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Targeting Endothelial Growth with Monoclonal Antibodies against Tie-1 Kinase in Mouse Models¹

Department of Pharmacology, Institute of Biomedicine, FIN-00014 University of Helsinki, Helsinki, Finland [P. K.], and Department of Nuclear Medicine, Uppsala University Hospital, S-751 85 Uppsala, Sweden [K. K.]

	ABSTRACT

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Purpose: Tie-1 is a transmembrane tyrosine kinase expressed in vascular endothelial cells during angiogenic processes and vasculogenesis. Here we evaluate targeting of rebuilding endothelium with ¹²⁵I-labeled Tie-1 monoclonal antibodies (mAbs) in mice.

Experimental Design: At first, activity of Tie-1 kinase during reforming of blood vessels was evaluated in melanoma allografts in transgenic mice with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining of the Tie-1 promoter gene. Subsequently, in vivo targeting of the healing wound was evaluated with iodinated Tie-1 mAbs in mice, and finally, after confirming the specificities for targeting, we evaluated the biodistribution of Tie-1 mAbs in a melanoma model.

Results: Tie-1 mAbs target epithelial skin wounds in mice. Biokinetics of ¹²⁵I-Tie-1 mAbs demonstrate a stabilized equilibrium between the blood and wound over 3 days. The accumulation in wound is 21% injected dose/gram (ID/g) and 17% ID/g at 48 h with the two clones of Tie-1 mAbs, 3c4c7 and 10f11g6. Tie-1 promoter is active in the melanoma model in mice. In melanomas, the tumor accumulation is 5% and 4.4% ID/g, and the tumor:liver values are 2.7 and 4.4, respectively, for the two clones of Tie-1 mAbs, 3c4c7 and 10f11g6. The clearance of ¹²⁵I-Tie-1 antibodies is slow when compared with that of the iodinated control antibody.

Conclusions: Targeting to wound is demonstrated with the Tie-1 mAbs. Accordingly, tumor targeting of melanoma is expertized with the same antibodies.

	Introduction

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Angiogenesis is an early step in the growth of both normal and malignant tissue formations. Changes in the expression rate of angiogenic growth factors and their receptors in vivo indicate enhanced growth (1 , 2) . Both normal tissues and abnormal growth depend on angiogenic signals which also deposit border lines for tissue growth. The tie gene encodes the endothelial receptor tyrosine kinase (Tie-1), which is expressed mainly during the embryonal vasculogenesis but also in adults during some angiogenic events. The main function of Tie-1 is to induce blood vessel stability during embryonal development (3) . Tie-1 cDNA (M_r 117,000) was first revealed from human leukemia cells, and its expression was detected in various endothelial cells (4) .

The expression of Tie-1 is associated with maturation of follicles in ovulation, and Tie-1 is detected in the granulation tissue of skin wounds in mice (5) . Enhanced Tie-1 is detected by mRNA expression from healing wounds at the seventh day of the healing process, from the granulation tissue of the skin. In regeneration of tissues, angiogenesis is essential to ensure a supply of nutrients to the newly formed and still-growing cells (6) . The endothelial cell differentiation depends on angiogenic signals which also deposit the normal level of signaling proteins in the mature state of endothelium.

Tie-1 promoter is active in vasculogenesis during mouse development. The activity is detected spatially in coronaries, capillaries, and cusps of the heart (7) and in some endothelial cells in the bone marrow. Some promoter activity also persists in the intra-alveolar capillaries of lung and in kidneys. The embryonal activity of Tie-1 is also demonstrated by mRNA expression in the dorsal aorta and endocardium (5) , and the activity pattern of Tie-1 promoter resembles the mRNA expression detected (7) in the later phases of angiogenesis and in the vasculogenesis of embryonal growth in mouse embryos and some human samples (8 , 9) . Tie-1 activity in adult tissues is, in general, down-regulated (10) and is restricted to the lung capillaries and to the heart and kidney vessels (8) . No ligand for Tie-1 has been identified thus far. The amino acid sequence of mouse Tie-1 is 92% identical to the human Tie-1 sequence.

Tie-1 is detected in human umbilical vein endothelial cells after VEGF³ stimulation in vitro (11) and in human blood samples (12) . The presence of Tie-1 receptor protein in connection with human tumor formation is not verified in melanomas, but Tie-1 mRNA expression has been detected by reverse transcription-PCR in human melanoma samples (13) . The activity of Tie-1 has been evaluated in many experiments regarding the neovascularization processes in mouse models (6 , 14 , 15) and in the growth of malignancies (16) to understand the behavior of diseases. The attempts to develop more accurate prognostic indicators for melanoma are limited within the correlation between aggressive growth rate and tumor tissue vascularization. The proliferation of endothelial cells in capillary sprouts promotes growth, and a direct tissue marker could relieve the malignant growth.

