The antibody-based targeted delivery of interleukin-4 and 12 to the tumor neovasculature eradicates tumors in three mouse models of cancer.

Preclinical studies with recombinant murine interleukin 4 (IL4) in models of cancer have shown potent tumor growth inhibition. However, systemic administration of human IL4 to cancer patients exhibited modest antitumor activity and considerable toxicities. To improve the therapeutic index and reduce side effects of this cytokine, we developed of a novel "immunocytokine" based on sequential fusion of murine IL4 with the antibody fragment F8 (specific to the alternatively spliced extra-domain A of fibronectin, a marker for tumor-angiogenesis) in diabody format. The resulting fusion protein, termed F8-IL4, retained full antigen-binding activity and cytokine bioactivity and was able to selectively localize on solid tumors in vivo. When used as single agent, F8-IL4 inhibited tumor growth in three different immunocompetent murine cancer models (F9 teratocarcinoma, CT26 colon carcinoma and A20 lymphoma). Furthermore, F8-IL4 showed synergistic effects when coadministered with immunocytokines based on IL2 and IL12. Indeed, combination therapy with an IL12-based immunocytokine yielded complete tumor eradication, in spite of the fact that IL4 and IL12 display opposite immunological mechanisms of action in terms of their polarization of T-cell based responses. No weight loss or any signs of toxicity were observed in treated mice, both in monotherapy and in combination, indicating a good tolerability of the immunocytokine treatment. Interestingly, mice cured from CT26 tumors acquired a durable protective antitumor immunity. Depletion experiments indicated that the antitumor activity was mediated by CD8+ T cells and by NK cells.

Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth.

Inactivation of the von Hippel-Lindau (VHL) gene is associated with the development of highly vascularized tumors. pVHL targets the alpha subunits of hypoxia inducible factor (HIF) for ubiquitin-mediated degradation in an oxygen-dependent manner. Although pVHL-deficient tumor cell lines demonstrate constitutive stabilization and activation of HIF, it has yet to be shown that loss of murine Vhl alone is sufficient to dysregulate HIF. We utilized a genetic approach to demonstrate that loss of Vhl is sufficient not only to stabilize HIF-alpha subunits under normoxia, but also fully activate HIF-mediated responses. These studies have implications for the hierarchy of signaling events leading to HIF stabilization, nuclear translocation, and target gene expression. We further demonstrate that loss of murine Vhl does not promote teratocarcinoma growth, indicating that other genetic changes must occur to facilitate Vhl-mediated tumorigenesis.

Targeted Vezf1-null mutation impairs vascular structure formation during embryonic stem cell differentiation.

OBJECTIVE: Vezf1 encodes an early zinc finger transcription factor that is essential for normal vascular development and functions in a dose-dependent manner. Here, we investigated the role of Vezf1 during processes of endothelial cell differentiation and maturation by studying mutant Vezf1 embryonic stem (ES) cells using the in vitro embryoid body differentiation model and the in vivo teratocarcinoma model. METHODS AND RESULTS: Vezf1-/- ES cell-derived embryoid bodies failed to form a well-organized vascular network and showed dramatic vascular sprouting defects. Our results indicate that the retinol pathway is an important mediator of Vezf1 function and that loss of Vezf1 results in reduced retinol/vitamin A signaling and aberrant extracellular matrix (ECM) formation. Unexpectedly, we also uncovered defects during in vitro differentiation of Vezf1-/- ES cells along hematopoietic cell lineages. Vezf1-/- ES cell-derived teratocarcinomas were able to spontaneously differentiate into cell types of all 3 germ layers. However, histological and immunohistochemical examination of these tumors showed decreased cell proliferation, delayed differentiation, and large foci of cells with extensive deposition of ECM. Embryoid bodies and teratocarcinomas derived from heterozygous ES cells displayed an intermediate phenotype. CONCLUSIONS: Together, these results suggest that Vezf1 is involved in early differentiation processes of the vasculature by regulating cell differentiation, proliferation, and ECM distribution and deposition.

