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Antiangiogenic therapy for the treatment of pediatric solid malignancies
Antiangiogenic Therapy for the Treatment of Pediatric Solid Malignancies By Andrew M. Davidoff and Jessica J. Kandel Memphis, Tennessee and New York, New York Although the past 30 years have seen remarkable progress in the treatment of childhood malignancies, not all types of cancer have enjoyed this improvement in prognosis. Because of this, clinical trials are ongoing in which novel treatment approaches are being evaluated, including immunotherapy, radionuclide therapy, and the use of agents that induce tumor apoptosis or differentiation. Additional treatment strategies are needed, however. One such strategy involves the use of angiogenesis inhibitors. Angiogenesis is the biologic process of new blood vessel formation. In addition to occurring as part of several normal, physiologic processes, angiogenesis is an essential component of a number of pathologic conditions, including cancer. Compelling data suggest that inhibition of angiogenesis can not only prevent tumor-associated neovascularization but also affect tumor growth and spread. An anticancer approach in which the tumor-induced new blood vessels are targeted is particularly appealing for several reasons. First, despite the extreme molecular and phenotypic heterogeneity of human cancer, it is likely that most, if not all, tumor types require neovascularization to achieve their full malignant phenotype. Therefore, antiangiogenic therapy may have broad applicability for the treatment of human cancer, as well as the many other pathologic processes that depend on angiogenesis. Second, the endothelial cells, although rapidly proliferating, are inherently normal with a very low rate of mutation. They are, therefore, unlikely to evolve an angiogenesis inhibitor-insensitive phenotype. This is in distinction to the rapidly proliferating tumor cells that do undergo a high rate of spontaneous mutation and therefore can readily generate drug-resistant clones. This review discusses progress in the development of antiangiogenic therapy for the treatment of pediatric solid tumors. © 2004 Elsevier Inc. All rights reserved.
I
T IS WIDELY accepted that acquiring the capacity to induce a new blood supply is a crucial step in the development of malignant tumors. Prior to the development of new blood vessels, a tumor in a “prevascular phase” can grow to only a limited size, approximately 2 to 3 mm3. At this point, the rapid cell proliferation is balanced by equally rapid cell death by apoptosis, and a nonexpanding tumor mass results. The switch to an angiogenic phenotype with tumor neovascularization results in a decrease in the rate of tumor cell apoptosis, thereby shifting the balance to proliferation and tumor growth.1,2 This occurs, in part, because of the increased perfusion resulting from the new, expanded blood supply, which permits improved nutrient and metabolite exchange. In addition, the proliferating endothelium supplies, in a paracrine manner, a variety of factors that promote tumor growth.3 The process of metastasis also appears to be angiogenesis-dependent.2,4 This is likely due to several factors. Seminars in Pediatric Surgery, Vol 13, No 1 (February), 2004: pp 53-60
First, there will be increased opportunities for tumor shedding into the circulation as new blood vessels penetrating the primary tumor provide sites of entry into the circulation. Also, disruption of the basement membrane by proteases elaborated by the proliferating endothelial cells may contribute to the metastatic potential of a tumor.5,6 Finally, successful growth of these metastatic cells in foreign target organs also depends on the stimulation and formation of new blood vessels, perhaps even when cells metastasize to the bone marrow. Clinically, the number and density of new microvessels within primary tumor sites have been shown to correlate with the likelihood of metastasis as well as the overall prognosis for patients with neoplasms of a wide variety of histologies, including pediatric tumors such as neuroblastoma and Wilms tumor.7,8 Inhibition of the development of these new tumor-induced blood vessels (antiangiogenesis) has, therefore, attracted great interest as a potential approach to the treatment of cancer. However, it has become increasingly evident that the regulation of tumor angiogenesis is complex: new blood vessel formation occurs as the result of competing pro- and antiangiogenic signals, originating in multiple tissues.9 Thus, angiogenesis appears to be critically affected by the particular context of each tumor. Specific genetic events in certain cancers, such as altered status of the p53 tumor suppressor10,11 or human epidermal growth factor receptor genes,12-14 play a role in angiogenesis by modulating key signals (eg, upregulating vascular endothelial growth factor [VEGF], or downregulating the endogenous angiogenesis inhibitor thrombospondin-1). The organ in which tumorigenesis takes place, with its unique population of cells and matrix, provides another set of modifying signals.15,16 Taken together, these observations suggest that angiogenesis in specific tumor types From the Department of Surgery, St. Jude Children’s Research Hospital, Memphis, TN and the Division of Pediatric Surgery, Columbia University, New York, NY. This work was supported by grants from the Assisi Foundation of Memphis 94-000, grant no. IRG-87-008-09 from the American Cancer Society, Cancer Center Support CORE grant, P30 CA 21765, American Lebanese Syrian Associated Charities (ALSAC) (A.M.D.), and the Pediatric Cancer Foundation and the Sorkin Fund (J.J.K.). Address reprint requests to Andrew Davidoff, MD, Department of Surgery, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. © 2004 Elsevier Inc. All rights reserved. 1055-8586/04/1301-0008$30.00/0 doi:10.1053/j.sempedsurg.2003.09.008 53
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and organs is potentially subject to equally specific regulation. Thus, there is probably value in studying angiogenesis and its critical components, in experimental models that utilize the growth of human tumor xenografts in orthotopic locations in immunodeficient mice. STRATEGIES FOR DEVELOPMENT OF ANTIANGIOGENIC AGENTS
A variety of different strategies are currently being used for effecting angiogenesis inhibition, including antibodies against angiogenic factors or their receptors, or soluble, truncated forms of these receptors. In addition, a number of endogenous inhibitors of angiogenesis and small molecules are in clinical development for use in cancer therapy. Several important concepts have emerged regarding angiogenesis inhibition as an anticancer strategy. First, the traditional scheduling of conventional cytotoxic therapy, the administration of a maximum tolerated dose followed by a recovery period, may permit the microvascular cells in a tumor bed, with their slower rate of cell division, to recover and proliferate, thereby again providing neovasculature to support tumor regrowth.17 A metronomic schedule in which cytotoxic drugs are administered more frequently, or even continuously, without a treatment-free interval and at a lower total dose, thereby more effectively targeting the endothelial cells, may be more effective at controlling tumor progression, even if the tumor cells are resistant to the drug.17 This appears to be true both for cytotoxic drugs and antiangiogenic agents. In addition, toxicity with this schedule may be decreased. Further studies have demonstrated that synergistic antitumor efficacy between angiogenesis inhibitors and conventional chemotherapeutic agents can be achieved, especially when these cytotoxic drugs are delivered with the metronomic dosing schedule just described.18 Second, there appear to be important distinctions between “direct” and “indirect” angiogenesis inhibitors, particularly with regard to the potential for the development of therapy resistance.19 An indirect inhibitor blocks production of, or neutralizes or blocks the endothelial cell receptor for, a tumor-elaborated angiogenic factor. It is now becoming accepted that resistance to indirect inhibitors can occur with the emergence of tumor cell clones that upregulate the factors being interfered with or stimulate angiogenesis through alternative pathways. Direct inhibitors, however, block endothelial cell activation through mechanisms independent of tumor cell-elaborated angiogenic factors and are, therefore, less likely to induce resistance because they target genetically stable endothelial cells rather than unstable mutating tumor cells.20 Finally, there is a need to establish reliable methods for evaluating efficacy of these various angiogenic ap-
