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Human epidermal growth factor 2 (HER2) is a potential driver oncogene. Although HER2-targeted precision therapy has been tested in non-small cell lung cancer (NSCLC), the demographic characteristics of HER2-positive NSCLC have not been systematically defined.

Background: Hemophilia B is an X‐linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma‐derived or recombinant FIX. Objective: In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Methods: Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitroand in vivo. Results: One variant containing three substitutions (V86A/E277A/R338A, FIX‐Triple) exhibited 13‐fold higher specific clotting activity and a 10‐fold increased affinity for human FVIIIa compared with FIX‐wild‐type (FIX‐WT) and was thus investigated systematically in vivo. Liver‐specific FIX‐Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5‐fold higher specific clotting activity compared with FIX‐WT. Human FIX‐Triple and FIX‐WT knock‐in mice were generated and it was confirmed that FIX‐Triple has 7‐fold higher specific clotting activity than FIX‐WT under normal physiological conditions. Protein infusion of FIX‐Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX‐WT. Moreover, tail‐vein administration of a serotype 8 recombinant Adeno‐associated vector (AAV8) expressing either FIX‐WT or FIX‐Triple in hemophilia B mice demonstrated a 7‐fold higher specific clotting activity of FIX‐Triple than FIX‐WT. Conclusions: Our results indicate that the FIX‐Triple variant exhibits significantly enhanced clotting activity relative to FIX‐WT due to tighter binding to FVIIIa, as demonstrated both in vitroand in vivo. Therefore, FIX‐Triple is a good candidate for further evaluation in protein replacement therapy as well as gene‐based therapeutic strategies.

To develop a suitable vector and an administration technique for in vivo gene transfer, the tissue distribution of plasmid DNA (pDNA) needs to be understood. In this study, a novel residualizing radiolabel for pDNA was developed. 4-[p-Azidosalicylamido]butylamine (ASBA) was coupled with diethylenetriaminepentaacetic acid (DTPA) anhydride, then the conjugate was reacted with pDNA by photoactivation, followed by labeling with [¹¹¹In]InCl3 to obtain ¹¹¹In-pDNA. The overall structure of pDNA was well preserved, and the retention of its transcriptional activity was 40−98%. After intravenous injection of ¹¹¹In-pDNA into mice, about 50% of the radioactivity was recovered in the liver within 3 min. The level remained stable for at least 2 h, followed by a very slow decrease to 45% at 24 h. This contrasted with the results obtained with ³²P-pDNA by nick translation, in which a rapid decrease in hepatic radioactivity was observed. The amount of radioactivity in the lung following the administration of polyethyleneimine/¹¹¹In-pDNA complexes correlates well with the transgene expression. These results indicate that the novel residualizing radiolabel clearly demonstrates the cells that have taken up pDNA and, therefore, gives us useful information about how to design a better approach for nonviral in vivo gene delivery.

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation's major effects were a 3-fold increase in kcat and a 2-fold decrease in Km both manifested only in the presence of FVIIIa. R338A-FIXa's increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin.

The antitumor effect of cisplatin-(CDDP)-encapsulated thermosensitive large unilamellar liposome (ThLip) administration with hyperthermia (HT) was examined in mice bearing Meth A fibrosarcoma. The tumor Pt levels after ThLip administration were increased in response to HT. The targeting index was approximately 3. The antitumor activity of ThLip + HT, as measured by tumor growth delay or tumor weight inhibition, was larger than that of ThLip without HT or a solution with or without HT. The CDDP dose in ThLip + HT to give equivalent tumor growth delay in solution (40 micrograms/mouse) + HT was about 10 micrograms/mouse, and therefore the targeted drug delivery enhancement ratio was about 4. The ratio correlates with the targeting index. The blood urea nitrogen level, as an indicator of CDDP nephrotoxicity, was increased 7 days after the administration of ThLip (40 micrograms CDDP/mouse) with HT. However, this blood urea nitrogen level rise was independent of the activity enhancement by the liposome. These findings suggest that the HT combined CDDP delivery system using ThLip can decrease the effective CDDP dose, thereby increasing its therapeutic index.