Vascularization of testicular germ cell tumours: evidence from experimental teratocarcinomas.

Testicular germ cell tumours (TGCTs) represent about 2% of male malignancies, being the most common cancer among adolescents and young adults. As in most neoplasias, TGCTs show a chaotic vascular architecture, altered blood supply and over-expression of pro-angiogenic factors, aspects closely related to tumour overgrowth and metastasis. Following this trend, our laboratory has analysed the effect of the hypoxic tumour microenvironment on cancer stem cells, particularly the expression of factors related to vascularization, such as matrix metalloproteinases, adhesion molecules, vascular endothelial growth factors (VEGF) and VEGF receptors. This review also summarizes our present knowledge on vascularization in the normal and neoplastic testis, the potential role of the factors involved in TGCT neovascularization and their importance as possible therapeutic targets.

Angiogenesis and vascular network of teratocarcinoma from embryonic stem cell transplant into seminiferous tubules.

BACKGROUND: Carcinoma in situ (CIS) of the testis is considered to be a precancerous germinal cell lesion, but the precise cellular and molecular mechanisms underlying transformation of CIS into invasive pluripotent cancer cells remain to be elucidated. Moreover, a satisfactory animal model for the experimental study of germinal tumours has not been developed to date. METHODS: We have developed a tumour model that involves the microinjection of green fluorescent protein-labelled embryonic stem (ES) cells (which are functionally equivalent to CIS cells) into syngenic mouse seminiferous tubules, a unique cell microenvironment in which germinal cells mature and CIS arise. To characterise the vascularisation of teratocarcinomas, which arise after cell transplant, we used immunohistochemistry, together with a qualitative and quantitative analysis of scanning electron microscopy images of corrosion casting samples. RESULTS: Embryonic stem cells transplanted into seminiferous tubules did not differentiate into germinal cells, but rather they behaved as invasive embryonal carcinoma (EC) stem cells. The vascular pattern of the experimental teratocarcinomas showed a highly disorganised architecture, and some of the neoplastic capillaries were derived, at least in part, from the original transplanted ES cells. CONCLUSION: The transplantation of pluripotent ES cells into seminiferous tubules efficiently recapitulates the early stages of development of teratocarcinomas. Consequently, this method constitutes a novel in vivo model to study the mechanisms of invasion and progression of experimental germinal tumours.

The antibody-mediated targeted delivery of interleukin-15 and GM-CSF to the tumor neovasculature inhibits tumor growth and metastasis.

Tumor-targeting immunocytokines represent a new class of anticancer pharmaceutical agents, which often display a superior therapeutic index compared with the corresponding unconjugated cytokines. In this article, we have studied the anticancer properties of interleukin-15 (IL-15) and granulocyte macrophage colony-stimulating factor (GM-CSF), fused to the human antibody fragment scFv(L19), specific to the EDB domain of fibronectin, a marker of angiogenesis. The immunocytokines L19-IL-15 and L19-GM-CSF were expressed in mammalian cells and purified to homogeneity, revealing no loss of cytokine activity in in vitro assays. Furthermore, the ability of the two immunocytokines to selectively localize to tumors in vivo was confirmed by biodistribution analysis with radioiodinated protein preparations. L19-IL-15 and L19-GM-CSF displayed a potent antitumor activity both in s.c. and in metastatic F9 and C51 murine models of cancer in immunocompetent mice. This therapeutic action was superior compared with IL-15-based and GM-CSF-based fusion proteins, containing antibodies of irrelevant specificity in the mouse, which were used as non-tumor-targeting controls. For both L19-IL-15 and L19-GM-CSF immunocytokines, CD8(+) T cells seemed to mostly contribute to the therapeutic action as shown by in vivo cell depletion experiments. The results presented in this article are of clinical significance, considering the fact that the sequence of EDB is identical in mouse and man and that the tumor-targeting ability of the L19 antibody has been extensively shown in clinical trials in patients with cancer.

