The metazoan parasitic blood f lukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism's complex molluscan-mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro. Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.Schistosoma mansoni, Schistosoma haematobium, and Schistosoma japonicum create widespread disease in tropical developing countries (1). These parasites thwart attempts to produce practical and effective vaccines, and pharmacological approaches are problematic (2). Schistosome eggs are laid by females paired with males and residing in the vasculature of parasitized vertebrate hosts. To be successfully infective, eggs must penetrate the intestinal wall and pass in feces to fresh water. From these released eggs hatch free-swimming miracidia, the first larval form. Successful miracidia encounter and penetrate the appropriate species of snail and develop into sporocysts, the second larval from. In the following weeks, these primary sporocysts asexually generate first-generation and second-generation daughter sporocysts in the snail. From the later-generation sporocysts develop the third larval form, free-swimming cercaria. To be successfully infective in the human or other vertebrate host, cercaria must encounter and penetrate the skin of the host and undergo further changes to form schistosomula. These move through several further developmental stages, ultimately leading to sexual pairing in the veins of the parasitized host and egg production.Approaches to elucidate the mechanisms schistosomes employ to escape host immunity, as well as molecular genetic and pharmacological investigations, have been limited by the lack of an in vitro system for the continuous proliferative culture of the parasite in the absence of the host. Recently, Yoshino and colleagues (3, 4) have reported significant success in limited culture of the intramolluscan stages of S. mansoni and S.japonicum, but these techniques do not allow extended culture of proliferating sporocysts.Here we describe long-term cultures of S. mansoni in vitro and the development from the snail-infective miracidial stage, through generations of mother and daughter sporocysts, t...

Despite major advances in the treatment of non-Hodgkin lymphoma (NHL), including the use of chemotherapeutic agents and the anti-CD20 antibody rituximab, the majority of patients eventually relapse, and salvage treatments with non-crossresistant compounds are needed to further improve patient survival. Here, we evaluated the antitumor effects of the microtubule destabilizing agent monomethyl auristatin E (MMAE) conjugated to the humanized anti-CD19 antibody hBU12 via a protease-sensitive valine-citrulline (vc) dipeptide linker. hBU12-vcMMAE induced potent tumor cell killing against rituximab-sensitive and -resistant NHL cell lines. CD19 can form heterodimers with CD21, and high levels of CD21 were reported to interfere negatively with the activity of CD19-targeted therapeutics. However, we observed comparable internalization, intracellular trafficking, and drug release in CD21 low and CD21 high , rituximab-sensitive and -refractory lymphomas treated with hBU12-vcMMAE. Furthermore, high rates of durable regressions in mice implanted with these tumors were observed, suggesting that both rituximab resistance and CD21 expression levels do not impact on the activity of hBU12-vcMMAE. Combined, our data suggest that hBU12-vcMMAE may represent a promising addition to the treatment options for rituximab refractory NHL and other hematologic malignancies, including acute lymphoblastic leukemia. (Blood. 2009;113:4352-4361)

Angiogenesis is impaired in aging. Delayed neovascularization is due, in part, to slowed endothelial cell migration. Migration requires an optimal level of adhesion to matrix proteins, a process mediated by matrix-degrading metalloproteases (MMPs) such as MMP1. To determine whether impaired angiogenesis in aging is associated with altered synthesis and activity of MMP1, we examined the expression of collagenase and tissue inhibitor of metalloprotease 1 (TIMP1) by immunostain of angiogenic sponge implants from young and aged mice. To characterize the relevance of MMP activity during the movement of aged endothelial cells, the secretion of MMP1 and TIMP1 by late-passage human microvascular endothelial cells (hmEC aged in vitro) and their non-aged (young) counterparts was quantified. The migration of aged human microvascular endothelial cells and the effect of inhibition of TIMP1 on the migration of aged hmEC or collagen I was also measured. Relative to young mice, granulation tissue from aged mice showed less expression of collagenase and increased expression of TIMP1. In vitro, aged hmEC were deficient in MMP1 secretion (55 +/- 13% relative to young cells) and activity but showed increased expression of TIMP1 (280 +/- 109% relative to young cells). Aged hmEC migrated significantly less distance than did young hmEC over a 5-day period (59 +/- 8% relative to young cells). In the presence of a blocking antibody to TIMP1, aged hmEC showed a significant increase in the distance migrated on collagen I over a 5 day period (142 +/- 11% relative to untreated aged hmEC). We propose that deficient MMP1 activity contributes to impaired cellular movement in aged microvascular endothelial cells and that perturbations that enhance collagenase activity increase their migratory ability and angiogenic potential.

