human tumor angiogenesis model was created by seeding 9 x 105 human dermal microvascular endothelial cells (HDMECs) and 1 x 105 Colo677 cells in a biodegradable poly-L-lactic acid sponge as described (43). Two sponges were implanted subcutaneously in each of 16 SCID mice. After 3 weeks, the mice were subjected to a single intravenous injection of {alpha}-human-CD106-sTF (20 µg) or control reagents (10 µg sTF or vehicle) in 0.2 mL. Mice were killed by CO2 inhalation 3 days after treatment, and weights and volumes of the excised tumors were measured. Tumor measurements had to be performed on excised tumors, because the tumors were not big enough for reliable caliper measurements in living animals.

Immunohistochemical Analysis of Treated Tumors

Staining for Bcl-2 and vimentin, both of which are expressed by Colo677 small-cell lung cancer cells, was performed on deparaffinized sections of regressed tumors in the Colo677 SCLC model. After antigen retrieval by autoclaving at 121 °C for 10 minutes or by microwave treatment at 600 W for 7 minutes in citrate buffer (10 mM citrate, pH 6.0), sections were incubated with monoclonal antibodies against bcl-2 (Dako Cytomation) diluted 1 : 20 in antibody diluent (Zymed, South San Francisco, CA) or monoclonal antibodies against vimentin (Dako Cytomation) diluted 1 : 200 in antibody diluent. Sections were then incubated with secondary antibody from the ChemMate Detection kit (Dako Cytomation), i.e., a biotinylated goat anti–mouse IgG antibody, followed by streptavidin–peroxidase, and subsequently developed with AEC substrate supplied in the kit according to the manufacturer's instructions. Tissue sections were also stained for macrophages, as follows. After antigen retrieval with proteinase K for 15 minutes at room temperature, the anti-F4/80 antibody was applied at a 1 : 500 dilution as described above. The sections were then incubated with biotinylated rabbit anti–rat IgG antibody at a 1 : 350 dilution (Dako Cytomation) and then with avidin–biotin complex (Vectastain ABC-kit, Vector Laboratories, Burlingame, CA). Sections were developed in diaminobenzidine at 0.7 mg/mL (DAB, Sigma) in PBS (pH 7.4) containing 0.02% H2O2.

Deparaffinized sections of negative control tumors from the human angiogenesis model were stained with an antibody against human von Willebrand factor after antigen demasking in 10 mM citrate (pH 6.0) for 4 minutes in a microwave at 650 W. Sections were blocked for 30 minutes with H2O2 (0.3% in methanol), incubated with a mouse anti-human monoclonal antibody against von Willebrand factor (diluted 1 : 50 in PBS containing 1% BSA), and incubated with rat anti–mouse IgG-peroxidase (Dianova) 4 µg/mL. The color reaction product was developed with AEC in carbazole buffer (3-amino-9-ethylcarbazole at 2.4 mg/mL in dimethylformamide diluted 1 : 10 in 25mM sodium acetate [pH 5.2] containing 0.15% H2O2).

Tumors from the human angiogenesis model treated with{alpha}-hCD106-sTF or control reagents were stained for fibrin with a polyclonal rabbit anti–human fibrin(ogen) antibody. Antigen retrieval was achieved by treating sections in 0.1% trypsin at 37 °C for 15 min. After incubation with the primary antibody at a 1 : 20 000 dilution in antibody dilution buffer, sections were incubated with secondary antibody and subsequently with AEC substrate, both from the ChemMate Detection kit (Dako Cytomation), according to the manufacturer's instructions.

Statistical Analysis

Statistical analysis was performed for the in vivo experiments to test the null hypothesis of no differences in tumor necrosis, tumor volumes, or tumor weights between treatment groups. For the short-term experiments, the percentage of necrosis in tumors was analyzed in one section from each mouse and means of percentage necrosis in six to 13 mice per treatment group were compared with the Mann–Whitney U test for unpaired groups. A possible correlation between TNF-{alpha} levels in the Colo677 tumor model and treatment outcome was investigated by calculating the Spearman–Rho correlation coefficient between TNF-{alpha} levels and the corresponding tumor necrosis values. For the long-term experiments, tumor volumes were measured, and mean volumes of tumors from all mice in each treatment group (i.e., five to 10 mice) were also compared with the Mann–Whitney U test for unpaired groups. The 95% confidence intervals for differences in mean tumor volume between groups were calculated using the normal approximation method without assuming equality of variances among groups (44). Thus, small discrepancies between (Mann–Whitney) P values and (normal) confidence intervals may arise. To test the effect of treatment with fusion protein or doxorubicin on the tumor volumes of mice treated with the combination treatment (fusion protein plus doxorubicin), logarithms of tumor volumes were calculated to equalize standard deviations, and an analysis of variance was performed.

