1. A biocompatible composition for treating, repairing, or replacing biological tissue comprising microspheres of a biodegradable polymer dispersed in a matrix of a non-biodegradable hydrogel, wherein the composition is formulated to have compatible mechanical properties and physical functionality with the biological tissue.
2. A composition according to claim 1, wherein upon biodegradation, the microspheres form open-celled pores.
3. A composition according to claim 1 where the composition has pre-existing pores throughout its structure.
4. A composition according to claim 1, wherein the non-biodegradable hydrogel is polyvinyl alcohol.
5. A composition according to claim 1, wherein the non-biodegradable hydrogel is polyvinyl alcohol and polyvinylpyrrolidone.
6. A composition according to claim 1, wherein the biodegradable polymer is poly lactic glycolic acid.
7. A composition according to claim 6, wherein the mole ratio of lactic to glycolic acid is about 50:50.
8. A composition according to claim 1, wherein the hydrogel further comprises a chemoattractant, chemokine, cytokine, adhesion molecules, or mixtures thereof.
9. A composition according to claim 1, wherein the microspheres further comprise a chemoattractant, chemokine, cytokine, adhesion molecules, or mixtures thereof.
10. A composition according to claim 9, wherein the chemoattractant is selected from the group consisting of IGF-1, FGF, BMP2, and mixtures thereof.
11. A composition according to claim 9, wherein the chemoattractant promotes the biosynthesis of extracellular-matrix.
12. A composition according to claim 9, wherein the type and amount of chemokine is compatable with the biological tissue.
13. A composition according to claim 1, wherein the microspheres have an average diameter between about 10 and about 100 microns.
14. A composition according to claim 1, wherein the composition is molded in the shape of a biological tissue defect.
15. A composition according to claim 1, wherein the composition is molded in the shape of a cylinder, cone, or ovoid for insertion into a biological tissue defect.
16. A composition according to claim 1, wherein the biological tissue is selected from the group consisting of cartilage, bone, bladder, liver, kidney, and mixtures thereof.
17. A composition according to claim 1, wherein the microspheres are unevenly dispersed in the matrix to form pores, avenues, or channels upon degradation.
18. A composition according to claim 17, wherein the pores, avenues, or channels formed upon degradation are sufficiently large to permit cellular migration into the remaining matrix of non-biodegradable hydrogel.
19. A composition according to claim 1, wherein the microspheres of biodegradable polymer are selected to degrade in vivo at a rate such that mechanical and functional characteristics of the implanted composition are maintained by the simultaneous ingrowth of biological material.
20. The composition according to claim 1, wherein the composition contains two or more discreet layers, wherein each layer has compatible mechanical properties and physical functionality for a distinct biological tissue.
21. The composition according to claim 20, wherein the composition has two layers, and further wherein one layer is compatible with bone and one layer is compatible with cartilage.
22. A method of making a composition according to claim 1, comprising forming microspheres containing a chemokine, dispersing the microspheres in a hydrogel matrix, and molding the matrix in the form of a biological tissue defect.
23. A method of treating, repairing, or replacing biological tissue comprising administering a composition according to claim 1 to a site of treatment, repair, or replacement in a patient.
24. The method of claim 23 wherein the biological tissue is selected from the group consisting of cartilage, bone, bladder, liver, kidney, and mixtures thereof.
25. A method of providing mechanical integrity to a biological defect comprising implanting a composition according to claim 1 to the site of a biological defect and allowing tissue ingrowth within biodegradable pores of the implanted composition, wherein the implanted composition provides mechanical integrity before, during, and after tissue ingrowth.
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 polypeptide comprising a single-domain of the variable region of the heavy chain of an antibody molecule, which is soluble and stable and capable of binding a specific antigen of interest, said polypeptide comprising a natural framework scaffold of a mammalian monoclonal antibody without induced mutations or modifications in the original VHVL interface framework residues, said VHVL interface comprising at least one charged residue.
2. The polypeptide of claim 1 wherein the polypeptide is substantially monomeric.
3. The polypeptide of claim 1, wherein the polypeptide is encoded by a polynucleotide isolated from a phage clone selected from a phage-display library comprising a plurality of recombinant phage, each of said recombinant phages having an expression vector encoding a single-domain of the variable region of the heavy chain of an antibody molecule comprising a natural framework scaffold of a mammalian monoclonal antibody without induced mutations or modifications in the original VHVL interface framework residues, having a unique VHVL interface comprising at least one charged residue and a randomized CDR3.
4. The polypeptide of claim 3 wherein the selected clone is produced in E. coli as insoluble inclusion bodies and the isolated polypeptide is subsequently refolded in-vitro and purified.
5. The polypeptide claim 3 wherein the scaffold element representing the VHVL interface comprises the sequence Lysine-44, Leucine-45, and Tryptophan-47.