The study here was performed to investigate the in vivo targeting of melanomas by radiolabeled mAbs against the transmembrane receptor Tie-1. Two different clones of Tie-1 mAbs are used to detect vascular angiogenesis.

	Materials and Methods

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Biodistribution of Tie-1 mAbs was investigated in vivo by measuring the organ accumulation of the ¹²⁵I-labeled antibodies at various time points. Activity of the Tie-1 gene was demonstrated by X-Gal staining of the ß-galactosidase markers in the melanoma allografts of the transgenic mice.

mAbs.
The mAbs were IgG1 subtype and generated against the transmembrane Tie-1 receptor with traditional methods (17) . The extracellular part of recombinant protein tie-1, which is equivalent to NH₂-terminal amino acids 24–760, serves as antigen for the Tie-1 mAbs (4) . In this study, we used two monoclonal clones, 3c4c7 and 10f11g6. Their affinities to soluble recombinant protein tie-1 are similar when determined by the Biacore method [3.8 x 10⁷ M^-1 (10f11) and 6.5 x 10⁷ M^-1 (3c4)]. The dissolution parameters of the binding coefficients were 4.6 x 10^-4 s^-1 (10f11) and 8.1 x 10^-4 s^-1 (3c4), which makes the dissolution slow and the in vivo binding experiments possible, even when the binding affinities as such are not very prominent.

Tie-1 mRNA was found in cells of megakaryoplastic origin (4) . HEL 92.1.7 cells and mouse LE GD 14-2 cells were used for in vitro assays to confirm the binding of Tie-1 mAbs. The HEL cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum. The mouse LE cells were transfected with tie-1 promoter vector (735-bp AflII-ApaI mouse/SDK LacZ) and grown as monolayers (7) . LE cells were received from Prof. Kari Alitalo. The immunoreactivities of Tie-1 mAbs were studied in competitive binding experiments. The overall binding capacity of 10f11g6 to LE cells is better than that seen with clone 3c4c7. The specifically bound amounts are 21 pmol of 10f11 and 7 pmol of 3c4 in 100,000 cells.

¹²⁵I -Tie-1 mAbs.
Antibodies were labeled by modified chloramine-T method (18) with 1 mCi of ¹²⁵I to 40 µg of protein. The radiolabeled antibodies were purified and concentrated using Sephadex-G25 column (Pharmacia AB, Uppsala, Sweden). The specific activity was 20 µCi/µg. Before in vivo studies, the immunoreactivities of ¹²⁵I-Tie-1 mAbs were determined in HEL cells (19) . The affinity of Tie-1 mAbs was 6 x 10⁹ M^-1 in the competitive binding method (20) . The human T-cell leukemia cells (MOLT4; American Type Culture Collection) were used as a nonspecific control of the antibody binding.

Animal Models.
Two in vivo models were used in targeting with the new Tie-1 mAbs. The growth of vascular endothelium is demonstrated in melanoma allografts (M3; American Type Culture Collection) and in the healing wound model in mice (C57BL/6; Bomholtgaard). The healing wound was selected because Tie-1 mRNA is detected in healing wounds of adult mice, in the granulation tissue of epidermal skin (5) . Maximal mRNA expression is restricted to the seventh day of the healing process, and the expression of Tie-1 is expected to be high at the time of biodistribution experiments of the wound model.

Biodistribution studies of Tie-1 mAbs in melanoma were performed after the activity of Tie-1 promoter was verified in the animal model. To perform these studies, we had permission from the local ethical committees and from the County Administration Board of Western Finland. The general principles of laboratory animal care (according to NIH Publication No. 86-23, as revised in 1985) were carefully followed in the experiments.

Biodistribution of Tie-1 mAbs.
In healing wound model, a minimum of 4 mice/time point were anesthetized with 3.6% chloralhydrate (0.0135 ml/g) i.p. Cutaneous wounding was performed by cutting a short epithelial wound of 1 inch in the caudal part of back skin. The ¹²⁵I-Tie-1 mAbs were injected i.v. at appropriate days to provide a healing wound of 8 days of age at required measuring times. The radioactivities in wound and main organs were measured at 24, 48, 72, 96, and 120 h.

Biodistribution in melanoma model was done similarly in 6–8-week-old female mice bearing melanoma allografts, which received i.v. injection with the ¹²⁵I-Tie-1 mAbs (100 µl, 20 µCi). Organ accumulation was determined at similar time points. Each measuring point consisted of three to six animals. The radioactivities were measured in a gamma counter ( Master 1277; Pharmacia, Wallac), and the specific organ activities were calculated after weighing the samples. As a control, we used iodinated mAbs of the same subtype IgG1 (Fc-3001; Orion Diagnostica) to define the clearance of ¹²⁵I-mAbs in the mouse model. The blocking of mouse thyroids for the accumulation of free iodine was started 1 day before the injections of ¹²⁵I-Tie-1 mAbs and continued throughout the experiment with potassium iodide solution (400 mg/100 ml) ad libitum.