In the human angiogenesis model, two tumors were implanted per mouse. We calculated the Spearman–Rho correlation coefficient between the two tumors and found no correlation, either for all mice combined (correlation coefficient = –0.06 at a statistical significance level of .8), or for any of the treatment groups when analyzed separately. Therefore, we analyzed the tumors as separate entities. Mean tumor volumes or mean tumor weights of different treatment groups were then compared with the Mann–Whitney U test for unpaired groups. We used SPSS software version 11.0 (SPSS Science Software, Erkrath, Germany) for the statistical calculations. All P values are two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of VCAM-1 in Human and Mouse Tumors

We analyzed tumors from four mouse models and primary tumor tissues from 17 Hodgkin lymphoma patients for VCAM-1 expression. In line with results of other studies (2123), all of 17 primary human Hodgkin tumors expressed VCAM-1 on the vasculature (Fig. 1). In contrast, VCAM-1 was expressed at low levels in tumor xenografts derived from human L540rec Hodgkin lymphoma cells (Fig. 1). To increase the expression of VCAM-1 in studies of the effect of fusion protein in L540rec tumors, we injected mice with LPS. In the Colo677 SCLC model, however, VCAM-1 expression was sufficient to allow for treatment studies without LPS pretreatment (Fig. 1). In the other two mouse models initially tested, F9 murine teratocarcinoma and H358 human non–small-cell lung carcinoma VCAM-1 was not expressed on tumor vessels, although human patients with non–small-cell lung carcinomas have been reported to express VCAM-1 on the tumor vasculature (25,26). Thus, two mouse models, i.e., the L540rec model plus low-dose LPS, and the Colo677 model without LPS, expressed VCAM-1 in sufficient amounts to be used for our experiments with VCAM-1-targeted fusion proteins.



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Fig. 1. Endothelial cell antigen expression in mouse xenograft tumors or human tumors. (A–C) Human Hodgkin lymphoma (L540rec) xenograft in mice. (D–F) Human Hodgkin lymphoma xenograft in mice treated with lipopolysaccharide to increase vascular cell adhesion molecule 1 (VCAM-1, also known as CD106) expression. (G–I) Human small-cell lung carcinoma (Colo677) xenograft in mice. (J–L) Primary tumor from a human patient with Hodgkin lymphoma. Panels A, D, G, and J: CD31 staining (positive control); B, E, H, and K: VCAM-1 staining. C, F, I, and L: staining with isotype-matched antibody (negative control). Arrows in B and E point to VCAM-1–positive vessels. Representative sections are shown at x100 magnification.

 
Generation of {alpha}-CD106-sTF Fusion Proteins and Control Proteins

We expressed and purified fusion proteins containing a scFv directed against either murine or human VCAM-1 as the targeting moiety and sTF as the effector moiety. We also expressed and purified the control proteins sTF and scFv. Analysis of recombinant proteins (data not shown) showed the expected molecular masses of 60 kDa (fusion proteins), 30 kDa (sTF), and 27 kDa (scFv). The identity of the proteins was confirmed by western blotting (data not shown). We determined levels of endotoxin LPS, a bacterial contaminant, in proteins used for the treatment experiments by the LAL assay. Endotoxin levels were 300 pg per µg of specific protein for {alpha}-CD106-sTF, 7 pg per µg for sTF, 90 pg per µg for scFv, and 0 pg/µg for vehicle. Because even low amounts of endotoxin can influence coagulation induction by sTF (16), we added endotoxin to control reagents so that levels would be equivalent to those in the fusion protein. In subsequent experiments (both long-term experiments and model for human angiogenesis), endotoxin levels were never greater than 3 pg per µg of specific protein; therefore, no adjustments were made.