proaches. Similarly, serum surrogate markers for evaluating response to therapy would be of great value but are problematic to select. Although monitoring of basic fibroblast growth factor, for example, to assess the response of hemangiomas to interferon therapy has been utilized, most tumors elaborate a wide variety of angiogenic factors, each of which might contribute to tumor angiogenesis. Another surrogate serum marker currently being evaluated as a marker for tumor angiogenesis is the level of circulating endothelial cell precursors derived from the bone marrow.21 Finally, there is a significant amount of interest is using noninvasive radiologic modalities such as magnetic resonance imaging, positron emission tomography, and ultrasound for the evaluation of antiangiogenic cancer therapy. ANGIOGENESIS INHIBITORS
The first clinical demonstration that an angiogenesis inhibitor could cause regression of a tumor was the use of interferon-␣ in a patient treated for life-threatening pulmonary hemangiomas.22 An increasing number of natural and synthetic inhibitors of angiogenesis have been identified recently, with each having demonstrated antitumor effects in murine models of malignancy (see Table 1). Several of these agents have been or are being used in clinical trials (see Table 2). Variable but generally lowlevel toxicity has been observed during initial trials of these agents. Although none of these clinical trials has included infants or children with malignancies, pediatric tumors would also be expected to be amenable to treatment with an antiangiogenic approach. For example, clinical neuroblastoma typically grows rapidly, is richly vascularized, and metastasizes early, features that appear to be highly angiogenesis-dependent. One study has demonstrated that increased vessel density within primary, untreated sites of neuroblastoma strongly correlated with widely disseminated disease and poor survival,7 while another study has shown that high-level expression of angiogenic factors is associated with advanced tumor stage in neuroblastoma.23 Similarly, tumor vascularity has been associated with relapsed, clinically aggressive Wilms tumors.8 Because of the evidence that growth of these tumors is angiogenesis-dependent, they are, therefore, predicted to be susceptible to antiangiogenic therapy. Consistent with this prediction, experimental neuroblastoma, Wilms tumor, and hepatoblastoma have been shown, in different mouse models, to be susceptible to the effects of a variety angiogenesis inhibitors, including inhibitors of the VEGF signaling pathway,24-27 antiangiogenically scheduled cytotoxic agents, either alone or in combination with VEGF blockade,18,28-30 pigment epithelium-derived factor31 and TNP-470 (a fumagillin derivative).17,32,33
ANTIANGIOGENIC THERAPY
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Table 1. Endogenous Inhibitors of Angiogenesis Name
Thrombospondin-1 and internal large, modular of thrombospondin-1 Angiostatin Endostatin Vasotatin Vascular endothelial growth factor inhibitor Fragment of platelet factor-4 Derivative of prolactin Restin Proliferin-related protein SPARC cleavage product Interferon ␣/ METH-1 and METH-2
Angiopoietin-2 Antithrombin III fragment Interferon-inducible protein-10
Description
Ref.
(180 kD) Extracellular matrix protein
47
38-kD fragment of plasminogen involving either kringle domains 1-3 or smaller kringle 5 fragments 20-kD zinc-binding fragment of type XVIII collagen NH2 terminal fragment (amino acids I-80) of calreticulin 174-amino acid protein with 20-30% homology to tumor necrosis factor superfamily N-terminal fragment of platelet factor-4 16-kD fragment of prolactin NC10 domain of human collagen XV Protein related to the proangiogenic molecule proliferin Fragments of secreted protein, acidic and rich in cystine Well-known antiviral proteins, may downregulate angiogenic factor expression Proteins containing metalloprotease and thrombospondin domains, and distintegrin domains in NH2 termini Antagonist of angiopoietin-1 that binds to Tie-2 receptor A fragment missing the carboxy-terminal loop of antithrombin III (a member of the serpin family) Upregulated by IFN-␥ and whose mechanism of antiangiogenic effect is unknown
48 49 50 51 52 53 54 55 56 57 58
59 60 61
From RS Kerbel, Tumor angiogenesis: past, present and the near future. Carcinogenesis 2000;21:505, adapted with permission.
TARGET SELECTION IN TUMOR ANGIOGENESIS AND VEGF
New blood vessel growth depends on the expression of proangiogenic cytokines, including the fibroblast growth factor, VEGF, ephrin, platelet-derived growth factor, and angiopoietin families. Of these, VEGF has received the most attention. It promotes endothelial cell proliferation, survival, vessel permeability, and appears to be nearly ubiquitous in human tumors. Thus, VEGF has been an attractive target for novel antiangiogenic strategies. This is reflected in the wide variety of agents that have been developed that target VEGF signaling. More recently, clinical testing appears to validate the choice of VEGF antagonism as a therapy in some adult cancers.34 Given the apparent central role for VEGF in human tumor angiogenesis, it is not surprising that agents that block VEGF function appear to be broadly effective in preventing growth of implanted tumors in a variety of experimental models. Yet there are clearly differences in responsiveness to anti-VEGF agents in individual tumor types, which likely reflect the influence of tumor-specific regulatory elements on vessel networks. Thus, VEGF antagonism may serve as a prototype for the development of antiangiogenic strategies that focus on specific cytokine targets in pediatric solid tumors. Investigators will need to determine both the relative efficacy of dif-
ferent blockade strategies and their specific utility in individual tumor types. APPROACHES TO VEGF BLOCKADE IN PEDIATRIC TUMOR MODELS
Development of a monoclonal humanized anti-VEGF antibody was first reported to be effective in blocking tumor growth and angiogenesis in murine models of human rhabdomyosarcoma and glioblastoma multiforme.35 The same antibody was subsequently demonstrated to inhibit development of vasculature and tumor progression in mice with intraabdominal implants of neuroblastoma, Wilms tumor, and hepatoblastoma,26,27,36 and metastases in Wilms tumor.27 In addition, selective inhibition of the VEGF165 isoform, which has been demonstrated to be principally responsible for stimulating tumor neoangiogenesis, was shown to have similar efficacy in Wilms tumor.37 Activation of the VEGFR2 receptor is critical for the proangiogenic effects of VEGF. Blockade of this receptor, using a specific antibody, also effectively inhibits tumor progression and angiogenesis in a murine model of pediatric leukemia.38 The combination of VEGF blockade with antiangiogenically scheduled cytotoxic drugs has demonstrated increased efficacy in pediatric solid tumor models. Klement et al reported that use of an anti-VEGF receptor antibody combined with antiangiogenically scheduled
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Table 2. Angiogenesis Inhibitors in Clinical Trials Drug
BMS-275291 Dalteparin (Fragmin) Suramin 2-methoxyestradiol (2-ME) CC-5013 (thalidomide analog)
LY317615 (protein kinase C  inhibitor)
Soy isoflavone (soy protein isolate) Thalidomide AE-941 (Neovastat) Anti-VEGF antibody (Bevacizumab; Avastin) Interferon-␣ VEGF-Trap ZD647 EMD 121974 Medi-522 (Vitaxin) Carboxyamidotriazole (CAI) Celecoxib (Celebrex) Halofuginone Hydrobromide (Tempostatin) Interleukin-12 Rofecoxib (VIOXX) CA
Phase
II II III I I I I II/III I II II II I-III III I-III I-III I I II II I I/II I I/II I II I-III I I-III III
Tumor Type
Prostate cancer Gliomablastoma multiforme Unresectable or metastatic pancreatic cancer Bladder cancer Advanced solid tumors Gliomas Refractory solid tumor and/or lymphomas Relapsed metastatic malignant melanoma Advanced solid tumors High-grade gliomas Refractory large B-cell lymphoma Breast cancer Multiple tumor types Non-small cell lung cancer Multiple tumor types Multiple tumor types Refractory solid tumors or non-Hodgkin’s lymphomas Refractory solid tumors or non-lymphomas-Hodgkin’s Metastatic non-small cell lung cancer Relapsed multiple myeloma Advanced solid tumors Recurrent malignant gliomas Advanced solid tumors or lymphoma Advanced colorectal cancer Advanced solid tumors or refractory lymphomas Recurrent ovarian epithelial, fallopian tube primary peritoneal Localized prostate cancer Progressive advanced solid tumors Multiple tumor types Previously resected stage I or III colorectal
Adapted from http://www.cancer.gov/clinicaltrials/development/anti-angio-table.
