Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin

doi: 10.1038/mtm.2015.5

Figure Lengend Snippet: Structural and functional studies with JO-4. ( a ) HAdV3 fiber knob amino acid sequence with β sheets A to J indicated by a line. JO-4 contains HAdV3 fiber knobs with a valine to aspartic acid substitution in position 239 of the HAdV3 fiber knob. Regions that have previously been found to be involved in DSG2 binding are boxed. ( b ) 3D-structure superimposition of the trimeric (wild type) HAdV3 fiber knob and the V239D mutated fiber knob. The common structure appears in grey, the EF loops of wild-type and mutant HAdV3 knob monomers are labeled in blue and green, respectively. ( c ) Shown is the interactions between D239 and K275 in the mutant and the new position of the L240 residue. Residue labels are colored green for the mutant and gray for wt. H-bonds between D239 and K275 are shown in dashes. ( d ) Affinity of JO-4 to DSG2 measured by surface plasmon resonance. JO-4 was immobilized on sensorchips, and background was automatically subtracted from the control flow cell. DSG2 were injected for 3 minutes at a concentration range from 12.5 to 200 nmol/l and kinetics and affinity parameters were evaluated using the BIAeval software ( k a = 3.3 × 10 5 M −1 s −1 ; k d = 4.7 × 10 −4 s −1 ; K D =1.4 nmol/l).

Article Snippet: Recombinant human DSG2 protein was from Leinco Technologies (St. Louis, MO).

Techniques: Functional Assay, Sequencing, Binding Assay, Mutagenesis, Labeling, Residue, SPR Assay, Control, Injection, Concentration Assay, Software

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin

doi: 10.1038/mtm.2015.5

Figure Lengend Snippet: Efficacy studies with JO-4 and Doxil in mouse models. ( a ) Efficacy study in mammary fat tumor pad model using primary ovarian cancer (ovc316) cells. Treatment was started when tumors reached a volume of 100 mm 3 . Mice were injected intravenously with Doxil (1 or 3 mg/kg) alone or in combination with 2 mg/kg JO-4. Treatment was repeated weekly. N = 10. ( b–f ) Studies in the spontaneous metastasis model based on MDA-MB-231-luc-D3H2LN cells. ( b ) In vivo luciferase imaging of a representative animal. The primary tumor in the mammary fat pad forms metastasis in regional lymph nodes (LN), e.g. , pancreatic and mesenteric lymph nodes. ( c ) DSG2 immunofluorescence analysis of sections from the primary tumor and mesenteric lymph node metastases. DSG2 signals are in red and appear to be membrane associated. Nuclei are stained in blue. ( d ) Western blot analysis of primary tumor and metastases with DSG2 antibodies. β-actin is used as a loading control. A representative animal is shown. ( e,f ) Comparison of in vivo luciferase signal in the primary tumor ( e ) and metastasis ( f ) 1 day before and 5 days after treatment with Doxil or JO-4 + Doxil. Shown is the total flux of the primary tumor or metastasis ROI after treatment divided by the ROI before treatment. Shown are averages of imaging sequences of five images each. The difference between the Doxil and Doxil+JO4 groups is significant ( P < 0.01) for both the primary tumor and the metastases. The difference between the untreated and Doxil groups is not significant.

Article Snippet: Recombinant human DSG2 protein was from Leinco Technologies (St. Louis, MO).

Techniques: Injection, In Vivo, Luciferase, Imaging, Immunofluorescence, Membrane, Staining, Western Blot, Control, Comparison

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin

doi: 10.1038/mtm.2015.5

Figure Lengend Snippet: Nonhuman primates (NHP) study #2 (JO-4+Doxil): Blood analysis. ( a ) Experimental design: Two sedated M. fascicularis , A11288 (A-88) (age: 4 years 11 months, weight: 5.0 kg) and A11293 (A-93) (age: 5 years 10 months, weight: 6.1 kg) were injected through the saphenous vein with 5 ml of saline (A-93) or 5 ml of JO-4 at a dose of 2 mg/kg (A-88) at an infusion rate of 2 ml/minute). One hour later, both animals received an intravenous injection of 20 ml of Doxil at a dose of 40 mg/m 2 (1.1 mg/kg). Blood was collected at the indicated time points. ( b ) One set of blood samples was submitted to the UW Clinical Laboratory for CBC and blood chemistry analysis. A second set of plasma samples was used for measuring the concentrations of JO-4, anti-JO-4 antibodies, Doxil, DSG2, and proinflammatory cytokines. Blood cell counts: Treatment-related changes were observed for white blood cells, neutrophiles, and lymphocytes. All other hematological parameters including red blood cell, platelet, monocyte, eosinophil, basophil counts were normal. ( c ) Blood chemistry: Treatment-related changes were observed for AST and ALT. Sodium, potassium, chloride, glucose, blood urea nitrogen, creatinine, total protein, albumin, globulin, A:G ratio, total bilirubin, calcium, magnesium, phosphate, cholesterol, alkaline phosphatase, and GGT levels were unremarkable. ( d ) Serum interferon-γ levels were measured by ELISA. For all analyzed serum proteins, at least two serum dilutions in duplicates were analyzed in two independent ELISAs. ( e ) Serum DSG2 (sDSG2) ECD concentrations.

Article Snippet: Recombinant human DSG2 protein was from Leinco Technologies (St. Louis, MO).

Techniques: Injection, Saline, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Preclinical safety and efficacy studies with an affinity-enhanced epithelial junction opener and PEGylated liposomal doxorubicin

doi: 10.1038/mtm.2015.5

Figure Lengend Snippet: Nonhuman primates (NHP) study #2 (JO-4+Doxil): Immunofluorescence analysis of tissue sections. ( a ) Presence of JO-4 in tissues. Staining for JO-4 appears in green, nuclei are blue. ( b ) Staining of adrenals sections with anti JO4 antibodies (green) and anti-DSG2 antibodies (red). ( c ) Staining of adrenals section with anti JO-4 antibodies (red) and anti-monkey IgG-FITC from NIH Nonhuman Primate Reagent Resource (green). ( d ) Staining for JO-4 (red) and the Kupffer cell marker F4/80 (green). ( e ) Staining of adrenal and kidney sections for Doxil using antibodies against PEG (red).

Article Snippet: Recombinant human DSG2 protein was from Leinco Technologies (St. Louis, MO).

Techniques: Immunofluorescence, Staining, Marker