Functional Characterization of {alpha}-CD106-sTF Fusion Proteins

Binding studies. Binding of {alpha}-mCD106-sTF to VCAM-1 was analyzed by flow cytometry of 2F2B and bEnd3 murine endothelial cells and by surface plasmon resonance using a sensor chip with immobilized VCAM-1. Binding of {alpha}-hCD106-sTF to VCAM-1 was analyzed by flow cytometry of HUVECs and by ELISA. Both recombinant fusion proteins bound specifically to VCAM-1–expressing endothelial cells and to purified VCAM-1 with nanomolar affinities. For {alpha}-mCD106-sTF, association and dissociation rates were also determined by surface plasmon resonance. The on rate was 9.2 x 104 · mole–1 · second–1 and the off rate was 1.5 x 103 · second–1, resulting in a dissociation constant of 16 nM (Fig. 2, A–C).



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Fig. 2. Functional analysis of fusion proteins containing soluble tissue factor (sTF) fused to monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) (A–C) Binding analysis. (A) Murine endothelial cells treated with {alpha}-mCD106-sTF (directed against mouse VCAM-1) and (B) human endothelial cells treated with {alpha}-hCD106-sTF (directed against human VCAM-1) were subjected to flow cytometry analysis of VCAM-1–positive endothelial cells. Broken lines = cells without antibody or cells with secondary antibody only. Boldface lines = Samples. (C) Real-time binding of {alpha}-mCD106-sTF to purified VCAM-1 immobilized on a CM5 sensor chip. Binding curves (from top to bottom) show binding at fusion protein concentrations of 20 µg/mL, 10 µg/mL, 5 µg/mL, 2 µg/mL, 1 µg/mL, and 0.5 µg/mL. (D–F) Coagulation analysis. (D) Factor X activation by {alpha}-mCD106-sTF in a cell-free assay. Filled circles: {alpha}-mCD106-sTF. Open circles: sTF (positive control). (E) Factor X activation after binding of {alpha}-mCD106-sTF to VCAM-1-positive endothelial cells. Filled circles = {alpha}-mCD106-sTF. Open circles = sTF (negative control, because sTF does not bind to endothelial cells). (F) Inhibition of factor X activation by addition of anti-mCD106 immunoglobulin G (IgG). VCAM-1–positive endothelial cells were incubated with {alpha}-mCD106-sTF at 5 nM and coagulation induction was analyzed in the presence of free competitor {alpha}-CD106 (IgG, open bars) at different concentrations. No inhibition was seen with an irrelevant IgG (solid bars). Data points in panels D–F represent means of triplicates of a representative experiment; error bars represent 95% confidence intervals.

 
Coagulation induction. Coagulation activity was measured by determining the ability of the sTF moiety in the fusion proteins to activate factor X to factor Xa. In a cell-free two-stage coagulation assay, both fusion proteins induced a concentration-dependent factor X activation with an activity similar to that of free sTF (Fig. 2, D and data not shown). In a cell-based two-stage coagulation assay, the {alpha}-mCD106-sTF fusion protein bound to 2F2B and bEND3 murine endothelial cells and induced coagulation. Specificity was confirmed by the lack of factor X activation by sTF alone, the lack of factor X activation by {alpha}-CD106-sTF on an irrelevant cell line (data not shown), and inhibition of coagulation activity by {alpha}-CD106-IgG (Fig. 2 F,). Similarly, the{alpha}-hCD106-sTF fusion protein bound to TNF-{alpha}–stimulated HUVECs and induced specific coagulation in the two-stage cell-based assay (data not shown).

Lack of direct toxicity on tumor cells and endothelial cells. We carried out cell viability assays to determine whether {alpha}-mCD106-sTF fusion protein was directly cytotoxic to tumor or endothelial cells. These assays measure the metabolic activity of cells and thereby their viability. In these assays, {alpha}-mCD106-sTF concentrations from 10–7 to 10–12 M did not cause direct cytotoxicity to L540rec, Colo677, 2F2B, or bEnd3 cells (data not shown).