A high-affinity human monoclonal antibody specific to the alternatively spliced EDA domain of fibronectin efficiently targets tumor neo-vasculature in vivo.

The alternatively spliced extra-domain B of fibronectin is one of the best characterized markers of tumor angiogenesis. Similarly, the extra-domain A (EDA), which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors, but this antigen has not been investigated so far as a target for antibody-based biomolecular intervention. We here describe the generation of 3 human monoclonal antibodies (named F8, B7 and D5), which recognize the same epitope of EDA, but which differ in terms of their dissociation constant to the human antigen (K(D) = 3.1, 16 and 17 nM, measured for monomeric preparations of the F8, B7 and D5 antibodies, respectively, in recombinant scFv format). When the 3 antibody fragments were cloned and expressed with a 5 amino acid linker, the 3 resulting homodimeric antibody preparations displayed comparable tumor: organ ratios in quantitative biodistribution studies, performed in immunocompetent 129SvEv mice, bearing subcutaneous syngeneic F9 murine tumors. The percent injected dose per gram (%ID/g) values in tumors 24 hr after intravenous injection were 9.3, 10.2 and 13 for F8, B7 and D5, respectively. The F8 antibody may serve as useful building block for the development of antibody-based targeted anti-cancer therapeutics. Preclinical and clinical investigations are facilitated by the fact that F8 recognizes the human and mouse antigen with comparable affinity, and by the observation that EDA over-expression is detectable not only in solid tumors, but also in hematological malignancies.

Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.

The oncofetal fibronectin (B-FN) isoform is present in vessels of neoplastic tissues during angiogenesis but not in mature vessels. B-FN could therefore provide a target for diagnostic imaging and therapy of cancer. Phage display libraries have been used to isolate human antibody fragments with pan-species recognition of this isoform. We describe the use of these fragments in nude mice to target an aggressive tumor (grafted F9 murine teratocarcinoma). Imaging in real time was done by infrared photodetection of a chemically coupled fluorophore. The targeting was improved by use of affinity-matured fragments with low kinetic dissociation rates (koff = 1.5 x 10(-4) s-1) and also by engineering dimeric fragments via a C-terminal amphipathic helix.

NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis.

BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.

Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis.

The formation of new blood vessels (angiogenesis) is an important step in tumor progression. Molecules capable of selectively targeting markers of angiogenesis may offer opportunities for the in vivo imaging of aggressive tumors and for the delivery of toxic agents to the tumoral vasculature. Using antibody phage display libraries and combinatorial mutagenesis, we isolated single-chain Fv antibody fragments, which recognize with different affinities the same epitope of the ED-B domain of fibronectin, a marker of angiogenesis. Two single-chain Fv fragments, E1 and L19, with dissociation constants of 41 nM and 0.054 nM, respectively, were investigated for their ability to target F9 murine teratocarcinoma grafted s.c. in nude mice when injected i.v. in either monomeric or homodimeric form (Mr 27,000 and 54,000, respectively). Biodistribution studies, performed at two time points (4 h and 24 h) with radiolabeled samples, showed that the higher affinity antibody targets the tumor significantly better than the lower affinity one, in terms both of tumor:organ ratios and of the amounts of antibody delivered to the tumor. In particular, more than 20% of the injected dose of dimeric L19 accumulated per gram of tumor at 4 h; the tumor:organ ratios at 4 h and 24 h were in the (2.1-8.6):1 and (10.3-29.4):1 range, respectively. This study demonstrates that, although vasculature represents only a small fraction of the total tumor mass, anti-ED-B antibodies can selectively target tumors in vivo and that this process is particularly efficient if very high-affinity binders are used.

Rescue of hypoxia-inducible factor-1alpha-deficient tumor growth by wild-type cells is independent of vascular endothelial growth factor.