cyclophosphamide improved the durability of the tumor suppressive response in experimental neuroblastoma.18 Similarly, the addition of antiangiogenically scheduled topotecan to anti-VEGF antibody improved efficacy in experimental Wilms tumor.29 These encouraging results must be considered in context with the observation that experimental neuroblastoma tumors recur during long-term antagonism with single-agent, anti-VEGFR2 antibody alone.18 It is possible that more potent agents, such as those that exploit the affinity of the VEGFR1 and VEGFR2 receptors for VEGF ligand, may improve results. For example, a novel agent based on the soluble portion of the VEGFR1 receptor completely inhibited rhabdomyosarcoma xenograft growth,39 whereas anti-VEGF antibody alone did not. The authors speculated that enhanced binding of stromal-cell derived VEGF, which contributes to stability of vasculature at very low levels, contributed to this differential response. Similarly, co-opted host vasculature can partly rescue tumor growth in experimental neuroblastoma when VEGF signaling is blocked by antiVEGF antibody. However, these host vessels can be destabilized and tumor growth more completely inhib-
ited when a high-affinity agent based on portions of the VEGFR1 and VEGFR2 receptor is used.25 LONG-TERM DELIVERY OF VEGF ANTAGONISTS: GENE THERAPY APPROACHES
A limitation of the above studies is that most focus on the effects of VEGF antagonism during a limited time frame. Long-term delivery of angiogenesis inhibitors is likely to be important, because treated tumor cells, although small and dormant, may maintain their capacity for growth, invasion, and metastasis if released from the restrictions of angiogenesis inhibition. An alternative to chronic administration of recombinant proteins or peptides is the use of a gene therapy approach in which genetic material encoding the therapeutic protein is transferred to a host target cell where it mediates longterm protein expression. This approach to the delivery of angiogenesis inhibitors is attractive for several reasons. (1) Long-term expression of an inhibitor could potentially be achieved following a single intervention or administration. (2) Difficulties with protein production and maintenance of function, especially when “scaling up” for clinical trials
ANTIANGIOGENIC THERAPY
may be avoided by in situ expression in host tissues. (3) Continuous, low-level expression of these proteins, as would be generated from gene-modified cells, may be the optimal delivery schedule. (4) Several angiogenic pathways can be targeted with a single vector through the delivery of multiple genes. (5) Potential side effects of antiangiogenic therapy could be avoided by either limited local expression or regulatable, intermittent, systemic expression of an inhibitor, both of which can be achieved through gene therapy mediated approaches. Gene therapy strategies for tumor antiangiogenesis are, in fact, already being tested in a number of different murine tumor models, with some success. However, most of these studies have used either retroviral vector producer cells, naked DNA, or adenoviral vectors.40 Unfortunately, retroviral vector producers may be impractical for human use and the transfer of naked DNA is typically an inefficient, transient process; adenoviralmediated gene transfer is also complicated by transient transgene expression as well as a host immune response to transduced target cells. Adeno-associated virus (AAV) is a nonpathogenic, helper-dependent member of the parvovirus family. A number of properties make AAVbased vectors promising for antiangiogenic gene therapy. Most importantly, unlike the other gene delivery systems just mentioned, recombinant AAV (rAAV) vectors have been shown to direct long-term transgene expression from nondividing cells. In addition, these vectors have an excellent safety profile. Also unlike other vectors of viral origin, AAV has never been associated with any human disease and is naturally replication-deficient, thereby providing an added measure of safety. In addition, rAAV is nonimmunogenic; the wild type viral genes have been removed, thus reducing the potential for invoking a cellmediated immune response due to the expression of foreign viral proteins. Over the past several years, different gene therapy approaches have been explored, utilizing different vectors and targets, for the delivery of angiogenesis inhibitors. One set of initial studies involved the in vitro transduction of tumor cells using retroviral vectors encoding a truncated, soluble form of the VEGF receptor-2 (tsFlk-1, a competitive inhibitor of VEGF). By targeting the tumor cells in an ex vivo setting, efficient transduction could be achieved with resultant high levels of transgene expression at all tumor sites in vivo following gene-modified tumor cell inoculation. Consistent with these predictions, restricted growth and angiogenesis was of experimental neuroblastoma were observed in tsFlk-1– expressing tumors,41 with measurable decreases in endothelial cell density. Ultimately, however ex vivo gene modification of tumor cells as part of an antiangiogenic strategy is clinically impractical. Therefore, it is rational to consider the
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possibility of in situ tumor cell transduction using retroviral vector-producer cell-mediated in vivo gene transfer to tumor cells. Investigators have reported that in situ tumor cell transduction could be efficiently performed and following transfer of the cDNA for tsFlk-1, significant restriction of tumor-induced angiogenesis and localized neuroblastoma growth resulted.42 More often, however, children who succumb to pediatric tumors have widespread, metastatic disease. Neuroblastoma, for example, is a systemic disease with multiple overt and occult sites of tumor, requiring systemic therapy. Therefore, a mechanism for long-term, systemic delivery of these angiogenesis inhibitors is required. One approach consistent with this requirement is to use retroviral vectors to modify murine bone marrow-derived cells, because these cells are among the most accessible, transducible, self-renewing cells. In addition, it is possible that, because some endothelial cells incorporated into tumor-induced new blood vessels appear to be derived from precursor cells of bone marrow origin,43 modifying these cells to express an inhibitor of endothelial cell activation might prevent endothelial cell precursor differentiation and/or create a milieu of angiogenesis inhibition at the sites of tumor neovascularization. Neuroblastoma growth, in mice challenged 3 months following transplantation with tsFlk-1-expressing bone marrow cells, was significantly inhibited when compared to tumor growth in controls.44 In addition, immunohistochemical analysis of tumors in each group demonstrated colocalization of vector-encoded Green Fluorescent Protein expression in cells immunopositive for endothelial cell markers, confirming that the endothelium of the tumorinduced neovasculature were derived, at least in part, from bone marrow precursors.44 These results confirmed that long-term expression of a functional angiogenesis inhibitor could be generated through gene-modified bone marrow-derived stem cells, and that this approach could have significant anticancer efficacy. Other gene therapy approaches using tsFlk-1 have been used. One alternative strategy has ultilized recombinant adeno-associated virus vectors to effect liver transduction in situ to establish long-term expression and systemic delivery of this angiogenesis inhibitor to provide the same antiangiogenic and antitumor efficacy. Following intraportal injection of rAAV tsFlk-1, highlevel, stable transgene expression was generated in mice peaking 3 to 5 weeks following vector administration. This established a systemic state of angiogenesis inhibition; sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug neovascularization in vivo.45 Significant antitumor efficacy was observed in two murine models of pediatric kidney tumors.45 Tumor development was prevented in a majority of the mice, with significant growth restriction of tumors in the re-