In Vivo Efficacy of {alpha}-CD106-sTF

Short-term experiments. In these experiments, groups of six to 13 mice bearing human Hodgkin lymphoma or SCLC xenograft tumors received a single injection of fusion protein or control reagent. We determined the amount of necrosis 3 days later by histologic analysis of several sections of the tumor from each mouse. In tumors of mice treated with the {alpha}-mCD106-sTF fusion protein, we saw thrombosed blood vessels and large areas of tumor necrosis (Fig. 3). Thrombosis was defined as complete occlusion of blood vessels with fibrin-containing plugs; mere erythrocyte aggregation was not considered to be thrombosis. There was some background necrosis in the tumors of control mice, but to a much lower extent than in fusion protein–treated mice. In the lymphoma model, up to 90% of the tumor area was necrotic (mean = 74%, 95% CI = 55 to 93) and in the SCLC model up to 50% of the area was necrotic (mean = 26%, 95% CI = 16 to 36) (Table 1). TNF-{alpha} levels in serum of treated mice in the Colo677 tumor model (24 samples analyzed) ranged from undetectable to 175 pg/mL and showed a similar distribution among treatment groups (FP: mean = 9.3 pg/mL, 95% CI = –4 to 23; sTF: 10.4 pg/mL, 95% CI = –12 to 33; scFv: mean = 52 pg/mL, 95% CI = –36 to 139; vehicle = 36.4 pg/mL, 95% CI = –8 to 80). There was no correlation between TNF-{alpha} level and the extent of necrosis (Spearman–Rho correlation coefficient = –0.021 at a statistical significance level of .9).



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Fig. 3. Histologic analysis of xenograft tumors after short-term treatment (i.e., 3 days) with fusion protein containing soluble tissue factor (sTF) fused to a mouse monoclonal antibody directed against vascular cell adhesion molecule-1 (VCAM-1 or CD106) ({alpha}-mCD106-sTF) or control reagent. Paraffin-embedded tumor sections were stained with hematoxylin and eosin. (A–D) L540rec Hodgkin lymphoma tumors. (A) Low-magnification view (x10) of a tumor from a mouse treated with {alpha}-mCD106-sTF plus lipopolysaccharide. Only a small area with vital tumor cells remains at the left edge of the tumor (darker stain). (B) Higher-magnification view (x40) of the same section. The viable rim at the left is followed by a linear inflammatory demarcation (white arrow), and necrotic tumor tissue is seen at right. Black arrows point to fibrin thrombi. (C) Necrotic tumor tissue (at left) in a tumor from a mouse treated with sTF plus lipopolysaccharide. Arrow points to a fibrin thrombus. (D) In a tumor from a mouse treated with vehicle (saline) plus lipopolysaccharide control, vital tumor tissue with numerous mitotic figures can be seen, reflecting a high proliferation rate of tumor cells. (E–H) Colo677 small-cell lung cancer tumors. (E) A tumor from a mouse treated with {alpha}-mCD106-sTF. A large area of necrotic tumor tissue (brighter stain) is evident. Arrow points to fibrin thrombus. (F) A tumor from a mouse treated with sTF shows a small subcapsular necrosis (arrow). (G) A tumor from a mouse treated with scFv shows microfocal necrosis (arrow) embedded in vital tumor tissue. (H) A tumor from a mouse treated with vehicle control with a small focal necrosis (arrow).

 

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Table 1.  Quantification of tumor tissue necrosis in xenograft tumors from mice treated with fusion proteins or control reagents in short-term experiments*

 
Long-term experiments. Tumor growth delay by multiple injections of {alpha}-CD106-sTF fusion protein was analyzed in the same xenograft models as used for the short-term experiments (Table 2 and Fig. 4). In both models, tumor growth appeared to be slowed by injection of the fusion protein. In the lymphoma model, low-dose LPS was added to upregulate VCAM-1 expression. In this model, tumor growth was statistically significantly slower in the group treated with fusion protein and LPS (FP/L, mean tumor volume on day 16 = 486 µL) than in the group treated with vehicle (mean tumor volume on day 16 = 701 µL; difference = 215 µL, 95% CI = –65 to 495, Mann–Whitney P = .02) or with scFv and LPS (mean tumor volume on day 16 = 847 µL; difference = 361, 95% CI = 189 to 533, Mann–Whitney P = .005). In this and another experiment (data not shown), tumors of FP/L-treated mice were smaller than those of sTF/L-treated mice, but statistical significance of the differences between tumor sizes was not reached at the group sizes that we tested.


