1. A semiconductor device comprising:
a semiconductor substrate including trenches defining active regions;
lower and upper device isolation patterns disposed in the trenches;
an intergate insulation pattern and a control gate electrode disposed on the semiconductor substrate to cross over the active regions;
a charge storage electrode between the control gate electrode and the active regions; and
a gate insulation pattern between the charge storage electrode and the active regions,
wherein the intergate insulation pattern directly contacts the upper device isolation pattern between the active regions.
2. The device of claim 1, wherein the lower device isolation pattern is a chemically vapor deposited silicon oxide film and the upper device isolation pattern is a high-density plasma silicon oxide film.
3. The device of claim 1, wherein the lower device isolation pattern includes a discontinuous interface extending toward a surface of the semiconductor substrate from a surface of the lower device isolation pattern, and wherein the upper device isolation pattern covers the discontinuous interface.
4. The device of claim 1, further comprising spacers disposed at sidewalls of the charge storage electrode under the intergate insulation pattern,
wherein the spacers expose a top surface of the upper device isolation pattern.
5. The device of claim 4, wherein the upper device isolation pattern comprises a recess region between the spacers, and wherein a bottom surface of the recess region is lower than a bottom surface of the spacers and higher than a top surface of the lower device isolation pattern.
6. The device of claim 1, wherein an upper surface of the lower device isolation pattern is substantially planar.
7. The device of claim 3, wherein the upper device isolation pattern is free of discontinuous interfaces.
8. A semiconductor device comprising:
a semiconductor substrate including trenches defining active regions;
lower and upper device isolation patterns disposed in the trenches;
an intergate insulation pattern and a control gate electrode disposed on the semiconductor substrate to cross over the active regions;
a charge storage electrode between the control gate electrode and the active regions; and
a gate insulation pattern between the charge storage electrode and the active regions,
wherein the intergate insulation pattern directly contacts the upper device isolation pattern between the active regions,
wherein the lower device isolation pattern includes a discontinuous interface extending toward a surface of the semiconductor substrate from a surface of the lower device isolation pattern, and
wherein the upper device isolation pattern covers the discontinuous interface.
9. A semiconductor device comprising:
a semiconductor substrate including trenches defining active regions;
lower and upper device isolation patterns disposed in the trenches;
an intergate insulation pattern and a control gate electrode disposed on the semiconductor substrate to cross over the active regions;
a charge storage electrode between the control gate electrode and the active regions; and
a gate insulation pattern between the charge storage electrode and the active regions,
wherein the intergate insulation pattern directly contacts the upper device isolation pattern between the active regions,
wherein the lower device isolation pattern includes a discontinuous interface extending toward a surface of the semiconductor substrate from a surface of the lower device isolation pattern,
wherein the upper device isolation pattern covers the discontinuous interface, and
wherein the upper device isolation pattern is free of discontinuous interfaces.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
What is claimed is:
1. A nucleic acid molecule, or its complement, that encodes an anti-platelet binding protein, wherein the nucleic acid is isolated from a phage display library by an in vitro selection process that comprises screening a diverse human antibody variable domain expression library against at least one human platelet antigen, and the human antibody variable domain expression library expresses single-chain proteins.
2. The nucleic acid molecule of claim 1, wherein the human antibody variable domain expression library is derived from human immune cell variable sequences and the in vitro selection process comprises building a human sFv phage display library and panning under conditions that select a nucleic acid molecule encoding an sFv able to bind to at least one human platelet antigen with high affinity.
3. The nucleic acid of claim 1 wherein the platelet antigen is selected from the group consisting of a human platelet glycoprotein IIb, human platelet glycoprotein IIIa, human platelet glycoprotein IIbIIIa complex, a human platelet alphavbeta3 vitronectin receptor, a peptide sequence of human leukocyte integrin alphaM I domain ranging from about Cys128 to about Ser172, a peptide having the amino acid sequence of GCPQEDSDIAFLIDGSGSIIPHDF (SEQ ID NO:1), a peptide having the amino acid sequence of DYPVDIYYLMDLSYSMKDDLWSIQN (SEQ ID NO:2) and combinations thereof.
4. An expression vector comprising the nucleic acid of claim 1 operably linked to a promoter.
5. The expression vector of claim 4 wherein the promoter is inducible.
6. The expression vector of claim 4 wherein the promotor is tissue-specific.
7. The expression vector of claim 6 wherein the promotor is tissue-specific for a tissue selected from the group consisting of arterial endothelial cells, mammary gland cells, bladder epithelium, and combinations thereof.
8. The expression vector of claim 6 wherein the promoter is effective for expression in plant cells.
9. The expression vector of claim 6, wherein said promoter is selected from the group consisting of a beta-lactamase promoter, a lac promoter, a trp promoter, a phoA promoter, an araBAD promoter, a T7 promoter, derivatives of the lambda PL and PR promotor and an OLPR hybrid promoter.