The ß-Galactosidase Staining of Melanomas.
The activity of the Tie-1 promoter in mice was verified with ß-galactosidase staining of melanoma allografts grown in transgenic mice. The offspring (F₂ and F₃) of LacZ-transfected NMRI versus DBA/2 animals were used in this experiment. The transfected construct with LacZ reporter gene linked to the murine tie promoter (the 735-bp mouse AflII-ApaI) has been studied previously in mice (7) , and, according to the experiments, the construct is active in the adult mouse.

For staining, samples of melanomas were transferred into 4% paraformaldehyde in PBS (pH 7.4) and incubated at 4°C for 20 min and then incubated in X-Gal reaction mixture [1 mg/ml X-Gal, 4 mM K₄Fe(CN)₆ x [³H]₂O, 4 mM K₃Fe(CN)₆, and 2 mM MgCl₂ in PBS] at 30°C for 2 days (21) . After incubation the specimens were washed in PBS for 5 h and transferred to 30% sucrose for cryopreservation. The samples were embedded in Tissue-Tek (Miles), and 15–20-µm sections were cut with a cryomicrotome. The sections were postfixed in 4% paraformaldehyde for 5 min, and nuclear fast red was used in counterstaining.

	Results

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The two Tie-1 mAbs, clones 3c4c7 and 10f11g6, show similar binding characteristics in biodistribution experiments and in the cell assays. In HEL cells, clone 3c4c7 is bound better than clone 10f11g6. The results are 8297 cpm with 3c4c7 and 4151 cpm with 10f11g6 for 10⁶ cells (cpm = cpm of ¹²⁵I-Tie-1 mAbs) in the direct binding measurements. The competitive binding assay in HEL cells reveals similar binding characteristics (Fig. 1) . The maximal bound amount (pmol of mAbs) is reached for the first binding site by adding <50 ng of mAbs in the cell assay.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. HEL cell assay with 10f11g6 (top) and 3c4c7 (bottom). The HEL cells are used in the competitive binding assay to determine the affinities of labeled Tie-1 mAbs. The specific binding with Tie-1 clone 10f11g6 is 3 pmol to 100,000 cells, whereas that with clone 3c4c7 is 10 pmol to 100,000 cells. A similar tendency of duplicating the maximally bound amounts with 3c4c7 is also noticed in other cell assays for TIE-1 antibodies and in the solid-phase RIA for the recombinant tie protein.

View larger version (65K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. A, expression of the LacZ gene is verified in the vascular endothelial cells of the transgenic mouse. The blue color stains the endothelial cells on the inner cavity of the three tiny blood vessels. Some peripheral vessels are visible, with the typical color spread in near proximity to the skin in this cross-section of the tail. B, a section of the tumor tissue of mouse melanomas. The tissue is very soft, and not many blood vessels are visible in the area, but the endothelial cells of blood vessels express ß-galactosidase and verify the activity of Tie-1 promoter in the vascular endothelium of the melanoma allografts.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The specific binding of Tie-1 mAb (clone 3c4c7) in the wound model is shown. Targeting the wound is observed, and the accumulation to the main organs is <5% ID/g at 2–4 days after injection of TIE-1 mAbs. The specific binding to wound is demonstrated with the equilibrium between the wound and blood.

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Biodistribution of Tie-1 mAbs in melanoma. The accumulation of Tie-1 mAbs to the target in the melanoma model is lower than that in the wound. Binding of TIE-1 mAbs to the tumor is higher than that to main organs and the maximal binding is detected at 2 days after the injection of Tie-1 mAbs, similarly to the wound model.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. The target:liver ratio is compared with the accumulation of two Tie-1 mAbs in main organs and in melanoma tumors. Targeting with Tie-1 mAb (clone 10f11) is best at 48 h. The tumor:liver ratios are, however, quite similar with both Tie-1 mAbs, and the targeting to tumor not enhanced if compared to main organs.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. The accumulation of activity is directly compared as cpm/g of tissue between the two Tie-1 mAbs separately in the melanoma (left panels) and wound models (right panels). Targeting with the clone 3c4c7 is similar in the melanoma and in the wound at 48 h after injection of the antibodies. Targeting with clone 10f11g6 is best in the wound model.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

 
In this study, we present targeting of the healing wound in vivo by Tie-1 mAbs (Fig. 3) . The expression of Tie-1 is connected to the maturation of vascular endothelium and to the rebuilding of wounded epithelium. Although Tie-1 protein expression is not directly shown with immunohistochemical staining in our experiments, Tie-1 expression is detected in the granulation tissue of the skin in healing wounds of adult mice with Tie-1 mRNA analyses (5) . In mouse tumors, Tie-1 protein expression is detected by Tie-1 mAbs (14) . The experiments examining Tie-1 activity in tissues are generally done by mRNA analyses (22) or by immunohistochemical methods for protein staining from frozen sections (23) .