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Table 2.  Statistical analysis of tumor volumes after treatment with fusion proteins alone or in combination with lipopolysaccharide or doxorubicin*

 


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Fig. 4. Growth analysis of Colo677 small-cell lung carcinoma xenograft tumors after long-term treatment with fusion proteins containing soluble tissue factor (sTF) fused to a mouse monoclonal antibody directed against vascular cell adhesion molecule 1 (VCAM-1 or CD106) ({alpha}-mCD106-sTF) or control reagent. (A) Growth of tumor xenografts in nude mice. Mice were treated with vehicle control (0.9% NaCl; V), with 20 µg of {alpha}-CD106-sTF (corresponding to 10 µg of sTF; FP), with 8 mg/kg doxorubicin (D), or with 20 µg of {alpha}-CD106-sTF plus 8 mg/kg doxorubicin (FP/D). Solid squares = V; triangles = FP; inverted triangles = D; diamonds = FP/D. (B) Treatment schedule is shown in this enlarged section of the tumor growth curves for D and FP/D; arrows below the panel indicate treatment days.

 
In the SCLC model, only mice treated with fusion protein and doxorubicin or with doxorubicin alone showed statistically significantly slower tumor growth over the course of the experiment (i.e., 2 weeks) than vehicle controls (Table 2). After the second fusion protein treatment, tumor volumes in the combined treatment group were smaller than those in the group treated with fusion protein alone or doxorubicin alone. Comparison of tumor sizes in the combination treatment group with those in each single treatment group by the Mann–Whitney U test showed that the difference between treatment groups was statistically significant on days 5–7. Mean tumor volumes on day 5 were 224 µL for FP/D-treated mice and 574 µL for vehicle-treated mice (difference = 350 µL, 95% CI = 194 to 506; Mann–Whitney P =.001), 454 µL for FP-treated mice (difference compared with FP/D = 230, 95% CI = 90 to 370, Mann–Whitney P = .02), and 285 µL for D-treated mice (difference compared with FP/D = 61 µL, 95% CI = –3 to 119, Mann–Whitney P = .04). An analysis of variance comparing the FP, FP/D, D, and vehicle groups showed that both fusion protein alone and doxorubicin alone led to statistically significant slower tumor growth than vehicle (P = .04 for FP and P = .001 for D), but the interaction between fusion protein and doxorubicin did not reach statistical significance.

In addition to growing more slowly, some of the tumors regressed in the groups treated with fusion protein alone or with the combination of fusion protein and doxorubicin. One tumor in eight tumors in fusion protein–treated mice, two of seven tumors in mice treated with combination therapy, and 0 of 22 tumors in control mice treated with vehicle, sTF or scFv regressed almost completely (i.e., were reduced to less than 50 µL in volume) and did not relapse during the 3-week observation period. In the group treated with doxorubicin alone, one mouse of seven had a small tumor (56 µL) at the end of the experiment. Histologic analysis revealed that the regressed tumors in the fusion protein–treated mice had a necrotic core and a viable rim, as is frequently seen with other vascular targeting agents. However, the cells of the rim did not have the appearance of tumor cells. Immunohistochemical staining of the regressed tumors for tumor cell surface markers (bcl-2 and vimentin) and for a macrophage marker (F4/80) confirmed that, in the mouse treated with fusion protein alone, tumor cells were almost exclusively located in the necrotic core and that the viable rim consisted mainly of macrophages (Fig. 5). A similar phenomenon was seen in the regressed tumors of mice treated with both fusion protein and doxorubicin, although there were slightly more vital tumor cells in the viable rim. In the small tumor of the doxorubicin-treated mouse, there was a large proportion of bcl2- and vimentin-positive vital tumor cells in the viable rim, and macrophages were scarce (data not shown).



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Fig. 5. Histologic analysis of tumor tissue remaining after macroscopic regression of Colo677 tumors from mice treated with fusion proteins containing soluble tissue factor (sTF) fused to a mouse monoclonal antibody directed against vascular cell adhesion molecule 1 (VCAM-1 or CD106) ({alpha}-mCD106-sTF). (A–C) Hematoxylin and eosin–stained tumor sections. (A) Low-magnification view (x20) shows central necrosis and viable rim. At higher magnifications (B, C; x100, x400), necrotic cells can be seen at the lower right. (D–F). Tumor sections stained for bcl-2, which is a marker for Colo677 tumor cells. (D) Low-magnification view shows immunostaining of the tumor cells in the center, but not in the rim. (E) Necrotic core (lower half) is stained with bcl-2. (F) At higher magnification, small cell clusters of viable tumor cells can be noted in the left side, on the right side a part of the necrotic core. (G–I) Tumor sections stained for vimentin, a different marker for Colo677 tumor cells. (G) Low-magnification view shows immunostaining of tumor cells in the necrotic core. (H) Lower half shows immunostained tumor cells in the necrotic area. (I) Higher magnification reveals small cell clusters of viable tumor cells next to the necrotic area (necrosis in right lower corner). (J–L) Tumor sections stained for the macrophage marker F4/80. Most cells in the viable rim are not tumor cells but macrophages. (M–O) Tumor sections stained with isotype-matched negative control antibody.