In tumors, rapid cell proliferation associated with deficient vascularization leads to areas of hypoxia. Tumor hypoxia has direct consequences on clinical and prognostic parameters and is a potential therapeutic target. The hypoxic response depends critically on hypoxia-inducible factor-1alpha (HIF-1alpha) in pathological (e.g., tumorigenesis) as well as physiological (e.g., development and wound healing) processes. By s.c. injection of HIF-1alpha(-/-) embryonic stem (ES) cells in nude mice, we were able to demonstrate the role of HIF-1alpha in cell differentiation of teratocarcinomas. HIF-1alpha(+/+) tumors grow fast and preferentially form neuronal tissue, whereas HIF-1alpha(-/-) tumors show delayed growth and favorably form mesenchyme-derived tissue. Mixing wild-type and HIF-1alpha(-/-) ES cells in the same tumor at a ratio as low as 1:100, we showed that HIF-1alpha(+/+) cells can rescue the growth of mixed tumors although these tumors are not significantly different phenotypically or genotypically from the original HIF-1alpha(-/-) tumors. Interestingly, these results are not restricted to teratocarcinomas: they were confirmed with mixtures of Hepa1/Hepa1C4 cells (where HIF-1beta is mutated), demonstrating that growth changes are not related to differences in differentiation observed within teratocarcinomas. We also showed that despite lower mRNA expression, vascular endothelial growth factor protein status in HIF-1alpha(-/-) and mixed tumors does not significantly differ from the HIF-1alpha(+/+) tumors. Moreover, we demonstrated that tumor vascularization remains proportional to vascular endothelial growth factor protein levels, but that hypoxic up-regulation of this growth factor is not the decisive factor influencing tumor growth. Differences in levels of apoptosis are not responsible for alteration in growth because poly(ADP-ribose) polymerase cleavage, a hallmark of the apoptotic process, was similar in HIF-1alpha(+/+), HIF-1alpha(-/-), and mixed tumors. Our data demonstrate that the HIF-1alpha-dependent response of a few cells is capable of sustaining the growth of the whole tumor, probably through the secretion of factors up-regulated under low oxygen conditions.

Reduced blood vessel formation and tumor growth in alpha5-integrin-negative teratocarcinomas and embryoid bodies.

Embryonic stem (ES) cells-wild-type, heterozygous, or null for alpha5-integrin-were injected ectopically into syngeneic mice to develop teratocarcinomas. alpha5-null-derived teratocarcinomas were significantly smaller than the wild-type or alpha5 heterozygous tumors. Histological analysis revealed the presence of tissues derived from all three germ layers, in all tumors. However, alpha5-null teratocarcinomas displayed less undifferentiated tissue than did the controls. Decreased proliferation and increased apoptosis were observed in the undifferentiated areas of the alpha5-null teratocarcinomas. The expression of extracellular matrix proteins, fibronectin and tenascin-C, and the basement membrane components, laminin, entactin/nidogen, and collagen IV, was similar in the different tumors, although the deposition of these molecules was more disorganized in alpha5-null teratocarcinomas. The absence of alpha5-integrin in the various tissues of the alpha5-null tumors was confirmed by immunohistochemistry. Many vessels, but not all, stained positively for alpha5-integrin, showing that they were host derived. Analysis of the area occupied by vessels revealed, on average, an 8-fold decrease in alpha5-null teratocarcinomas compared with control tumors. Staining for smooth muscle alpha-actin showed that pericytes and smooth muscle cells were recruited around the vessels in all tumors, suggesting similar vessel differentiation. Deposition of EIIIA and EIIIB and fibronectin around the vessels was observed in all tumors. The fact that some, although few, alpha5-integrin-negative vessels existed in alpha5-null tumors indicated that alpha5-/- ES cells could differentiate into endothelial cells. Endothelial cell differentiation and vessel formation were analyzed also in vitro. alpha5-null ES cells were differentiated into embryoid bodies, although they were delayed in growth and attachment. Differentiation into endothelial cells was achieved, but the organization into a complex vasculature was delayed compared with controls. We conclude that alpha5beta1-integrin plays a significant role in vessel formation both in ES cell cultures and in teratocarcinomas. Reduced vascularization likely contributed to the reduced proliferation and increased apoptosis observed in alpha5-null teratocarcinomas.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

Soluble tissue factor induces coagulation on tumor endothelial cells in vivo if coadministered with low-dose lipopolysaccharides.

