| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 31.12.02 |
| PATENT TITLE |
Insulin-like growth factor binding protein (IGFBF-5) |
| PATENT ABSTRACT |
A purified insulin-like growth factor binding protein (IGFBP) selected from the group consisting of insulin-like growth factor binding protein having an amino acid sequence that, preferably, is at least 70% homologous to the amino acid sequence of FIG. 1 and fragments thereof that are capable of binding to an antibody specific for the protein or to an insulin-like growth factor is described. This new IGFBP is designated herein as IGFBP-5. Recombinant DNA molecules encoding the binding proteins and subsequences thereof are also described along, with recombinant microorganisms and cell lines containing the DNA molecules and methods for producing the binding proteins using recombinant hosts containing the relevant DNA molecules. Antibodies to the protein, useful in various diagnostic applications, are also described. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | November 28, 1994 |
| PATENT REFERENCES CITED |
Shimasaki et al. 1991 J. Biol. Chem. 266(16): 10646.* Keifer et al 1991. Biochem. Biophys. Res. Commun. 176 (11): 219.* R.S. Fuller et al., Yeast Prohormone Processing Enzyme . . . , (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 1434. D. Julius et al., Glycosylation and Processing of Prepro-x-Factor Through the Yeast Secretory Pathway, (1984) Cell, 36:309. Bathurst et al, Yeast KEX2 Protease has the Properties of Human Proalbumin Converting Enzyme, (1987) Science 235:348-350. Thomas et al., Yeast KEX2 Endopeptidase Correctly Cleaves a Neuroendocrine Prohormone in Mammalian Cells, (1988), Science 241:226. A.M.W. van den Ouweland et al., Structural Homology Between the Human Fur Gene Product and the Subtilisin-Like Protease Encoded by Yeast KEX2, (1990), Nuc. Acids Res. 18:664. R.S. Fuller, et al., Intracellular Targeting and Structural Conservation of a Prohormone-Processing Endoprotease, (1989), Science 246:482. S.P. Smeekens et al., Identification of a Human Insulinoma cDNA Encoding a Novel Mammalian Protein . . . , (1990), J. Biol. Chem. 265:2997. A.J.M. Roebroek, Evolutionary Conserved Close Linkage of the c-fes/fps Proto-Oncogene and Genetic Sequences Encoding a Receptor-Like Protein, (1986), EMBO J. 5:2197. A.J.M. Roebroek et al., Characterization of Human c-fes/fps Reveals a New Transcription Unit (FUR) . . . , (1986), Molec. Biol. Rep. 11:117. R.S. Fuller et al., The Saccharomyces Cerevisiae KEX2 Gene . . . , (1986) Microbiol., 1986:273. S. Shimasaki et al., 2nd International Symposium on Insulin-Like Growth Factors/Somatomedins entitled Isolation and Molecular Characterization of Three Novel IGFBPs. G. Povoa, et al., Eur. J. Biochem (1984) 144:199-204.* R. Koistinen, et al. Endocringology (1986) 118:1375-1378.* D. Powell, et al. J. Chromatogr. (1987) 420:163-170.* Y. L. Lee, et al. Mol Endocrinol. (1988) 2:404-411.* A. Brinkman, et al. The EMBO Journal (1988) 7:2417-2423.* M. Brewer, et al. Bioch. Biophys. Res. Com. (1988) 152:1289-1297.* C. Binkert, et al. The EMBO Journal (1989) 8:2497-2502.* R. Baxter, et al. Bioch. Biopys. Res. Com. (1986). 139:1256-1261.* W. Wood, et al. Mol. Endocrinol. (1988) 2:1176-1185.* C. Mottola, et al. Journ. of Biol. Chem. (1986) 261:11180-11188.* R. Lyons, et al. Mol. Cell. Endocrinol. (1986) 45:263-270. S. Mohan, et al. Proc. Natl. Acad. Sci. (1989) 86:8338-8342. J. Zapf, et al. J. of Biol. Chem. (1990) 265:14892-14898. S. Shimasaki, et al. Mol. Endocrinology (1990) 4:1451-1458. S. Shimasaki, et al. Second International IGF Symposium Abstract (1991). Andress et al., "Human Osteoblast-derived Insulin-like growth Factor (IGF) Binding Protein-5-Stimulates Osteoblast Mitogenesis and Potentiates IGF Action," The Journal of Biological Chemistry 267(31):22467-22472 (1992). Andress et al., "A Novel Human Insulin-like Growth Factor Binding Protein Secreted by Osteoblast-like Cells," Biochemical and Biophysical Research Communications 276(1):213-218 (1991). Shimasaki et al., "Isolation and Molecular Cloning of Insulin-like Growth Factor-Binding Protein-6," Mol. Endocrin 5:938-948 (1991). Shimasaki et al., "Identification of Five Different Insulin-like Growth Factor Binding Proteins (IGFBPs) from Adult Rat Serum and Molecular Cloning of a Novel IGFBP-5 in Rat and Human," Journal of Biological Chemistry 