Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells.

Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.

Thrombin cleavage of osteopontin initiates osteopontin's tumor-promoting activity.

BACKGROUND: Osteopontin (OPN) is a multifunctional proinflammatory matricellular protein overexpressed in multiple human cancers and associated with tumor progression and metastases. Thrombin cleavage of OPN reveals a cryptic binding site for α4 ß1 and α9 ß1 integrins. METHODS: Thrombin cleavage-resistant OPNR153A knock-in (OPN-KI) mice were generated and compared to OPN deficient mice (OPN-KO) and wild type (WT) mice in their ability to support growth of melanoma cells. Flow cytometry was used to analyze tumor infiltrating leukocytes. RESULTS: OPN-KI mice engineered with a thrombin cleavage-resistant OPN had reduced B16 melanoma growth and fewer pulmonary metastases than WT mice. The tumor suppression phenotype of the OPN-KI mouse was identical to that observed in OPN-KO mice and was replicated in WT mice by pharmacologic inhibition of thrombin with dabigatran. Tumors isolated from OPN-KI mice had increased tumor-associated macrophages with an altered activation phenotype. Immunodeficient OPN-KI mice (NOG-OPN-KI) or macrophage-depleted OPN-KI mice did not exhibit the tumor suppression phenotype. As B16 cells do not express OPN, thrombin-cleaved fragments of host OPN suppress host antitumor immune response by functionally modulating the tumor-associated macrophages. YUMM3.1 cells, which express OPN, showed less tumor suppression in the OPN-KI and OPN-KO mice than B16 cells, but its growth was suppressed by dabigatran similar to B16 cells. CONCLUSIONS: Thrombin cleavage of OPN, derived from the host and the tumor, initiates OPN's tumor-promoting activity in vivo.

The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa.

Tissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa·TFPI encounter complex is followed by slow rearrangement into a tight FXa·TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gla-domain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1-161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXa- inhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa·TFPI* complex, but also inhibits FXa·TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.