BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein
FIG. 1 is an amino acid sequence alignment showing sequence similarity between human Bst2 and mouse Damp 1;
FIG. 2 shows the locations of PCR primers used in a process for cloning a human Bst2 soluble fragment and a mouse Damp 1 soluble fragment into an expression vector;
FIGS. 3A-3B show the results of electrophoresis analysis of a human Bst2 soluble fragment and a mouse Damp 1 soluble fragment;
FIG. 4 shows the expression pattern of Bst2 gene during homotypic aggregation of U937 cells;
FIG. 5 shows the promoting effect of Bst2 overexpression on homotypic aggregation of U937 cells;
FIG. 6 shows the effect of a Bst2 soluble fragment on homotypic aggregation of U937 cells;
FIG. 7 shows the effect of a Bst2 soluble fragment on intercellular adhesion between human vascular endothelial (HUVEC) cells and U937 cells;
FIG. 8 shows the dose-dependent effect of a Bst2 soluble fragment on intercellular adhesion between HUVEC cells and U937 cells;
FIG. 9 shows the effect of Bst2 siRNA on intercellular adhesion between HUVEC cells and U937 cells;
FIG. 10 shows the effect of Bst2 siRNA on intercellular adhesion between HUVEC cells and U937 cells upon Bst2 overexpression;
FIGS. 11A-11B show the effect of Bst2 overexpression on aggregation of Jurkat cells and interleukin-2 (IL-2) production in Jurkat cells;
FIG. 12 shows the effect of a Bst2 soluble fragment and Bst2 siRNA on aggregation of Jurkat cells;
FIGS. 13A-13B shows graphs showing the effect of a Bst2 soluble fragment on aggregation of Jurkat cells and IL-2 production;
FIG. 14 shows the change in the number of sedimented immune cells upon treatment of a Bst2 soluble fragment;
FIG. 15 shows the decreased levels of cytokines upon treatment of a Bst2 soluble fragment;
FIG. 16 shows the functional similarity between human Bst2 and mouse Damp 1;
FIG. 17 shows the inhibitory effect of a mouse Damp 1 soluble fragment on asthma induced in mice;
FIG. 18 shows PEG moieties used in preparation of PEG-conjugated forms of a Bst2 soluble fragment;
FIG. 19 shows the improved metabolic degradation of PEG-conjugated Bst2; and
FIG. 20 shows the expression and distribution of Bst2 in inflammation-associated diseases.
FIGS. 21A-21D show schematics of Bst2 decoy fused to Fc region. A, the Bst2 decoy itself, B, the Bst2 decoy fused to the hinge-CH2-CH3 portion of an IgG heavy chain Fc; C, Bst2 fusion protein that is stabilized through the naturally-occuring IgG kappa chain-heavy chain disulfide bonding; D, Bst2 decoy-IgG Fc is expressed without other Bst2 dimerization counterparts.
FIGS. 22A-22D show representative vector maps of Bst2 decoy-IgG Fc fusion proteins of FIG. 21.
FIG. 23 shows PCR-cloning and fusion strategy.
FIGS. 24A-24B show PAGE of purified Bst2 decoy and other Fc fusions. A, representative PAGE gel (4˜12% gradient gel, Invitrogen) stained with Coomassie depicting various Bst2 fusion proteins following affinity purification. B. Page after size-exclusion chromatography of the sample from lane 6 in FIG. 24A.
FIGS. 25A-25B show direct binding of Bst2 decoy to immune cells on A, Bst2 coated plate; and B, BSA coated plate.
FIG. 26 shows plasma half-life of Bst2 decoy or Fc fusions.
FIG. 27 shows inhibition of Bst2 decoy-Fc fusions in the binding between Bst2 decoy and cells.
FIGS. 28A-28D show H & E staining of tissue, which show the effect of Bst2 decoy-Fc fusions on a mouse model of asthma. A. Normal mouse, B. asthma mouse untreated, C. asthma mouse treated with dBst2:dBst2-IgG1 Fc, D. asthma mouse treated with dBst2-IgG1 Fc.
FIG. 29 shows binding of phage clones to Bst2/Damp 1 decoy.
FIGS. 30A-30B show anti-Bst2/Damp 1 monoclonal antibody. (A) Heavy chain variable regions; and (B) kappa chain variable regions. CDR1, CDR2 and CDR3 regions are boxed as well as indicated by asterisks.
FIGS. 31A-31B show anti-Bst2 monoclonal antibodies transiently expressed and purified on a PAGE gel. (A) under non-reducing conditions; (B) under reducing conditions.
Patent Application
20070154479