266(16):10646-10653 (1991). |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A purified polypeptide capable of binding to an insulin-like growth factor selected from the group consisting of human insulin-like growth factor I and human insulin-like growth factor II, said polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272, of SEQ ID NO:8; (b) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272, of SEQ ID NO:8; (c) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272, of SEQ ID NO:8; and (d) a polypeptide comprising a fragment of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8 wherein said fragment comprises at least 10 consecutive amino acids thereof. 2. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 3. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 4. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 5. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 6. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 7. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 8. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 9. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 10. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 11. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 12. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 13. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 14. The polypeptide of claim 1, wherein the polypeptide comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 15. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 16. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 17. The polypeptide of claim 1, wherein the polypeptide consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 18. The polypeptide of claim 1, wherein the polypeptide consists of the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 19. The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 20. The polypeptide of claim 1, wherein the polypeptide comprises a fragment of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8 and wherein said fragment comprises at least 25 consecutive amino acids thereof. 21. A composition comprising a pharmaceutically acceptable carrier or diluent, and the purified polypeptide of claim 1. 22. A pharmaceutical composition for treating a physiological disorder that results from an excessive production of free IGF comprising: (a) the polypeptide of claim 1, and (b) a pharmaceutically acceptable carrier or diluent. 23. A polypeptide produced by a process comprising the steps of (a) providing a host cell transformed by a nucleic acid sequence encoding a polypeptide capable of binding to an insulin-like growth factor selected from the group consisting of human insulin-like growth factor I and human insulin-like growth factor II, said polypeptide selected from the group consisting of (a) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272, of SEQ ID NO:8; (b) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272, of SEQ ID NO:8; (c) a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272, of SEQ ID NO:8; and (d) a polypeptide comprising a fragment of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8 wherein said fragment comprises at least 10 consecutive amino acids thereof, wherein the nucleic acid sequence is under the control of a promoter capable of providing for expression of the polypeptide in the host cell; and (b) culturing the host cell under conditions that permit expression of the polypeptide. 24. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 25. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 26. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 27. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 28. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 29. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 30. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 31. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 32. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 33. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 34. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide consists of the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 35. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 36. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 37. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 38. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 39. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 40. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 41. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 42. The polypeptide of claim 23, wherein said nucleic acid sequence encodes a polypeptide comprising a fragment of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8 and wherein said fragment comprises at least 25 consecutive amino acids thereof. 43. A purified insulin-like growth factor binding protein-5 (IGFBP-5) having the amino acid sequence of SEQ ID NO:8. 44. A recombinant DNA molecule comprising a nucleic acid sequence encoding a polypeptide according to claim 1. 45. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 46. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 47. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 48. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 49. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 50. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8. 51. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 52. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 53. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 54. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 55. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 56. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 16-272 of SEQ ID NO:8. 57. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that comprises a polypeptide comprising an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 58. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 80% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 59. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 90% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 60. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of an amino acid sequence that has at least 95% sequence identity to the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 61. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide that consists of the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 62. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide comprising the amino acid sequence depicted at positions 21-272 of SEQ ID NO:8. 63. The recombinant DNA molecule of claim 44, wherein the nucleic acid sequence encodes a polypeptide comprising a fragment of the amino acid sequence depicted at positions 1-272 of SEQ ID NO:8 and wherein said fragment comprises at least 25 consecutive amino acids thereof. 64. The recombinant DNA molecule of claims 44, wherein the molecule comprises the nucleic acid sequence depicted at nucleotide positions 1-859 of SEQ ID NO:7. 65. The recombinant DNA molecule of claim 44, wherein the molecule comprises the nucleic acid sequence depicted at nucleotide positions 89-859 of SEQ ID NO:7. 66. The recombinant DNA molecule of claim 44, wherein the molecule comprises the nucleic acid sequence depicted at nucleotide positions 104-859 of SEQ ID NO:7. 67. The recombinant DNA molecule of claim 44, wherein the sequence is a human DNA sequence. 68. The recombinant DNA molecule of claim 44, wherein the sequence is a genomic sequence. 69. The recombinant DNA molecule of claim 44, wherein the sequence is a cDNA sequence. 70. A recombinant DNA expression vector comprising the DNA of claim 44, wherein the vector is capable of effecting the expression of the polypeptide in a microorganism. 71. The recombinant DNA molecule of claim 44, wherein the DNA molecule is contained in pBsBP6.1. 72. An expression vector comprising the DNA molecule of claim 44 and a regulatory sequence for expression of the polypeptide. 73. The expression vector as claimed in claim 72, wherein the vector is operable for expression in an insect cell. 74. The expression vector as claimed in claim 72, wherein the vector is operable for expression in a yeast host. 75. The expression vector as claimed in claim 72, wherein the vector is operable for expression in a bacterial host. 76. The expression vector as claimed in claim 72, wherein the regulatory sequence comprises a promoter sequence selected from the group consisting of ADH2/GAPDH and GAPDH promoter sequences. 77. The expression vector as claimed in claim 76 wherein the vector further comprises east a fragment of a pre-pro alpha-factor leader sequence sufficient for secretion. 78. A host cell transformed with the expression vector of claim 72. 