OBJECTIVE: This study was performed to evaluate the mechanisms leading to tumor vessel occlusion by tissue factor-based drugs, which are used in vascular targeting approaches for the treatment of malignant tumors. METHODS AND RESULTS: The effects of nontargeted soluble tissue factor were evaluated in vitro and in vivo. Tumor-bearing mice were treated with (1) the extracellular portion of tissue factor (soluble tissue factor), (2) low nontoxic doses of lipopolysaccharides, or (3) a combination thereof. The combination treatment showed the best effects and resulted in selective thrombosis of tumor vessels. On the basis of our data from subsequent in vitro analyses, including surface plasmon resonance measurements and endothelial cell based coagulation assays, we propose a model on how soluble tissue factor, although lacking its membrane anchor, can promote selective tumor vessel occlusion. CONCLUSIONS: To our knowledge, this is the first report to describe the molecular mechanisms of coagulation induction by untargeted soluble tissue factor in vivo. Combination treatments including soluble tissue factor might represent an alternative vascular targeting approach for the treatment of malignant tumors.

Extra cellular matrix derived metabolite regulates angiogenesis by FasL mediated apoptosis.

OBJECT: Antiangiogenic treatments are beginning to give promising outcomes in many vascular diseases including tumor angiogenesis. In this current study the antiangiogenic and pro-apoptotic actions of α1(IV)NC1 and its N- and C- peptides α1S1(IV)NC1, α1S2(IV)NC1 were investigated in-vitro and in-vivo. STUDY METHOD: Endothelial cells (ECs) were treated with α1(IV)NC1, α1S1(IV)NC1, α1S2(IV)NC1 and in-vitro proliferation, migration, tube formation and apoptotic assays were executed. FasL, Fas, Caspase-8, -3 and PARP activations were studied using immunoblotting analysis using specific antibodies. Also the in-vivo antiangiogenic and pro-apoptotic effects were tested using α1(IV)NC1 in a mice model. RESULTS: Like α1(IV)NC1, its N- and C- terminal α1S2(IV)NC1 and α1S1(IV)NC1 domains posses anti-proliferative, pro-apoptotic activity and inhibit ECs migration and tube formation in-vitro. Both α1S1(IV)NC1 and α1S2(IV)NC1 domains promote apoptosis by activating FasL and down stream apoptotic events including activation of caspase-8, -3 and PARP cleavage in a dose dependent manner in-vitro in ECs. Tumors in mice showed apoptotic TUNEL positive microvasculature upon α1(IV)NC1 treatment, indicating inhibition of tumor angiogenesis and tumor growth. Further, the antitumor activity of α1(IV)NC1 was abrogated when caspase-3 inhibitor was used. These results conform additional properties of α1(IV)NC1 as an endogenous angioinhibitor that induces apoptosis in-vitro and in-vivo by activating FasL mediated caspase-3. SIGNIFICANCE: α1(IV)NC1 and its N- and C- terminal α1S1(IV)NC1 and α1S2(IV)NC1 domains also posses pro-apoptotic and angioinhibitory activity in-vitro and in-vivo. α1(IV)NC1 regulates tumor angiogenesis by activating FasL mediated apoptosis in-vitro and in-vivo. These results demonstrate that α1(IV)NC1 and its peptides inhibit neo-vascular diseases.

Microvascular biodistribution of L19-SIP in angiogenesis targeting strategies.