6. The polypeptide of claim 1 wherein the specific antigen of interest is an immunoglobulin molecule.
7. The polypeptide of claim 3 wherein the CDR3 sequence between residues 95 and 100 C comprises the consensus sequence: Gly-X-Ser-Pro-Gln, wherein X represents any amino acid.
8. The polypeptide of claim 3 wherein the CDR3 sequence between residues 95 and 100 C is selected from the sequences: Gln-Ser-Gly-Gln-Ser-Pro-Gln-Ser-Ile, and Asn-Gly-Lys-Ser-Pro-Gln-Ala-Ala-Trp.
9. The polypeptide of claim 1 wherein the specific antigen of interest is tumor necrosis factor.
10. The polypeptide of claim 9 wherein the CDR3 sequence between residues 95 and 100 C comprises the sequence: Phe-Pro-Thr-Gly-Asp-Leu-Ala-Glu-Lys.
11. The polypeptide of claim 1 wherein the specific antigen of interest is Streptavidin.
12. The polypeptide of claim 11 wherein the CDR3 sequence between residues 95 and 100 C is selected from the sequences: His-Ala-Gln-Arg-Arg-Pro-Trp-Ile-Arg, and Glu-Asp-Pro-His-Pro-Gln-Arg-Gly-Tyr.
13. A peptide capable of binding a specific antigen of interest, said peptide being derived from the randomized sequence of the CDR3 region of a polypeptide comprising a single-domain of the variable region of the heavy chain of an antibody molecule, which is soluble and stable and capable of binding said specific antigen of interest, said polypeptide comprising a natural framework scaffold of a mammalian monoclonal antibody without induced mutations or modifications in the original VHVL interface framework residues, said VHVL interface comprising at least one charged residue.
14. The peptide of claim 13 wherein the polypeptide is encoded by a polynucleotide isolated from a phage clone selected from a phage-display library comprising a plurality of recombinant phage, each of said recombinant phages having an expression vector encoding a single-domain of the variable region of the heavy chain of an antibody molecule comprising a natural framework scaffold of a mammalian monoclonal antibody without induced mutations or modifications in the original VHVL interface framework residues, having a unique VHVL interface comprising at least one charged residue and a randomized CDR3.
15. The peptide of claim 13 wherein the peptide comprises 4-20 amino acids.
16. The peptide of claim 13 wherein the peptide comprises 7-15 amino acids.
17. The peptide of claim 13 wherein the specific antigen of interest is an immunoglobulin molecule.
18. The peptide of claim 13 wherein the specific antigen of interest is tumor necrosis factor.
19. A pharmaceutical composition comprising as an active ingredient the polypeptide of claim 1, and a physiologically acceptable diluent or carrier.
20. A pharmaceutical composition comprising as an active ingredient the peptide of claim 13, and a physiologically acceptable diluent or carrier.
21. A phage-display library comprising a plurality of recombinant phage, each of said recombinant phage having an expression vector encoding a single-domain of the variable region of the heavy chain of an antibody molecule comprising a natural framework scaffold of a mammalian monoclonal antibody without any mutations or modifications in the original interface framework residues, having a unique VHVL interface comprising at least one charged residue, and a randomized CDR3.
22. The phage display library of claim 21 wherein the single-domain variable region of the heavy chain is substantially monomeric.
23. The phage display library of claim 21 wherein the scaffold element representing the VHVL interface comprises the sequence Lysine-44, Leucine-45, and Tryptophan-47.
24. An isolated phage clone which binds selectively to a specific antigen of interest, said clone being selected from a phage-display library comprising a plurality of recombinant phage, each of said recombinant phage having an expression vector encoding a single-domain of the variable region of the heavy chain of an antibody molecule comprising a natural framework scaffold of a mammalian monoclonal antibody without any mutations or modifications in the original interface framework residues, having a unique VHVL interface and a randomized CDR3.
25. The isolated phage clone of claim 24 wherein the phage display library comprises a scaffold element representing the VHVL interface comprising the sequence Lysine-44, Leucine-45, and Tryptophan-47.
26. The isolated phage clone of claim 24 encoding a polypeptide wherein the specific antigen of interest is an immunoglobulin molecule.
27. The isolated phage clone of claim 24 encoding a polypeptide wherein the specific antigen of interest is tumor necrosis factor.
28. A method of treatment of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the polypeptide of claim 1.
29. A method of diagnosis of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient the polypeptide of claim 1 in an amount which is effective to diagnose the disease.
30. A method of treatment of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the polypeptide of claim 13.
31. A method of diagnosis of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient the polypeptide of claim 13 in an amount which is effective to diagnose the disease.
32. A method of treatment of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the polypeptide of claim 9.
33. A method of diagnosis of a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising as an active ingredient the polypeptide of claim 9 in an amount which is effective to diagnose the disease.