79. The host cell as claimed in claim 78, wherein the cell is selected from the group consisting of a bacterial cell, a yeast cell, a mammalian cell and an insect cell. 80. A recombinant microorganism or cell line containing the DNA molecule of claim 44. 81. The microorganism of claim 80, wherein the microorganism is a yeast. 82. The cell line of claim 80, wherein the cell line is a CHO cell line. 83. A method for producing a recombinant polypeptide comprising the steps of culturing a recombinant host transformed by the DNA molecule of claim 44 under conditions that allow the expression of the polypeptide; and isolating the expressed polypeptide. 84. The method of claim 83, wherein the host is a microorganism. 85. The method of claim 83, wherein the host is a eucaryotic cell. # SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 8 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 14 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCTGTAGATC TCCG # # # 14 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AATTCGGAGA TCTACAGG # # # 18 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGATCTGAAT TCGCCAATGA # # # 20 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: AGATCTAAGC TTCACCACCA CA # # 22 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCAAAGGATT CTACAAGAG # # # 19 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc #= "oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CAAACCTTCC CGTGGCCGC # # # 19 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1612 base #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCTCCTGCC CCACCCCGAG GTAAAGGGGG CGACTAAGAG AAGATGGTGT T #GCTCACCGC 60 GGTCCTCCTG CTGCTGGCCG CCTATGCGGG GCCGGCCCAG AGCCTGGGCT C #CTTCGTGCA 120 CTGCGAGCCC TGCGACGAGA AAGCCCTCTC CATGTGCCCC CCCAGCCCCC T #GGGCTGCGA 180 GCTGGTCAAG GAGCCGGGCT GCGGCTGCTG CATGACCTGC GCCCTGGCCG A #GGGGCAGTC 240 GTGCGGCGTC TACACCGAGC GCTGCGCCCA GGGGCTGCGC TGCCTCCCCC G #GCAGGACGA 300 GGAGAAGCCG CTGCACGCCC TGCTGCACGG CCGCGGGGTT TGCCTCAACG A #AAAGAGCTA 360 CCGCGAGCAA GTCAAGATCG AGAGAGACTC CCGTGAGCAC GAGGAGCCCA C #CACCTCTGA 420 GATGGCCGAG GAGACCTACT CCCCCAAGAT CTTCCGGCCC AAACACACCC G #CATCTCCGA 480 GCTGAAGGCT GAAGCAGTGA AGAAGGACCG CAGAAAGAAG CTGACCCAGT C #CAAGTTTGT 540 CGGGGGAGCC GAGAACACTG CCCACCCCCG GATCATCTCT GCACCTGAGA T #GAGACAGGA 600 GTCTGAGCAG GGCCCCTGCC GCAGACACAT GGAGGCTTCC CTGCAGGAGC T #CAAAGCCAG 660 CCCACGCATG GTGCCCCGTG CTGTGTACCT GCCCAATTGT GACCGCAAAG G #ATTCTACAA 720 GAGAAAGCAG TGCAAACCTT CCCGTGGCCG CAAGCGTGGC ATCTGCTGGT G #CGTGGACAA 780 GTACGGGATG AAGCTGCCAG GCATGGAGTA CGTTGACGGG GACTTTCAGT G #CCACACCTT 840 CGACAGCAGC AACGTTGAGT GATGCGTCCC CCCCCAACCT TTCCCTCACC C #CCTCCCACC 900 CCCAGCCCCG ACTCCAGCCA GCGCCTCCCT CCACCCCAGG ACGCCACTCA T #TTCATCTCA 960 TTTAAGGGAA AAATATATAT CTATCTATTT GAGGAAACTG AGGACCTCGG A #ATCTCTAGC 1020 AAGGGCTCAA CTTCGAAAAT GGCAACAACA GAGATGCAAA AAGCTAAAAA G #ACACCCCCC 1080 CCCTTTAAAT GGTTTTCTTT TTGAGGCAAG TTGGATGAAC AGAGAAGGGA A #GAGAGGAAG 1140 AACGAGAGGA AGAGAAGGGA AGGAAGTGTT TGTGTAGAAG AGAGAGAAAG A #CGAATAGAG 1200 TTAGGAAAAG GAAGACAAGC AGGTGGGCAG GAAGGACATG CACCGAGACC A #GGCAGGGGC 1260 CCAACTTTCA CGTCCAGCCC TGGCCTGGGG TCGGGAGAGG TGGGCGCTAG A #AGATGCAGC 1320 CCAGGATGTG GCAATCAATG ACACTATTGG GGTTTCCCAG GATGGATTGG T #CAGGGGGAG 1380 AAAGGAAAAG GCAAAACACT CCAGGACCTC TCCCGGATCT GTCTCCTCCT C #TAGCCAGCA 1440 GTATGGACAG CTGGACCCCT GAACTTCCTC TCCTCTTACC TGGGCAGAGT G #TTGTCTCTC 1500 CCCAAATTTA TAAAAACTAA AATGCATTCC ATTCCTCTGA AAGCAAAACA A #ATTCATAAT 1560 TGAGTGATAT TAAATAGAGA GGTTTTCGGA AGCAGATCTG TGAATATGAA A #T 1612 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 272 amino #acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Val Leu Leu Thr Ala Val Leu Leu Leu L #eu Ala Ala Tyr Ala Gly 1 5 # 10 # 15 Pro Ala Gln Ser Leu Gly Ser Phe Val His C #ys Glu Pro Cys Asp Glu 20 # 25 # 30 Lys Ala Leu Ser Met Cys Pro Pro Ser Pro L #eu Gly Cys Glu Leu Val 35 # 40 # 45 Lys Glu Pro Gly Cys Gly Cys Cys Met Thr C #ys Ala Leu Ala Glu Gly 50 # 55 # 60 Gln Ser Cys Gly Val Tyr Thr Glu Arg Cys A #la Gln Gly Leu Arg Cys 65 #70 #75 #80 Leu Pro Arg Gln Asp Glu Glu Lys Pro Leu H #is Ala Leu Leu His Gly 85 # 90 # 95 Arg Gly Val Cys Leu Asn Glu Lys Ser Tyr A #rg Glu Gln Val Lys Ile 100 # 105 # 110 Glu Arg Asp Ser Arg Glu His Glu Glu Pro T #hr Thr Ser Glu Met Ala 115 # 120 # 125 Glu Glu Thr Tyr Ser Pro Lys Ile Phe Arg P #ro Lys His Thr Arg Ile 130 # 135 # 140 Ser Glu Leu Lys Ala Glu