 
SCID Mouse Model of Human Angiogenesis

To determine whether the {alpha}-human CD106-sTF fusion protein can lead to the occlusion of human tumor vasculature, we used a previously published model (43) in which human endothelial cells are cotransplanted with human tumor cells into a SCID mouse host, resulting in a tumor that consists of human tumor cells and both human and murine tumor vessels. This model has been characterized extensively, and the presence of functional human blood vessels positive for human CD31, CD34, VCAM-1, intercellular adhesion molecule 1 (ICAM-1), and vWF has been demonstrated previously (43). At the treatment time point (i.e., 3 weeks after implantation) most microvessels are of human origin (43). For this study, we used Colo677 cells as the tumor cells. The resulting tumors had the same morphology as the subcutaneous Colo677 tumors (data not shown) and had both human and murine vessels, as confirmed by immunohistochemistry for human von Willebrand factor (data not shown).

In an exploratory study with groups of five or six mice each, fusion protein–treated mice had statistically significantly smaller tumor volumes after 3 days than control mice treated with vehicle or sTF. The data on the tumor volumes is given in Table 2. We also measured tumor weights in this experiment. The mean tumor weights were: 42 mg for fusion protein–treated mice, 71 mg for vehicle-treated mice (difference = 29 mg, 95% CI = 8 to 100, Mann–Whitney P = .008) and 68 mg for sTF-treated mice (difference compared with fusion protein–treated mice = 26 mg, 95% CI = 7 to 101, Mann–Whitney P = .01). Histologic analysis did not reveal large thrombotic areas in the tumors, possibly because of fast clearance of necrotic material by the many giant cells that are a typical feature of this mouse model. However, immunohistochemical analysis revealed fibrin clots in the tumors of fusion protein–treated mice and sTF-treated mice, but not in vehicle-treated mice (data not shown). Thus, although tumor growth was statistically significantly slowed, the exact mechanism of action is still under investigation.

Tolerability of the Fusion Protein

Clinical observation. Mice were observed for clinical signs of toxicity at prespecified times (5, 10, 15, 30, 60 and 120 minutes and then daily) after treatment. Apart from a slight hypoactivity 10 minutes after injection that occurred in all treatment groups and was probably due to the volume increase of the blood, we observed no clinical signs of toxicity in fusion protein–treated mice in either the short-term or long-term experiments. Mice treated with any of the LPS-containing regimens showed slight hypoactivity and mild diarrhea. In the long-term experiments, one of the nine mice treated with sTF and LPS experienced body weight loss and decrease of leukocyte and platelet counts and died after the third treatment. Of the 14 mice treated with a doxorubicin-containing regimen, eight (57%) developed diarrhea, body weight loss, and epidermolysis at mechanically stressed skin areas. Body weight loss greater than 10% compared with the initial weight, recorded at any time in the experiment, was observed in one of the nine lymphoma-bearing mice treated with sTF and LPS (see above) and in one of the five lymphoma-bearing mice treated with scFv and LPS. Among the SCLC-bearing mice, such a weight loss was seen in five of the seven mice treated with doxorubicin and three of the seven mice treated with doxorubicin plus fusion protein. All other mice had stable body weights.

Histologic analysis of organs. Histologic sections of all major organs were analyzed by light microscopy for thrombotic or necrotic events. No necrosis was evident in any organ tissues in the several hundred sections that were analyzed (Table 3). We detected a few thrombotic events in the lungs (one to three thromboses per whole organ section in some of the mice), with extravasation of erythrocytes. Such events were seen in all treatment groups and might have been caused by a procoagulant state induced by the tumor burden. We occasionally saw small fibrin plugs in kidney, liver, pancreas, and heart sections from all treatment groups, but, in contrast with the lung, there was no associated tissue reaction. The lack of tissue reaction suggests that the latter events occurred after death and therefore are most likely artifacts. All other organs were free of thromboses or necroses. Thus, the fusion protein was very well tolerated when given alone. Multiple doses of LPS and high-dose doxorubicin were toxic, and future combination treatments will have to use lower doses or less toxic formulations of chemotherapy.