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maining mice. Thus, long-term, in vivo expression of a functional angiogenesis inhibitor was established using rAAV, with resultant anticancer efficacy in a relevant, orthotopic tumor model. This study was the first to demonstrate the feasibility of using liver-targeted rAAV vectors in antiangiogenic gene therapy. Next, this antitumor strategy was tested in a murine model of neuroblastoma, a tumor known to be more aggressive and to elaborate a spectrum of proangiogenic factors.23 Despite the capacity of neuroblastoma to activate different angiogenic pathways, monotherapy with rAAV-mediated delivery of tsFlk-1, as with the renal tumor models, had significant efficacy against localized and metastatic neuroblastoma. Localized retroperitoneal tumors were significantly smaller in tsFlk-1-expressing mice and survival for mice with metastatic disease was greater (AM Davidoff, unpublished data). In an effort to improve antineuroblastoma efficacy, larger doses of the vector were administered to cohorts of mice. Although enzyme-linked immunosorbent assay confirmed that higher levels of tsFlk-1 were generated in these mice, antitumor efficacy was not improved. In fact, paradoxically, the tumors were more aggressive than in control mice. Further investigation revealed that mice expressing the higher levels of tsFlk-1 also had significantly higher levels of circulating mouse VEGF (mVEGF) than those mice expressing lower but more effective levels of tsFlk-1; these mice were expressing levels of VEGF similar to control mice. An additional surprising finding was that the tumor cells were not solely responsible for this apparent compensatory increase in VEGF expression, but that host cells were contributing to the higher circulating levels of VEGF. Binding of mVEGF
by tsFlk-1 appeared to influence mVEGF bioavailability. Although a greater amount of mVEGF was bound in high tsFlk-1 expressing mice, more mVEGF was also unbound and available to support angiogenesis. This novel resistance mechanism impaired the effectiveness of angiogenesis inhibition at higher doses of the inhibitor. These results suggest that careful titering of angiogenesis inhibitors, such as tsFlk-1, may be required to achieve maximal antitumor efficacy and avoid therapy resistance mediated, in part, by affecting ligand bioavailability. This has important implications for therapeutic strategies that use decoy receptors and other agents, such as antibodies, to bind angiogenic factors, in an attempt to inihibit tumor neovascularization. CONCLUSIONS
These results suggest that antitumor strategies that focus on proangiogenic cytokines in general, and VEGF in particular, hold promise for the treatment of children with aggressive malignancies. However, several issues remain to be addressed. First, the potential for the development of resistance must be evaluated, especially when chronic VEGF antagonism is used as monotherapy. This may involve several mechanisms, including co-option of host vasculature, as documented in murine models of glioblastoma and neuroblastoma,25,46 or the increased expression of VEGF by either tumor cells or host tissues. Second, the potential consequences of long-term angiogenesis inhibition must be defined, especially when considering administering this therapy to growing children. Finally, synergy between angiogenesis inhibitors and other treatment modalities should be explored.
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kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: Angiogenic implications for signal transduction therapy of solid tumors. Am J Pathol 151:1523-1530, 1997 15. Essler M, Ruoslahti E: Molecular specialization of breast vasculature: A breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc Natl Acad Sci USA 99:2252-2257, 2002 16. Ruoslahti E, Rajotte D: An address system in the vasculature of normal tissues and tumors. Annu Rev Immunol 18:813-827, 2000 17. Browder T, Butterfield CE, Kraling BM, et al: Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878-1886, 2000 18. Klement G, Baruchel S, Rak J, et al: Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105:R15R24, 2000 19. Folkman J, Browder T, Palmblad J: Angiogenesis research: guidelines for translation to clinical application. Thromb Haemost 86:23-33, 2001 20. Kerbel RS, Yu J, Tran J, et al: Possible mechanisms of acquired resistance to anti-angiogenic drugs: Implications for the use of combination therapy approaches. Cancer Metastasis Rev 20:79-86, 2001 21. Celletti FL, Waugh JM, Amabile PG, et al: Vascular endothelial growth factor enhances atherosclerotic plaque progression. Nat Med 7:425-429, 2001 22. Ezekowitz RA, Mulliken JB, Folkman J: Interferon alfa-2a therapy for life-threatening hemangiomas of infancy. N Engl J Med 326:1456-1463, 1992 23. Eggert A, Ikegaki N, Kwiatkowski J, et al: High-level expression of angiogenic factors is associated with advanced tumor stage in human neuroblastomas. Clin Cancer Res 6:1900-1908, 2000 24. Davidoff AM, Leary MA, Ng CY, et al: Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo. J Pediatr Surg 36:30-36, 2001 25. Kim ES, Serur A, Huang J, et al: Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma. Proc Natl Acad Sci USA 99:11399-11404, 2002 26. McCrudden KW, Hopkins B, Frischer J, et al: Anti-VEGF antibody in experimental hepatoblastoma: Suppression of tumor growth and altered angiogenesis. J Pediatr Surg 38:308-314, 2003 27. Rowe DH, Huang J, Kayton ML, et al: Anti-VEGF antibody suppresses primary tumor growth and metastasis in an experimental model of Wilms’ tumor. J Pediatr Surg 35:30-32, 2000 28. McCrudden KW, Yokoi A, Thosani A, et al: Topotecan is anti-angiogenic in experimental hepatoblastoma. J Pediatr Surg 37: 857-861, 2002 29. Soffer SZ, Moore JT, Kim E, et al: Combination antiangiogenic therapy: increased efficacy in a murine model of Wilms tumor. J Pediatr Surg 36:1177-1181, 2001 30. Soffer SZ, Kim E, Moore JT, et al: Novel use of an established agent: Topotecan is anti-angiogenic in experimental Wilms tumor. J Pediatr Surg 36:1781-1784, 2001 31. Abramson LP, Stellmach V, Doll JA, et al: Wilms’ tumor growth is suppressed by antiangiogenic pigment epithelium-derived factor in a xenograft model. J Pediatr Surg 38:336-342, 2003 32. Matsusaka S, Nakasho K, Terada N, et al: Inhibition by an angiogenesis inhibitor, TNP-470, of the growth of a human hepatoblastoma heterotransplanted into nude mice. J Pediatr Surg 35:11981204, 2000 33. Nagabuchi E, VanderKolk WE, Une Y, et al: TNP-470 antian-