INTRODUCTION: Various strategies using L19-mediated fibronectin targeting have become useful clinical tools in anti-tumour therapy and diagnostics. The aim of our study was to characterise the microvascular biodistribution and binding process during tumour angiogenesis and after anti-angiogenic therapy. MATERIALS AND METHODS: SF126 glioma and F9 teratocarcinoma cells were implanted into dorsal skin fold chambers (SF126: n = 4; F9: n = 6). Using fluorescence and confocal intravital microscopy the biodistribution process was assessed at t = 0 h, t = 4 h and t = 24 h after intravenous application of Cy3-L19-SIP. Sunitinib treatment was applied for six days and microscopy was performed 2 and 6 days after treatment initiation. Analysed parameters included: vascular and interstitial binding, preferential binding sites of L19-SIP, microvascular blood flow rate, microvascular permeability. Histological analysis included CD31 and DAPI. RESULTS: L19-SIP showed a specific and time-dependent neovascular binding with a secondary extravasation process reaching optimal vascular/interstitial binding ratio 4 hours after iv administration (F9: L19-SIP: vascular binding: 74.6 ± 14.5; interstitial binding: 46.8 ± 12.1; control vascular: 22,2 ± 16.6). Angiogenic sprouts were preferred binding sites (F9: L19-SIP: 188 ± 15.5; RTV: 90.6 ± 13.5). Anti-angiogenic therapy increased microvascular hemodynamics (SF126: Su: 106.6 ± 13.3 µl/sec; Untreated: 19.7 ± 9.1 µl/sec) and induced increased L19-SIP accumulation (SF 126: t24; Su: 92.6 ± 2.7; Untreated: 71.9 ± 5.9) in therapy resistant tumour vessels. CONCLUSION: L19-SIP shows a time and blood-flow dependent microvascular biodistribution process with angiogenic sprouts as preferential binding sites followed by secondary extravasation of the antibody. Microvascular biodistribution is enhanced in anti-angiogenic-therapy resistant tumour vessels.

Antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation.

The antibody-mediated targeted delivery of cytokines, growth factors and immunomodulators offers great potential for the therapy of cancer and other serious conditions. Interferon-alpha has long been used in the clinic for the treatment of patients with certain malignancies or with viral disease. Promising anticancer activity has recently been reported for two fusion proteins consisting of immunoglobulins bearing the interferon-alpha polypeptide at the C-terminal end of the molecule. Here we describe the design, production and characterization of a novel immunocytokine, in which murine interferon-alpha2 was sequentially fused with the tumor-targeting antibody fragment scFv(F8), specific to the alternatively-spliced EDA domain of fibronectin. The resulting fusion protein (F8-IFNa) could be produced to homogeneity and was shown to retain both antigen binding activity and interferon-alpha activity. Biodistribution studies in tumor-bearing mice with radioiodinated protein preparations confirmed the ability of F8-IFNa to selectively localize at the tumor site. However, using two different murine models of cancer (F9 teratocarcinomas and Cloudman S91 melanomas in immunocompetent mice), we could not detect a striking superiority for the therapeutic performance of F8-IFNa as compared to KSF-IFNa, a fusion protein of irrelevant specificity in the mouse which was used as negative control. In the paper, we present hypotheses why the antibody-based pharmacodelivery of interferon-alpha fails to eradicate tumors, in contrast to the situation observed by our group for other immunocytokines, which benefit from a selective localization at the tumor site.

Tumor cells secrete galectin-1 to enhance endothelial cell activity.