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Table 3.  Number of mice with thrombotic (T) and necrotic (N) events in organ tissues*

 
Laboratory Parameters of Treated Mice

As more sensitive assays of systemic coagulation, we used blood counts and evaluated several coagulation parameters (i.e., thrombin–antithrombin complexes, antithrombin III, and soluble fibrin) obtained by vena cava puncture 3 days after treatment (Table 4). To identify early changes in coagulation parameters, we also measured these parameters 15 minutes, 30 minutes, 4 hours, and 24 hours after treatment (in three mice each) for fusion protein–treated mice. Except for a slight and reversible decrease of antithrombin III and hemoglobin in the fusion protein–treated SCLC-bearing mice at 30 minutes, laboratory parameters were within the reference range. Thus, it seems unlikely that ongoing systemic activation of the coagulation system is occurring.


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Table 4.  Laboratory parameters of treated mice*

 

    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The goal of this study was to generate a recombinant specific vascular occluding agent directed against a luminal tumor endothelial cell marker and to critically evaluate its clinical applicability. This is the first report, to our knowledge, to demonstrate specific thrombosis-induced necrosis and tumor growth delay in vivo with a recombinant sTF-based fusion protein directed against a luminal endothelial marker (i.e., VCAM-1), to study potential side effects by measuring coagulation parameters in treated mice, and to evaluate targeted sTF-based drugs in a tumor model for human angiogenesis.

One strength of the approach we used is that we were able to test, in parallel, agents directed against a murine antigen and a homologous human antigen. Such parallel testing allows the agent to be studied in multiple well-characterized mouse models and to combine the results with those from the few models containing human endothelial cells. Therefore, one is not restricted to the rare cases of antibodies recognizing antigens across different species for the in vivo characterization.

In vitro analysis of the fusion proteins revealed that both their targeting (anti–VCAM-1) and effector (sTF) moieties were functional and specific. We ruled out the possibility that in vivo effects were due to direct toxicity of the drugs on endothelial cells or tumor cells. Factors that are known to bias coagulation induction, e.g., trace amounts of LPS or cytokines, were carefully controlled.

In vivo efficacy was demonstrated in several mouse models. In the L540rec human lymphoma model, the addition of LPS was necessary to increase VCAM-1 expression. LPS is a pleiotropic molecule, and repeated long-term administration produces several different effects (45); administration of multiple doses of LPS also resulted in toxicity in some of the mice in this study. Therefore we also used a different model, the Colo677 SCLC model, in which VCAM-1 is expressed constitutively on the tumor endothelium at levels sufficient to allow targeting of the fusion protein. We observed in vivo efficacy under carefully controlled conditions but also found that tumor growth was best controlled when the regimen was combined with doxorubicin. We have previously found that tumor vessels can be sensitized by LPS for coagulation induction with tissue factor–based drugs (16). In this study, we found some evidence that pretreatment with doxorubicin seemed to render the tumor endothelium more sensitive to fusion protein treatment. Further studies are under way to investigate this phenomenon with various treatment schedules and lower doses of chemotherapeutic agents.

We carried out an extensive evaluation of the tolerability of the fusion protein, and we believe that our study provides the most comprehensive information to date about potential side effects of sTF-based constructs. In particular, we established assays that would indicate whether systemic coagulation induction takes place in the mouse. Analyzing this effect is especially important because mice are not the ideal species in which to evaluate thrombembolic complications (46). Consequently, more sensitive assays than previously applied (i.e., clinical observation and histological evaluation of organs) are necessary to judge possible thrombembolic side effects. In separate experiments(Unruh, Grunow, Gottstein, unpublished data) we determined that measurements of blood counts, thrombin–antithrombin complexes, antithrombin III, and soluble fibrin monomers would be suited for evaluating systemic coagulation activation. We found no indication that the fusion proteins induce ongoing systemic coagulation. We suggest that other sTF-based drugs under development be evaluated for toxicity in a similar fashion.