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giogenic therapy for advanced murine neuroblastoma. J Pediatr Surg 32:287-293, 1997 34. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al: Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 21:60-65, 2003 35. Kim KJ, Li B, Winer J, et al: Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 362:841-844, 1993 36. Rowe DH, Huang J, Li J, et al: Suppression of primary tumor growth in a mouse model of human neuroblastoma. J Pediatr Surg 35:977-981, 2000 37. Huang J, Moore J, Soffer S, et al: Highly specific antiangiogenic therapy is effective in suppressing growth of experimental Wilms tumors. J Pediatr Surg 36:357-361, 2001 38. Zhu Z, Hattori K, Zhang H, et al: Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity. Leukemia 17:604-611, 2003 39. Gerber HP, Kowalski J, Sherman D, et al: Complete inhibition of rhabdomyosarcoma xenograft growth and neovascularization requires blockade of both tumor and host vascular endothelial growth factor. Cancer Res 60:6253-6258, 2000 40. Feldman AL, Libutti SK: Progress in antiangiogenic gene therapy of cancer. Cancer 89:1181-1194, 2000 41. Davidoff AM, Leary MA, Ng CY, et al: Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo. J Pediatr Surg 36:30-36, 2001 42. Davidoff AM, Leary MA, Ng CY, et al: Retroviral vectorproducer cell mediated angiogenesis inhibition restricts neuroblastoma growth in vivo. Med Pediatr Oncol 35:638-640, 2000 43. Asahara T, Masuda H, Takahashi T, et al: Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85:221228, 1999 44. Davidoff AM, Ng CY, Brown P, et al: Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express an angiogenesis inhibitor, can restrict tumor growth in mice. Clin Cancer Res 7:2870-2879, 2001 45. Davidoff AM, Nathwani AC, Spurbeck WW, et al: rAAVmediated long-term liver-generated expression of an angiogenesis inhibitor can restrict renal tumor growth in mice. Cancer Res 62:30773083, 2002 46. Holash J, Maisonpierre PC, Compton D, et al: Vessel cooption, regression, and growth in tumors mediated by angiopoietins and VEGF. Science 284:1994-1998, 1999 47. Tolsma SS, Volpert OV, Good DJ, et al: Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J Cell Biol 122:497-511, 1993 48. O’Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315-328, 1994 49. O’Reilly MS, Boehm T, Shing Y, et al: Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277-285, 1997 50. Pike SE, Yao L, Jones KD, et al: Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth. J Exp Med 188:2349-2356, 1998 51. Zhai Y, Ni J, Jiang GW, et al: VEGI, a novel cytokine of the tumor necrosis factor family, is an angiogenesis inhibitor that suppresses the growth of colon carcinomas in vivo. FASEB J 13:181-189, 1999
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52. Gupta SK, Hassel T, Singh JP: A potent inhibitor of endothelial cell proliferation is generated by proteolytic cleavage of the chemokine platelet factor 4. Proc Natl Acad Sci USA 92:7799-7803, 1995 53. Clapp C, Martial JA, Guzman RC, et al: The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis. Endocrinology 133:1292-1299, 1993 54. Ramchandran R, Dhanabal M, Volk R, et al: Antiangiogenic activity of restin, NC10 domain of human collagen XV: Comparison to endostatin. Biochem Biophys Res Commun 255:735-739, 1999 55. Jackson D, Volpert OV, Bouck N, et al: Stimulation and inhibition of angiogenesis by placental proliferin and proliferin-related protein. Science 266:1581-1584, 1994 56. Hasselaar P, Sage EH: SPARC antagonizes the effect of basic fibroblast growth factor on the migration of bovine aortic endothelial cells. J Cell Biochem 49:272-283, 1992
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57. Sidky YA, Borden EC: Inhibition of angiogenesis by interferons: effects on tumor- and lymphocyte-induced vascular responses. Cancer Res 47:5155-5161, 1987 58. Sage EH: Pieces of eight: Bioactive fragments of extracellular proteins as regulators of angiogenesis. Trends Biol Sci 7:182, 1999 59. Maisonpierre PC, Suri C, Jones PF, et al: Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277:55-60, 1997 60. O’Reilly MS, Pirie-Shepherd S, Lane WS, et al: Antiangiogenic activity of the cleaved conformation of the Serpin Antithrombin III. Science 285:1926-1928, 1999 61. Strieter RM, Kunkel SL, Arenberg DA, et al: Interferon gammainducible protein 10 (IP-10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis. Biochem Biophys Res Commun. 210:51-57, 1997
I
T IS WIDELY accepted that acquiring the capacity to induce a new blood supply is a crucial step in the development of malignant tumors. Prior to the development of new blood vessels, a tumor in a “prevascular phase” can grow to only a limited size, approximately 2 to 3 mm3. At this point, the rapid cell proliferation is balanced by equally rapid cell death by apoptosis, and a nonexpanding tumor mass results. The switch to an angiogenic phenotype with tumor neovascularization results in a decrease in the rate of tumor cell apoptosis, thereby shifting the balance to proliferation and tumor growth.1,2 This occurs, in part, because of the increased perfusion resulting from the new, expanded blood supply, which permits improved nutrient and metabolite exchange. In addition, the proliferating endothelium supplies, in a paracrine manner, a variety of factors that promote tumor growth.3 The process of metastasis also appears to be angiogenesis-dependent.2,4 This is likely due to several factors. Seminars in Pediatric Surgery, Vol 13, No 1 (February), 2004: pp 53-60
First, there will be increased opportunities for tumor shedding into the circulation as new blood vessels penetrating the primary tumor provide sites of entry into the circulation. Also, disruption of the basement membrane by proteases elaborated by the proliferating endothelial cells may contribute to the metastatic potential of a tumor.5,6 Finally, successful growth of these metastatic cells in foreign target organs also depends on the stimulation and formation of new blood vessels, perhaps even when cells metastasize to the bone marrow. Clinically, the number and density of new microvessels within primary tumor sites have been shown to correlate with the likelihood of metastasis as well as the overall prognosis for patients with neoplasms of a wide variety of histologies, including pediatric tumors such as neuroblastoma and Wilms tumor.7,8 Inhibition of the development of these new tumor-induced blood vessels (antiangiogenesis) has, therefore, attracted great interest as a potential approach to the treatment of cancer. However, it has become increasingly evident that the regulation of tumor angiogenesis is complex: new blood vessel formation occurs as the result of competing pro- and antiangiogenic signals, originating in multiple tissues.9 Thus, angiogenesis appears to be critically affected by the particular context of each tumor. Specific genetic events in certain cancers, such as altered status of the p53 tumor suppressor10,11 or human epidermal growth factor receptor genes,12-14 play a role in angiogenesis by modulating key signals (eg, upregulating vascular endothelial growth factor [VEGF], or downregulating the endogenous angiogenesis inhibitor thrombospondin-1). The organ in which tumorigenesis takes place, with its unique population of cells and matrix, provides another set of modifying signals.15,16 Taken together, these observations suggest that angiogenesis in specific tumor types From the Department of Surgery, St. Jude Children’s Research Hospital, Memphis, TN and the Division of Pediatric Surgery, Columbia University, New York, NY. This work was supported by grants from the Assisi Foundation of Memphis 94-000, grant no. IRG-87-008-09 from the American Cancer Society, Cancer Center Support CORE grant, P30 CA 21765, American Lebanese Syrian Associated Charities (ALSAC) (A.M.D.), and the Pediatric Cancer Foundation and the Sorkin Fund (J.J.K.). Address reprint requests to Andrew Davidoff, MD, Department of Surgery, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. © 2004 Elsevier Inc. All rights reserved. 1055-8586/04/1301-0008$30.00/0 doi:10.1053/j.sempedsurg.2003.09.008 53