10. The expression vector of claim 4, selected from the group consisting of a plasmid, a phage, a cosmid, a retrovirus vector, a DNA virus vector and a chromosomally integrated vector.
11. A host cell comprising the vector of claim 4.
12. A non-human mammal comprising cells transformed by the vector of claim 4 that produces the anti-platelet binding protein expressed by the vector.
13. A non-human transgenic mammal that comprises the vector of claim 4 that produces the anti-platelet binding protein expressed by the vector.
14. An anti-platelet binding protein encoded by the nucleic acid molecule of claim 1 that binds to activated or non-activated human platelet glycoprotein IIbIIIa receptor and inhibits platelet aggregation or thrombus formation.
15. The anti-platelet binding protein of claim 14 that is an sFv protein that has a molecular weight ranging from about 24 kDa to about 30 kDa, per sFv subunit.
16. The anti-platelet binding protein of claim 14 that is an sFv protein that binds to a human platelet glycoprotein IIbIIIa receptor with a Kd ranging from about 1 pM to about 10 nM and a koff from about 105 sec1 to about 103 sec1.
17. A composition comprising the anti-platelet binding protein of claim 14 and a pharmaceutically acceptable carrier.
18. A substantially isolated and purified human antibody that binds to a platelet antigen that comprises an active antigen-binding site of the anti-platelet binding protein of claim 14.
19. The substantially isolated and purified human antibody of claim 18 that is a monoclonal antibody.
20. A human anti-platelet binding protein that comprises a fragment of the human antibody of claim 18 selected from the group consisting of an Fab fragment, an Fv fragment, a light chain fragment, a heavy chain fragment, and combinations thereof.
21. A conjugate comprising a non-antigenic polymer covalently linked to the single-chain antigen-binding polypeptide of claim 15.
22. The conjugate of claim 21, wherein said non-antigenic polymer is selected form the group consisting of dextran, polyvinyl pyrrolidones, polyacryl amides, polyvinyl alcohols and carbohydrate-based polymers.
23. The conjugate of claim 21, wherein said non-antigenic polymer is a polyalkylene oxide.
24. The conjugate of claim 23, wherein said polyalkylene oxide comprises an alkyl terminal.
25. The conjugate of claim 23, wherein said polyalkylene oxide is polyethylene glycol.
26. The conjugate of claim 24, wherein said alkyl-terminated polyalkylene oxide is a monomethyl-terminated polyethylene glycol, (mPEG).
27. The conjugate of claim 21, wherein said non-antigenic polymer has a molecular weight ranging from about 200 to about 60,000.
28. The conjugate of claim 21, wherein said non-antigenic polymer has a molecular weight ranging from about from about 1,000 to about 40,000.
29. The conjugate of claim 21, wherein said non-antigenic polymer has a molecular weight ranging from about 2,000 to about 12,500.
30. The conjugate of claim 21, comprising a thiol linkage between said single-chain antigen-binding polypeptide and said non-antigenic polymer.
31. The conjugate of claim 21, comprising a urethane linkage between said single-chain antigen-binding polypeptide and said non-antigenic polymer.
32. A method of producing an anti-platelet binding protein by culturing a host cell transformed with the vector of claim 4.
33. A method of producing an anti-platelet binding protein by administering the vector of claim 4 to a mammal, under conditions effective to allow cells of said mammal to be productively transfected with said vector.
34. A method of producing an anti-platelet binding protein by producing a transgenic mammal expressing the vector of claim 4 in a tissue selected from the group consisting of arterial endothelial cells, mammary gland, bladder epithelium, and combinations thereof.
35. A method of producing an anti-platelet binding protein by producing a transgenic plant expressing the vector of claim 4 in a plant tissue selected from the group consisting of seeds, fruit, leaves, stems, roots, and combinations thereof.
36. A method of inhibiting platelet aggregation or platelet mediated thrombus formation in blood comprising contacting said blood with an effective amount of the anti-platelet binding protein of claim 14.
37. A method of inhibiting platelet aggregation or platelet mediated thrombus formation in blood comprising contacting said blood with host cells that produce the anti-platelet binding protein of claim 14.
38. The method of claim 36 wherein said blood is present in a mammal.
39. The method of claim 37 wherein said host cells are autologous to said mammal.
40. The method of claim 36 wherein said anti-platelet binding protein is present in said blood in a concentration ranging from about 1 pg to 1 mg per ml of whole blood.
41. A method of inhibiting platelet aggregation or platelet mediated formation of fibrin in a blood vessel, said blood vessel having an endothelial lining in need of treatment thereof, comprising contacting the endothelial lining of said blood vessel with the vector of claim 4.
42. The method of claim 41 wherein said blood vessel is an autologous graft that is contacted with the vector before or after being grafted into a mammal in need thereof.
43. The method of claim 38 wherein said mammal is a human.