Purpose: Individually targeting B-cell antigens with monoclonal antibody therapeutics has improved the treatment of non-Hodgkin lymphoma (NHL). We examined if the antitumor activity of rituximab, CD20-specific antibody, could be improved by simultaneously targeting CD40 with the humanized monoclonal antibody dacetuzumab (SGN-40).Experimental Design: Dacetuzumab was dosed with rituximab to determine the in vivo activity of this combination in a subcutaneous Ramos xenograft model of non-Hodgkin lymphoma (NHL). The effect of dacetuzumab on rituximab antibody-dependent cell mediated-cytotoxicity (ADCC), antiproliferative, and apoptotic activities were evaluated in vitro using NHL cell lines. Western blotting and flow cytometry were used to contrast the signaling pathways activated by dacetuzumab and rituximab in NHL cells.Results: The dacetuzumab-rituximab combination had significantly improved antitumor activity over the equivalent dose of rituximab in the Ramos xenograft model (P ¼ 0.0021). Dacetuzumab did not augment rituximab-mediated ADCC activity; however, these antibodies were additive to synergistic in cellproliferation assays and produced increased apoptosis in combination. Rituximab signaling downregulated BCL-6 oncoprotein in a cell line-specific manner, whereas dacetuzumab strongly downregulated BCL-6 in each cell line. Dacetuzumab induced expression of the proapoptotic proteins TAp63 and Fas, whereas rituximab did not affect basal expression of either protein. Finally, rituximab partially blocked dacetuzumab-mediated upregulation of the prosurvival protein BCL-x L .Conclusions: Targeting CD40 with dacetuzumab enhanced the antitumor activity of rituximab in cell line and xenograft NHL models. The distinct but complementary apoptotic signal transduction profiles of dacetuzumab and rituximab are an important mechanism behind the improved activity of this combination.

Seattle Genetics is utilizing the highly potent DNA cross-linking molecule, pyrrolobenzodiazepine (PBD) dimer, as the payload across multiple antibody drug conjugate (ADC) platforms. Previous studies have demonstrated that PBD dimers bind to the minor groove of DNA causing an interstrand crosslink; however, it is poorly understood how the cells recognize and respond to this insult. The DNA damage pathway exists to ensure genomic fidelity by either repairing damaged DNA or forcing programmed cell death if the damage is too extensive. Cells do this in an orchestrated manner that involves sensing the damage and eliciting a signaling cascade to induce repair and cell cycle arrest and ultimately either cell cycle re-start, death, or senescence. Using SGN-CD70A, an ADC consisting of a CD70-directed engineered cysteine antibody linked to two PBD dimers, we have examined the DNA damage response and cell death kinetics in a panel of renal cell carcinoma (RCC) and non-Hodgkin lymphoma (NHL) cell lines after treatment with free PBD and SGN-CD70A. We examined the central DNA damage sensing protein kinases, ATM, ATR, and DNA-PK and find that all three are phosphorylated on activation residues within 24 hours at EC50 doses in both RCC and NHL cell lines. Similarly, the downstream checkpoint kinases, Chk1 and Chk2 are also activated, within 24 hours after treatment with SGN-CD70A. The kinetics of pChk2 activation mirror Caspase 3/7 activation, with both peaking 48-72 hours after treatment. To confirm the involvement of the DNA damage pathway in the PBD mechanism, we used small molecules to inhibit ATM (KU-60019), ATR (VE-821), DNA-PK (NU-7441), and Chk1/2 (AZD7762, LY2603618) and found that all were synergistic with SGN-CD70A and accelerated cell death kinetics. We also conducted a small molecule screen to identify other processes involved in SGN-CD70A's activity. The Mps1 inhibitor (AZ 3146), which shortens mitosis and abrogates the mitotic checkpoint, was strongly synergistic. Conversely, molecules that cause mitotic arrest, such as microtubule inhibitors and Plk1 inhibitors did not show the same degree of synergy. To follow up on this finding we looked at cell cycle kinetics. We found that SGN-CD70A treatment alone causes a G2 arrest and a modest increase in polyploid cells whereas synergistic combinations with SGN-CD70A and DNA damage kinase and Mps1 inhibitors decrease the number of cells in G2 while markedly increasing polyploid cells. These data suggest induction of mitotic catastrophe may be part of the cellular response to SGN-CD70A treatment. Based on these results, we are currently testing whether sequencing SGN-CD70A and various mitotic inhibitors can increase synergy. Citation Format: Sharsti L. Sandall, Renee McCormick, Jamie Miyamoto, Travis Biechele, Che-Leung Law, Timothy S. Lewis. SGN-CD70A, a pyrrolobenzodiazepine (PBD) dimer linked ADC, mediates DNA damage pathway activation and G2 cell cycle arrest leading to cell death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 946. doi:10.1158/1538-7445.AM2015-946