Tumor angiogenesis is a key event in cancer progression. Here, we report that tumors can stimulate tumor angiogenesis by secretion of galectin-1. Tumor growth and tumor angiogenesis of different tumor models are hampered in galectin-1-null (gal-1(-/-)) mice. However, tumor angiogenesis is less affected when tumor cells express and secrete high levels of galectin-1. Furthermore, tumor endothelial cells in gal-1(-/-) mice take up galectin-1 that is secreted by tumor cells. Uptake of galectin-1 by cultured endothelial cells specifically promotes H-Ras signaling to the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) kinase (Mek)/Erk cascade and stimulates endothelial cell proliferation and migration. Moreover, the activation can be blocked by galectin-1 inhibition as evidenced by hampered membrane translocation of H-Ras.GTP and impaired Raf/Mek/Erk phosphorylation after treatment with the galectin-1-targeting angiogenesis inhibitor anginex. Altogether, these data identify galectin-1 as a proangiogenic factor. These findings have direct implications for current efforts on galectin-1-targeted cancer therapies.

VEGF antibody plus cisplatin reduces angiogenesis and tumor growth in a xenograft model of ovarian cancer.

In our previous study, PAb, a VEGF polyclonal antibody was found to inhibit murine tumor growth significantly. The main objective of this study was to investigate the efficacy of combination therapy of (PAb) and cisplatin on human ovarian cancer xenograft. Effect of VEGF, PAb, PAb-cisplatin combination, and cisplatin alone on cultured human ovarian teratocarci-noma cell line PA-1 were assessed by measuring cell proliferation, matrigel invasion, MMP-9 expression, and MMP-9 secretion. In vivo, effect of PAb was observed in a xenograft model of ovarian cancer. Antitumor efficacy was monitored by assessment of tumor volume, MVD, serum NO, serum VEGF, and p53 expression. VEGF increased proliferation of PA-1 cell in a dose-dependent manner while addition of PAb inhibited cell proliferation, cell invasion as well as MMP-9 secretion in vitro. Tumor burden in PAb and PAb-cisplatin combination group was reduced by 41% (p < 0.05) and 66% (p < 0.01), respectively. A significant decrease in MVD, serum NO, serum VEGF, and p53 expression was also observed after PAb and PAb-cisplatin combination treatment when compared to normal mouse serum IgG-treated control mice. Thus, it was concluded that VEGF immunoneutralization may enhance cisplatin-in-duced apoptosis in human ovarian cancer and thus may be an effective way to reduce tumor growth in ovarian carcinoma.

Regulation of COX-2 mediated signaling by alpha3 type IV noncollagenous domain in tumor angiogenesis.

Human alpha3 chain, a noncollagenous domain of type IV collagen [alpha3(IV)NC1], inhibits angiogenesis and tumor growth. These biologic functions are partly attributed to the binding of alpha3(IV)NC1 to alphaVbeta3 and alpha3beta1 integrins. alpha3(IV)NC1 binds alphaVbeta3 integrin, leading to translation inhibition by inhibiting focal adhesion kinase/phosphatidylinositol 3-kinase/Akt/mTOR/4E-BP1 pathways. In the present study, we evaluated the role of alpha3beta1 and alphaVbeta3 integrins in tube formation and regulation of cyclooxygenase-2 (COX-2) on alpha3(IV)NC1 stimulation. We found that although both integrins were required for the inhibition of tube formation by alpha3(IV)NC1 in endothelial cells, only alpha3beta1 integrin was sufficient to regulate COX-2 in hypoxic endothelial cells. We show that binding of alpha3(IV)NC1 to alpha3beta1 integrin leads to inhibition of COX-2-mediated pro-angiogenic factors, vascular endothelial growth factor, and basic fibroblast growth factor by regulating IkappaBalpha/NFkappaB axis, and is independent of alphaVbeta3 integrin. Furthermore, beta3 integrin-null endothelial cells, when treated with alpha3(IV)NC1, inhibited hypoxia-mediated COX-2 expression, whereas COX-2 inhibition was not observed in alpha3 integrin-null endothelial cells, indicating that regulation of COX-2 by alpha3(IV)NC1 is mediated by integrin alpha3beta1. Our in vitro and in vivo findings demonstrate that alpha3beta1 integrin is critical for alpha3(IV)NC1-mediated inhibition of COX-2-dependent angiogenic signaling and inhibition of tumor progression.