EXPLOITING THE USE OF PLASMINOGEN KRINGLE DOMAINS FOR CANCER GENE THERAPY Sabrina R. Perri Division of Experimental Medicine, Deparlment of Medicine Faculty of Medicine, McGiII University Montreal, Quebec, Canada February 2007 A thesis submitled to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Doctor of Philosophy (Ph. O.). ©2007 Sabrina R. 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While these forms may be included Bien que ces formulaires in the document page count, aient inclus dans la pagination, their removal does not represent il n'y aura aucun contenu manquant. any loss of content from the thesis. ••• Canada ABSTRACT It is well characterized that tumors depend upon angiogenesis in order to progress and form metastases. In this regard, strategies to inhibit neovascularization have been developed either aimed to target endothelial cells directly or indirectly via suppression of pro-angiogenic growth factors produced by the tumor or surrounding stromal cells. Several anti-angiogenic agents are present endogenously in our body as cleavage products of larger precursor proteins. Angiostatin is one of the most widely studied inhibitors of angiogenesis and encompasses the first 3 or 4 kringle domains of human plasminogen (PIg). Of particular interest, is the fifth kringle of human Plg (K5), which displays higher anti-angiogenic potency than angiostatin. In fact, kringles 1 to 5 exhibit greater inhibitory activity against endothelial cells than angiostatin, and this finding suggests that K5 domain acts synergistically to enhance the anti-angiogenic effect of angiostatin. Employing a gene-based approach for the delivery of an anti-angiogenic agent provides sustained, biologically relevant levels of the therapeutic protein of interest and circumvents the limitations associated with direct recombinant protein use, such as the necessity for prolonged repeated administration, high dosages, high expense and risk of toxicity. Therefore, we proposed that the K5 domain --Qn its OWll- could act as a potent anti-angiogenic and anti-tumor protein within the context of a cancer gene therapy strategy. In our first study, we assessed the angiostatic properties of the K5 peptide domain III an orthotopic brain cancer model. We demonstrated that the disulfide bridging conformation of K5, necessary to maintain its functionality, is conserved upon secretion by gene-modified mammalian cells. Kringle 5 retrovirally gene-engineered human U87 11 glioma celIs produced functional soluble KS protein capable of suppressing growth factor-induced endothelial celI migration in vitro and inhibiting glioma-induced angiogenesis in vivo. Interestingly, secreted KS prote in blocked the recruitment of tumor­ associated CD4S+Mac3+0rl" macrophages in vivo and inhibited the migration of CD206+ human monocyte-derived macrophages in vitro. Moreover, in a clinicalIy relevant orthotopic glioma model, soluble KS induced long-term survival in a majority of test animaIs. Thus, these findings validate the use of a gene therapy approach to deliver Plg KS protein and suggest that KS acts as a novel 2-pronged anti-tumor agent, mediating its inhibitory effect via its action on host-derived endothelial ceUs and tumor-associated macrophages. To determine if KS mediated its anti-tumor effect by modulating other immune effector cells, we tested the use of soluble KS in a murine DAl3 mammary adenocarcinoma mode!. Soluble KS produced by retrovirally gene-modified DAl3 ceUs led to long-term survival (over 1 year) of immunocompetent BALB/c mice however, we failed to observe tumor rejection in immunodeficient NOD-SCID and BALB/c nude mice. Further analysis revealed that KS enhances the recruitment of tumor-infiltrating CD3+ lymphoid cells, in particular the natural killer T (NKT)-lymphocyte phenotype. Consistent with our previous findings, we demonstrated that KS led to a significant decrease in tumor-associated microvessel length and density. Interestingly, KS tumors were characterized by a robust neutrophilic infiltrate. This may be explained by the ability of KS to act as a strong chemotactic agent for human neutrophils in vitro as weU as its ability to promote CD64+ activation within the CD 11 b+ neutrophil phenotype. These findings confirm that KS acts as a potent angiostatic agent and possesses a novel 111 pro-inflammatory role via its ability to recruit tumor-associated neutrophils and NKT­ lymphocytes, leading to a strong anti-tumor response. Tumor-associated macrophages (TAM) are key immune effector cells implicated in promoting tumor progression and metastasis. It would thus be desirable to explore strategies to reduce T AM infiltration within the tumor microenvironment. In our first study, we demonstrated that soluble K5 protein blocks macrophage recruitment. In addition, the recent observation that angiostatin reduces macrophage infiltration in an atherosc1erosis model prompted our laboratory to further explore the use of angiostatin as an anti-macrophage agent. We demonstrated that angiostatin suppresses the in vitro migration of both murine peritoneal macrophages and human monocyte-derived CD206+ macrophages. Furthermore, we showed that angiostatin led to a decrease in the gelatinolytic activity of macrophage-produced matrix metalloproteinase-9, which may explain, in part, the observed angiostatin-mediated inhibition of migration. Additionally, we detected the presence of the /3-subunit of A TP synthase on the cell-surface of macrophages. A TP synthase was previously found to be a receptor for angiostatin on the cell-surface endothelial cells. We propose that the presence of A TP synthase on the surface of macrophages may promote interaction with angiostatin and prevent migration, similar to what has been reported with endothelial cells. Our findings suggest that angiostatin holds promise as an inhibitory agent against macrophages. Overall, our data supports the use of Plg K5 protein as a potent anti-angiogenic agent and reveals that K5 as weIl as angiostatin possess novel immune modulatory properties that potentially enhance their utility as therapeutic agents against cancer. IV RÉSUMÉ Il a été largement cerné que les tumeurs dépendent de l'angiogenèse afin d'accomplir leur progression et former des métastases. À cet effet, des méthodes visant à inhiber la néovascularisation ont été élaborées afin de cibler les cellules endothéliales de façon directe ou indirecte via la suppression des facteurs de croissance pro-angiogéniques produits par la tumeur ou les cellules stromales environnantes. Plusieurs agents anti­ angiogéniques demeurent présents endogéniquement dans notre organisme en tant que produits afférents à de plus vastes protéines et ce en tant que précurseurs. L' angiostatine s'avère être un des inhibiteurs les plus étudiés et englobe les trois ou quatre premiers domaines kringle du plasminogène humain. II importe de noter qu'il s'agit du cinquième kringle du Plg humain (K5) qui démontre une capacité anti-angiogénique plus élevée que l'angiostatine. En effet, les kringles 1 à 5 présentent une activité inhibitrice supérieure face aux cellules endothéliales que ne le démontre l' angiostatine. De plus, cette découverte suggère que la région K5 agit synergiquement afin d'augmenter l'effet anti­ angiogénique de l'angiostatine. L'utilisation d'une approche de thérapie génique dans le transfert d'un agent anti-angiogénique fournit un niveau biologique significatif et soutenu de la protéine concernée et circonvient les limites associées à l'usage direct du recombinant de protéines telle que par exemple la nécessité d'administrer le produit de façon prolongée et répétée, en dosage élevé et en tenant compte du risque élevé de toxicité. Nous proposons donc que le domaine K5 agit de lui-même comme puissant agent anti-angiogénique et comme protéine anti-tumeur dans