Targeting with iodinated antibodies is not the best possible solution for in vivo experiments. We ended up with the techniques because ¹²⁵I-Tie-1 mAbs in the cell experiments were stable, and the affinity was unchanged for 2 months. In pilot in vivo experiments with freshly labeled ¹²⁵I-Tie-1 mAbs, the dissociation of the label was also under control.

Tie-1 mRNA is detected in primary cutaneous melanomas by immunohistochemical methods (13) . Before proceeding to the biodistribution experiments, it was necessary to determine Tie-1 expression in the melanoma model in mice. The activity of Tie-1 promoter is detected in the vascular endothelium of the melanoma allografts of transgenic mice. This experiment verified the activity of Tie-1 in the tumor formation and, moreover, the origin of the blood vasculature in the melanoma model.

Tumor targeting with antibodies that bind to transmembrane cell receptor is a competent instrument for tumor detection in vivo. Markers correlating with endothelial cell proliferation are needed for detection of malignant growth. The formation of vasculature in tumor progression is an important step for independent tissue growth and is a known sign for the malignancy of solid tumors. Tie-1 mAbs are a fine tool for tumor targeting, but the limitation is the density of the vasculature in solid tumors. The low density of new vessels in tumor samples is detected in the immunohistochemical staining of Tie-1 in human mammary carcinoma samples (23) . The targeting of melanomas with Tie-1 mAbs reflects, in part, the low density of growing vessels (Fig. 2B) .

In addition to angiogenesis in tumor formation, a role for the growth pattern of cells has recently been suggested to affect the detection of malignant growth. The tubular pattern of growth that is typical to embryonic vascular networks is also significant in the growth of cutaneous melanomas (15) . The targeting of melanomas by Tie-1 mAbs may be due in part to binding to melanoma cells. In an earlier study (15) , Tie-1 expression was detected in the highly aggressive cells of melanoma lines with Western blotting. In the less aggressive cells, Tie-1 was not expressed. The result suggests that direct targeting of melanomas by ¹²⁵I-Tie-1 mAbs is possible. However, the specimens for the Western blot experiment in that study were whole-cell lysates of different cell cultures, and we might argue that melanoma cells grown in culture represent a different, in vitro cell type, and Tie-1 protein is not necessarily detected similarly in melanomas in vivo.

The accumulation to wound is higher with the Tie-1 mAb clone 10f11g6 than with 3c4c7. The growth pattern in wound is also not similar to tumor growth, suggesting that clone 10f11g6 targets better to the rebuilding vascular endothelium under conditions in which the growth rate is more intense, and mature endothelial cells dominate. In tumor growth, new vasculature may not yet be mature, and both Tie-1 mAbs target melanomas similarly.

In the transgenic mouse model of prostate adenocarcinoma (22) , total RNA was analyzed from prostate tumors, with various markers associated with angiogenesis and detected by Northern blotting. In those results, Tie-1 mRNA was detected at a higher level than that seen in the normal prostates of C57BL/6 mice. Moreover, an 8-fold induction in Tie-1 mRNA level was detected in melanoma xenografts grown in transgenic mice overexpressing VEGF. The vascular growth factor VEGF is detected in other studies also in association with the expression of angiogenic factors and their receptors.

Targeting melanoma in vivo with the Tie-1 mAbs is important for the detection of aggressive tumor growth. This targeting with Tie-1 is supported by studies on the growing pattern of cutaneous melanoma, which resembles the embryonal growth pattern of the vasculature. The straightforward experiments with iodinated Tie-1 mAbs targeting the healing wound suggest that Tie-1 mAbs are capable of immunodetection in vivo. Accordingly, tumor targeting with the same Tie-1 mAbs is expertized.

	FOOTNOTES

 
¹ Presented at the "Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates," October 24–26, 2002, Princeton, NJ. This study was supported by TEKES (National Technology Agency of Finland).

² To whom requests for reprints should be addressed, at Department of Nuclear Medicine, Entrance 78, Uppsala University Hospital, S-751 85 Uppsala, Sweden. Phone: 46-18-6111006; Fax: 46-18-6114124; E-mail: kalevi.kairemo{at}onkologi.uas.lul.se

³ The abbreviations used are: VEGF, vascular endothelial growth factor; mAb, monoclonal antibody; X-Gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside; ID/g, injected dose/gram; HEL, human erythroleukemia; LE, lung endothelial.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Karnani, P. Articles by Kairemo, K. Search for Related Content PubMed PubMed Citation Articles by Karnani, P. Articles by Kairemo, K.