To test the fusion proteins directed against human antigens, we used a previously established mouse model for human angiogenesis (43), in which the mice bear human tumors nourished by human vessels that are connected to the mouse vascular system. The {alpha}-hCD106-sTF fusion protein had a statistically significant antitumor effect compared with either vehicle or sTF in this system.

One limitation of the experiments reported in this article is that the long-term experiments are still at an early stage, because further mouse models should tested, including those that typically are very sensitive to tumor-selective coagulation induction, e.g., sarcomas (47). A second limitation is directly related to one of the strengths of this study, i.e., the effort to carefully control factors that might bias the results and to include all relevant negative controls. In consequence, we observed that, at the group sizes tested, the fusion protein by itself did not always show a statistically significant advantage over all possible negative controls. Nevertheless, a statistically significant treatment effect was observed under certain conditions (e.g., short-term experiments, human angiogenesis model), which justifies further work to refine sTF-based drugs.

To markedly improve the efficacy of sTF-based drugs without increasing their potential for side effects, one must understand their molecular mechanisms of action. Full-length TF physiologically serves as a cofactor within the membrane-bound extrinsic factor Xase complex to activate factors VII, IX, and X. sTF has lost the ability to activate factor VII (48), and therefore factor VIIa becomes a limiting factor as long as this function is not restored. We have shown previously that even untargeted sTF can support coagulation if sufficient amounts of factor VIIa are present, because factor VIIa can also mediate the binding of sTF to membranes via its glutamic acid–rich (Gla) domain (16). However, the specificity of binding will then be conferred via the Gla domain and not by the ligand. For sTF-based drugs to work effectively, the required amounts of factor VIIa can be provided by applying the sTF drug in an environment with preexisting vascular damage and coagulation (16,39,40), by adding factor VIIa exogenously (41), or by restoring the ability of the sTF drug to activate factor VII to VIIa. We believe that the last approach should be most advantageous, because in this case the coagulation process is effectively triggered by the binding of the specific ligand.

Useful targets for this type of specific vascular occluding agent should 1) be expressed at sufficient levels for efficient targeting of the agent on tumor endothelial cells, 2) be expressed at low levels on nontumor endothelium, 3) be located on the luminal side of the vessel, and 4) have a low internalization rate. Of the well-characterized endothelial cell antigens, VCAM-1 is among the best targets to meet these specifications. Nevertheless, with the advent of powerful technologies to screen for new luminal endothelial cell targets (49), additional suitable targets will likely be identified in the near future.

Although selectivity of the target antigen for the tumor endothelium is important, low-level expression of the target in other tissues might not pose a major problem because the tumor vascular endothelium has been shown to support coagulation better than normal vessels. Indeed, Ran et al. (17) demonstrated binding of a biochemical conjugate of sTF and anti–VCAM-1 to murine lung and heart vasculature without accompanying thrombosis or necrosis. However, it remains to be determined whether, in certain pathologic conditions, such as inflammation or atherosclerosis, nontumor vessels would act similarly to tumor vessels in supporting increased coagulation induction.

The observation that cancer patients are in a prothrombotic or hypercoagulable state (50) supports the concept of specific vascular occluding agents as a treatment strategy, because this approach uses a physiological system that is not impaired by either the underlying disease or previous therapy. In theory, the localization of the coagulation process within the tumor vasculature through efficient targeting should result in a decrease of the systemic procoagulant tendency. To achieve safe and efficient targeting, it is crucial to identify the trigger parameters for this type of coagulation induction and to characterize their presence in human vasculature (tumor, normal, or nontumor pathologic vasculature).


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We thank Anke Wagener and Tanja Roth for excellent technical assistance, Samir Tawadros for care and monitoring of laboratory animals, and Wendy Song for technical assistance with the human angiogenesis model. Aiko Ferrari contributed to the immunohistological studies and the in vivo analyses, and Britta Engelhardt kindly provided bEnd3 cells.

Financial support was obtained from the Deutsche Forschungsgesellschaft (DFG SFB502-T6) to C.G., the Novartis Foundation for Therapeutic Research to C.G., the NIH/NIDCR (DE14601) to J.E.N., and the Köln Fortune Program at the Faculty of Medicine of the University Cologne to C.G.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Manuscript received September 24, 2004; revised March 10, 2005; accepted March 15, 2005.


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