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and organs is potentially subject to equally specific regulation. Thus, there is probably value in studying angiogenesis and its critical components, in experimental models that utilize the growth of human tumor xenografts in orthotopic locations in immunodeficient mice. STRATEGIES FOR DEVELOPMENT OF ANTIANGIOGENIC AGENTS
A variety of different strategies are currently being used for effecting angiogenesis inhibition, including antibodies against angiogenic factors or their receptors, or soluble, truncated forms of these receptors. In addition, a number of endogenous inhibitors of angiogenesis and small molecules are in clinical development for use in cancer therapy. Several important concepts have emerged regarding angiogenesis inhibition as an anticancer strategy. First, the traditional scheduling of conventional cytotoxic therapy, the administration of a maximum tolerated dose followed by a recovery period, may permit the microvascular cells in a tumor bed, with their slower rate of cell division, to recover and proliferate, thereby again providing neovasculature to support tumor regrowth.17 A metronomic schedule in which cytotoxic drugs are administered more frequently, or even continuously, without a treatment-free interval and at a lower total dose, thereby more effectively targeting the endothelial cells, may be more effective at controlling tumor progression, even if the tumor cells are resistant to the drug.17 This appears to be true both for cytotoxic drugs and antiangiogenic agents. In addition, toxicity with this schedule may be decreased. Further studies have demonstrated that synergistic antitumor efficacy between angiogenesis inhibitors and conventional chemotherapeutic agents can be achieved, especially when these cytotoxic drugs are delivered with the metronomic dosing schedule just described.18 Second, there appear to be important distinctions between “direct” and “indirect” angiogenesis inhibitors, particularly with regard to the potential for the development of therapy resistance.19 An indirect inhibitor blocks production of, or neutralizes or blocks the endothelial cell receptor for, a tumor-elaborated angiogenic factor. It is now becoming accepted that resistance to indirect inhibitors can occur with the emergence of tumor cell clones that upregulate the factors being interfered with or stimulate angiogenesis through alternative pathways. Direct inhibitors, however, block endothelial cell activation through mechanisms independent of tumor cell-elaborated angiogenic factors and are, therefore, less likely to induce resistance because they target genetically stable endothelial cells rather than unstable mutating tumor cells.20 Finally, there is a need to establish reliable methods for evaluating efficacy of these various angiogenic ap-
proaches. Similarly, serum surrogate markers for evaluating response to therapy would be of great value but are problematic to select. Although monitoring of basic fibroblast growth factor, for example, to assess the response of hemangiomas to interferon therapy has been utilized, most tumors elaborate a wide variety of angiogenic factors, each of which might contribute to tumor angiogenesis. Another surrogate serum marker currently being evaluated as a marker for tumor angiogenesis is the level of circulating endothelial cell precursors derived from the bone marrow.21 Finally, there is a significant amount of interest is using noninvasive radiologic modalities such as magnetic resonance imaging, positron emission tomography, and ultrasound for the evaluation of antiangiogenic cancer therapy. ANGIOGENESIS INHIBITORS
The first clinical demonstration that an angiogenesis inhibitor could cause regression of a tumor was the use of interferon-␣ in a patient treated for life-threatening pulmonary hemangiomas.22 An increasing number of natural and synthetic inhibitors of angiogenesis have been identified recently, with each having demonstrated antitumor effects in murine models of malignancy (see Table 1). Several of these agents have been or are being used in clinical trials (see Table 2). Variable but generally lowlevel toxicity has been observed during initial trials of these agents. Although none of these clinical trials has included infants or children with malignancies, pediatric tumors would also be expected to be amenable to treatment with an antiangiogenic approach. For example, clinical neuroblastoma typically grows rapidly, is richly vascularized, and metastasizes early, features that appear to be highly angiogenesis-dependent. One study has demonstrated that increased vessel density within primary, untreated sites of neuroblastoma strongly correlated with widely disseminated disease and poor survival,7 while another study has shown that high-level expression of angiogenic factors is associated with advanced tumor stage in neuroblastoma.23 Similarly, tumor vascularity has been associated with relapsed, clinically aggressive Wilms tumors.8 Because of the evidence that growth of these tumors is angiogenesis-dependent, they are, therefore, predicted to be susceptible to antiangiogenic therapy. Consistent with this prediction, experimental neuroblastoma, Wilms tumor, and hepatoblastoma have been shown, in different mouse models, to be susceptible to the effects of a variety angiogenesis inhibitors, including inhibitors of the VEGF signaling pathway,24-27 antiangiogenically scheduled cytotoxic agents, either alone or in combination with VEGF blockade,18,28-30 pigment epithelium-derived factor31 and TNP-470 (a fumagillin derivative).17,32,33
ANTIANGIOGENIC THERAPY
55
Table 1. Endogenous Inhibitors of Angiogenesis Name
Thrombospondin-1 and internal large, modular of thrombospondin-1 Angiostatin Endostatin Vasotatin Vascular endothelial growth factor inhibitor Fragment of platelet factor-4 Derivative of prolactin Restin Proliferin-related protein SPARC cleavage product Interferon ␣/ METH-1 and METH-2
Angiopoietin-2 Antithrombin III fragment Interferon-inducible protein-10
Description
Ref.
(180 kD) Extracellular matrix protein
47
38-kD fragment of plasminogen involving either kringle domains 1-3 or smaller kringle 5 fragments 20-kD zinc-binding fragment of type XVIII collagen NH2 terminal fragment (amino acids I-80) of calreticulin 174-amino acid protein with 20-30% homology to tumor necrosis factor superfamily N-terminal fragment of platelet factor-4 16-kD fragment of prolactin NC10 domain of human collagen XV Protein related to the proangiogenic molecule proliferin Fragments of secreted protein, acidic and rich in cystine Well-known antiviral proteins, may downregulate angiogenic factor expression Proteins containing metalloprotease and thrombospondin domains, and distintegrin domains in NH2 termini Antagonist of angiopoietin-1 that binds to Tie-2 receptor A fragment missing the carboxy-terminal loop of antithrombin III (a member of the serpin family) Upregulated by IFN-␥ and whose mechanism of antiangiogenic effect is unknown
48 49 50 51 52 53 54 55 56 57 58
59 60 61
From RS Kerbel, Tumor angiogenesis: past, present and the near future. Carcinogenesis 2000;21:505, adapted with permission.
