A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial.

BACKGROUND: Although the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)-compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials. METHODS: Genetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product. RESULTS: Two hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites). CONCLUSION: Large-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

ZEB1 drives epithelial-to-mesenchymal transition in lung cancer.

Increased expression of zinc finger E-box binding homeobox 1 (ZEB1) is associated with tumor grade and metastasis in lung cancer, likely due to its role as a transcription factor in epithelial-to-mesenchymal transition (EMT). Here, we modeled malignant transformation in human bronchial epithelial cells (HBECs) and determined that EMT and ZEB1 expression are early, critical events in lung cancer pathogenesis. Specific oncogenic mutations in TP53 and KRAS were required for HBECs to engage EMT machinery in response to microenvironmental (serum/TGF-ß) or oncogenetic (MYC) factors. Both TGF-ß- and MYC-induced EMT required ZEB1, but engaged distinct TGF-ß-dependent and vitamin D receptor-dependent (VDR-dependent) pathways, respectively. Functionally, we found that ZEB1 causally promotes malignant progression of HBECs and tumorigenicity, invasion, and metastases in non-small cell lung cancer (NSCLC) lines. Mechanistically, ZEB1 expression in HBECs directly repressed epithelial splicing regulatory protein 1 (ESRP1), leading to increased expression of a mesenchymal splice variant of CD44 and a more invasive phenotype. In addition, ZEB1 expression in early stage IB primary NSCLC correlated with tumor-node-metastasis stage. These findings indicate that ZEB1-induced EMT and associated molecular changes in ESRP1 and CD44 contribute to early pathogenesis and metastatic potential in established lung cancer. Moreover, TGF-ß and VDR signaling and CD44 splicing pathways associated with ZEB1 are potential EMT chemoprevention and therapeutic targets in NSCLC.