TARGET SELECTION IN TUMOR ANGIOGENESIS AND VEGF
New blood vessel growth depends on the expression of proangiogenic cytokines, including the fibroblast growth factor, VEGF, ephrin, platelet-derived growth factor, and angiopoietin families. Of these, VEGF has received the most attention. It promotes endothelial cell proliferation, survival, vessel permeability, and appears to be nearly ubiquitous in human tumors. Thus, VEGF has been an attractive target for novel antiangiogenic strategies. This is reflected in the wide variety of agents that have been developed that target VEGF signaling. More recently, clinical testing appears to validate the choice of VEGF antagonism as a therapy in some adult cancers.34 Given the apparent central role for VEGF in human tumor angiogenesis, it is not surprising that agents that block VEGF function appear to be broadly effective in preventing growth of implanted tumors in a variety of experimental models. Yet there are clearly differences in responsiveness to anti-VEGF agents in individual tumor types, which likely reflect the influence of tumor-specific regulatory elements on vessel networks. Thus, VEGF antagonism may serve as a prototype for the development of antiangiogenic strategies that focus on specific cytokine targets in pediatric solid tumors. Investigators will need to determine both the relative efficacy of dif-
ferent blockade strategies and their specific utility in individual tumor types. APPROACHES TO VEGF BLOCKADE IN PEDIATRIC TUMOR MODELS
Development of a monoclonal humanized anti-VEGF antibody was first reported to be effective in blocking tumor growth and angiogenesis in murine models of human rhabdomyosarcoma and glioblastoma multiforme.35 The same antibody was subsequently demonstrated to inhibit development of vasculature and tumor progression in mice with intraabdominal implants of neuroblastoma, Wilms tumor, and hepatoblastoma,26,27,36 and metastases in Wilms tumor.27 In addition, selective inhibition of the VEGF165 isoform, which has been demonstrated to be principally responsible for stimulating tumor neoangiogenesis, was shown to have similar efficacy in Wilms tumor.37 Activation of the VEGFR2 receptor is critical for the proangiogenic effects of VEGF. Blockade of this receptor, using a specific antibody, also effectively inhibits tumor progression and angiogenesis in a murine model of pediatric leukemia.38 The combination of VEGF blockade with antiangiogenically scheduled cytotoxic drugs has demonstrated increased efficacy in pediatric solid tumor models. Klement et al reported that use of an anti-VEGF receptor antibody combined with antiangiogenically scheduled
56
DAVIDOFF AND KANDEL
Table 2. Angiogenesis Inhibitors in Clinical Trials Drug
BMS-275291 Dalteparin (Fragmin) Suramin 2-methoxyestradiol (2-ME) CC-5013 (thalidomide analog)
LY317615 (protein kinase C  inhibitor)
Soy isoflavone (soy protein isolate) Thalidomide AE-941 (Neovastat) Anti-VEGF antibody (Bevacizumab; Avastin) Interferon-␣ VEGF-Trap ZD647 EMD 121974 Medi-522 (Vitaxin) Carboxyamidotriazole (CAI) Celecoxib (Celebrex) Halofuginone Hydrobromide (Tempostatin) Interleukin-12 Rofecoxib (VIOXX) CA
Phase
II II III I I I I II/III I II II II I-III III I-III I-III I I II II I I/II I I/II I II I-III I I-III III
Tumor Type
Prostate cancer Gliomablastoma multiforme Unresectable or metastatic pancreatic cancer Bladder cancer Advanced solid tumors Gliomas Refractory solid tumor and/or lymphomas Relapsed metastatic malignant melanoma Advanced solid tumors High-grade gliomas Refractory large B-cell lymphoma Breast cancer Multiple tumor types Non-small cell lung cancer Multiple tumor types Multiple tumor types Refractory solid tumors or non-Hodgkin’s lymphomas Refractory solid tumors or non-lymphomas-Hodgkin’s Metastatic non-small cell lung cancer Relapsed multiple myeloma Advanced solid tumors Recurrent malignant gliomas Advanced solid tumors or lymphoma Advanced colorectal cancer Advanced solid tumors or refractory lymphomas Recurrent ovarian epithelial, fallopian tube primary peritoneal Localized prostate cancer Progressive advanced solid tumors Multiple tumor types Previously resected stage I or III colorectal
Adapted from http://www.cancer.gov/clinicaltrials/development/anti-angio-table.
cyclophosphamide improved the durability of the tumor suppressive response in experimental neuroblastoma.18 Similarly, the addition of antiangiogenically scheduled topotecan to anti-VEGF antibody improved efficacy in experimental Wilms tumor.29 These encouraging results must be considered in context with the observation that experimental neuroblastoma tumors recur during long-term antagonism with single-agent, anti-VEGFR2 antibody alone.18 It is possible that more potent agents, such as those that exploit the affinity of the VEGFR1 and VEGFR2 receptors for VEGF ligand, may improve results. For example, a novel agent based on the soluble portion of the VEGFR1 receptor completely inhibited rhabdomyosarcoma xenograft growth,39 whereas anti-VEGF antibody alone did not. The authors speculated that enhanced binding of stromal-cell derived VEGF, which contributes to stability of vasculature at very low levels, contributed to this differential response. Similarly, co-opted host vasculature can partly rescue tumor growth in experimental neuroblastoma when VEGF signaling is blocked by antiVEGF antibody. However, these host vessels can be destabilized and tumor growth more completely inhib-
ited when a high-affinity agent based on portions of the VEGFR1 and VEGFR2 receptor is used.25 LONG-TERM DELIVERY OF VEGF ANTAGONISTS: GENE THERAPY APPROACHES
A limitation of the above studies is that most focus on the effects of VEGF antagonism during a limited time frame. Long-term delivery of angiogenesis inhibitors is likely to be important, because treated tumor cells, although small and dormant, may maintain their capacity for growth, invasion, and metastasis if released from the restrictions of angiogenesis inhibition. An alternative to chronic administration of recombinant proteins or peptides is the use of a gene therapy approach in which genetic material encoding the therapeutic protein is transferred to a host target cell where it mediates longterm protein expression. This approach to the delivery of angiogenesis inhibitors is attractive for several reasons. (1) Long-term expression of an inhibitor could potentially be achieved following a single intervention or administration. (2) Difficulties with protein production and maintenance of function, especially when “scaling up” for clinical trials
ANTIANGIOGENIC THERAPY
may be avoided by in situ expression in host tissues. (3) Continuous, low-level expression of these proteins, as would be generated from gene-modified cells, may be the optimal delivery schedule. (4) Several angiogenic pathways can be targeted with a single vector through the delivery of multiple genes. (5) Potential side effects of antiangiogenic therapy could be avoided by either limited local expression or regulatable, intermittent, systemic expression of an inhibitor, both of which can be achieved through gene therapy mediated approaches. Gene therapy strategies for tumor antiangiogenesis are, in fact, already being tested in a number of different murine tumor models, with some success. However, most of these studies have used either retroviral vector producer cells, naked DNA, or adenoviral vectors.40 Unfortunately, retroviral vector producers may be impractical for human use and the transfer of naked DNA is typically an inefficient, transient process; adenoviralmediated gene transfer is also complicated by transient transgene expression as well as a host immune response to transduced target cells. Adeno-associated virus (AAV) is a nonpathogenic, helper-dependent member of the parvovirus family. A number of properties make AAVbased vectors promising for antiangiogenic gene therapy. Most importantly, unlike the other gene delivery systems just mentioned, recombinant AAV (rAAV) vectors have been shown to direct long-term transgene expression from nondividing cells. In addition, these vectors have an excellent safety profile. Also unlike other vectors of viral origin, AAV has never been associated with any human disease and is naturally replication-deficient, thereby providing an added measure of safety. In addition, rAAV is nonimmunogenic; the wild type viral genes have been removed, thus reducing the potential for invoking a cellmediated immune response due to the expression of foreign viral proteins. Over the past several years, different gene therapy approaches have been explored, utilizing different vectors and targets, for the delivery of angiogenesis inhibitors. One set of initial studies involved the in vitro transduction of tumor cells using retroviral vectors encoding a truncated, soluble form of the VEGF receptor-2 (tsFlk-1, a competitive inhibitor of VEGF). By targeting the tumor cells in an ex vivo setting, efficient transduction could be achieved with resultant high levels of transgene expression at all tumor sites in vivo following gene-modified tumor cell inoculation. Consistent with these predictions, restricted growth and angiogenesis was of experimental neuroblastoma were observed in tsFlk-1– expressing tumors,41 with measurable decreases in endothelial cell density. Ultimately, however ex vivo gene modification of tumor cells as part of an antiangiogenic strategy is clinically impractical. Therefore, it is rational to consider the
57
possibility of in situ tumor cell transduction using retroviral vector-producer cell-mediated in vivo gene transfer to tumor cells. Investigators have reported that in situ tumor cell transduction could be efficiently performed and following transfer of the cDNA for tsFlk-1, significant restriction of tumor-induced angiogenesis and localized neuroblastoma growth resulted.42 More often, however, children who succumb to pediatric tumors have widespread, metastatic disease. Neuroblastoma, for example, is a systemic disease with multiple overt and occult sites of tumor, requiring systemic therapy. Therefore, a mechanism for long-term, systemic delivery of these angiogenesis inhibitors is required. One approach consistent with this requirement is to use retroviral vectors to modify murine bone marrow-derived cells, because these cells are among the most accessible, transducible, self-renewing cells. In addition, it is possible that, because some endothelial cells incorporated into tumor-induced new blood vessels appear to be derived from precursor cells of bone marrow origin,43 modifying these cells to express an inhibitor of endothelial cell activation might prevent endothelial cell precursor differentiation and/or create a milieu of angiogenesis inhibition at the sites of tumor neovascularization. Neuroblastoma growth, in mice challenged 3 months following transplantation with tsFlk-1-expressing bone marrow cells, was significantly inhibited when compared to tumor growth in controls.44 In addition, immunohistochemical analysis of tumors in each group demonstrated colocalization of vector-encoded Green Fluorescent Protein expression in cells immunopositive for endothelial cell markers, confirming that the endothelium of the tumorinduced neovasculature were derived, at least in part, from bone marrow precursors.44 These results confirmed that long-term expression of a functional angiogenesis inhibitor could be generated through gene-modified bone marrow-derived stem cells, and that this approach could have significant anticancer efficacy. Other gene therapy approaches using tsFlk-1 have been used. One alternative strategy has ultilized recombinant adeno-associated virus vectors to effect liver transduction in situ to establish long-term expression and systemic delivery of this angiogenesis inhibitor to provide the same antiangiogenic and antitumor efficacy. Following intraportal injection of rAAV tsFlk-1, highlevel, stable transgene expression was generated in mice peaking 3 to 5 weeks following vector administration. This established a systemic state of angiogenesis inhibition; sera from these mice inhibited endothelial cell activation in vitro and Matrigel plug neovascularization in vivo.45 Significant antitumor efficacy was observed in two murine models of pediatric kidney tumors.45 Tumor development was prevented in a majority of the mice, with significant growth restriction of tumors in the re-
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DAVIDOFF AND KANDEL
maining mice. Thus, long-term, in vivo expression of a functional angiogenesis inhibitor was established using rAAV, with resultant anticancer efficacy in a relevant, orthotopic tumor model. This study was the first to demonstrate the feasibility of using liver-targeted rAAV vectors in antiangiogenic gene therapy. Next, this antitumor strategy was tested in a murine model of neuroblastoma, a tumor known to be more aggressive and to elaborate a spectrum of proangiogenic factors.23 Despite the capacity of neuroblastoma to activate different angiogenic pathways, monotherapy with rAAV-mediated delivery of tsFlk-1, as with the renal tumor models, had significant efficacy against localized and metastatic neuroblastoma. Localized retroperitoneal tumors were significantly smaller in tsFlk-1-expressing mice and survival for mice with metastatic disease was greater (AM Davidoff, unpublished data). In an effort to improve antineuroblastoma efficacy, larger doses of the vector were administered to cohorts of mice. Although enzyme-linked immunosorbent assay confirmed that higher levels of tsFlk-1 were generated in these mice, antitumor efficacy was not improved. In fact, paradoxically, the tumors were more aggressive than in control mice. Further investigation revealed that mice expressing the higher levels of tsFlk-1 also had significantly higher levels of circulating mouse VEGF (mVEGF) than those mice expressing lower but more effective levels of tsFlk-1; these mice were expressing levels of VEGF similar to control mice. An additional surprising finding was that the tumor cells were not solely responsible for this apparent compensatory increase in VEGF expression, but that host cells were contributing to the higher circulating levels of VEGF. Binding of mVEGF
by tsFlk-1 appeared to influence mVEGF bioavailability. Although a greater amount of mVEGF was bound in high tsFlk-1 expressing mice, more mVEGF was also unbound and available to support angiogenesis. This novel resistance mechanism impaired the effectiveness of angiogenesis inhibition at higher doses of the inhibitor. These results suggest that careful titering of angiogenesis inhibitors, such as tsFlk-1, may be required to achieve maximal antitumor efficacy and avoid therapy resistance mediated, in part, by affecting ligand bioavailability. This has important implications for therapeutic strategies that use decoy receptors and other agents, such as antibodies, to bind angiogenic factors, in an attempt to inihibit tumor neovascularization. CONCLUSIONS
These results suggest that antitumor strategies that focus on proangiogenic cytokines in general, and VEGF in particular, hold promise for the treatment of children with aggressive malignancies. However, several issues remain to be addressed. First, the potential for the development of resistance must be evaluated, especially when chronic VEGF antagonism is used as monotherapy. This may involve several mechanisms, including co-option of host vasculature, as documented in murine models of glioblastoma and neuroblastoma,25,46 or the increased expression of VEGF by either tumor cells or host tissues. Second, the potential consequences of long-term angiogenesis inhibition must be defined, especially when considering administering this therapy to growing children. Finally, synergy between angiogenesis inhibitors and other treatment modalities should be explored.
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