Ala Val Lys Lys A #sp Arg Arg Lys Lys Leu 145 #150 #155 #160 Thr Gln Ser Lys Phe Val Gly Gly Ala Glu A #sn Thr Ala His Pro Arg 165 # 170 # 175 Ile Ile Ser Ala Pro Glu Met Arg Gln Glu S #er Glu Gln Gly Pro Cys 180 # 185 # 190 Arg Arg His Met Glu Ala Ser Leu Gln Glu L #eu Lys Ala Ser Pro Arg 195 # 200 # 205 Met Val Pro Arg Ala Val Tyr Leu Pro Asn C #ys Asp Arg Lys Gly Phe 210 # 215 # 220 Tyr Lys Arg Lys Gln Cys Lys Pro Ser Arg G #ly Arg Lys Arg Gly Ile 225 #230 #235 #240 Cys Trp Cys Val Asp Lys Tyr Gly Met Lys L #eu Pro Gly Met Glu Tyr 245 # 250 # 255 Val Asp Gly Asp Phe Gln Cys His Thr Phe A #sp Ser Ser Asn Val Glu 260 # 265 # 270 86. A method of producing a recombinant polypeptide comprising: (a) providing a cell that comprises the isolated DNA molecule of claim 44 that is operatively connected to control sequences that allows expression of the polypeptide; and (b) allowing the cells to produce the polypeptide. 87. An expression vector comprising the DNA molecule of claim 50 and a regulatory sequence for expression of the polypeptide. 88. An expression vector comprising the DNA molecule of claim 56 and a regulatory sequence for expression of the polypeptide. 89. An expression vector comprising the DNA molecule of claim 62 and a regulatory sequence for expression of the polypeptide. 90. An expression vector comprising the DNA molecule of claim 63 and a regulatory sequence for expression of the polypeptide. 91. A host cell transformed with the expression vector of claim 87. 92. A host cell transformed with the expression vector of claim 88. 93. A host cell transformed with the expression vector of claim 89. 94. A host cell transformed with the expression vector of claim 90. 95. A method for producing a recombinant polypeptide comprising the steps of culturing the host cell of claim 91 under conditions that allow the expression of the polypeptide; and isolating the expressed polypeptide. 96. A method for producing a recombinant polypeptide comprising the steps of culturing the host cell of claim 92 under conditions that allow the expression of the polypeptide; and isolating the expressed polypeptide. 97. A method for producing a recombinant polypeptide comprising the steps of culturing the host cell of claim 93 under conditions that allow the expression of the polypeptide; and isolating the expressed polypeptide. 98. A method for producing a recombinant polypeptide comprising the steps of culturing the host cell of claim 94 under conditions that allow the expression of the polypeptide; and isolating the expressed polypeptide. 99. An isolated DNA molecule encoding insulin-like growth factor binding protein-5 (IGFBP-5) having the sequence of SEQ ID NO:8. 100. An antibody which specifically binds to a polypeptide according to claim 1. 101. The antibody of claim 100, wherein said antibody is a monoclonal antibody. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION 1. Field of the Disclosure The present invention relates generally to production of polypeptides from recombinant DNA molecules encoding such polypeptides. More specifically, this invention relates to a new insulin-like growth factor binding protein (designated herein as IGFBP-5), recombinant DNA molecules encoding this polypeptide, and methods for producing IGFBP-5 from recombinant host cells. 2. Description of the Related Art Insulin-like growth factors (IGFS) are low molecular weight polypeptide hormones with structural homology to proinsulin. Two different IGFs are known, namely IGF-I and IGF-II, which are mitogenic in vitro for a wide variety of cells in tissue culture. Both IGFs stimulate in vitro the growth of various tissues and in particular they induce collagen synthesis. IGF-I mediates the growth promoting effect of growth hormone in chondrogenesis and bone formation and is therefore essential for normal growth of an individual. This is demonstrated by the fact that pygmies and toy poodles are deficient in IGF-I but have normal growth hormone level in their serum. IGF-II is believed to play a key role in fetal development and nerve growth. In addition to their primary effect on skeletal tissue IGFs also exhibit growth-stimulating functions on other tissues. Wound fibroblasts are known to produce IGFs which are effective in stimulating fibroblasts to grow and synthesize collagen, a structural protein normally required for wound healing. Vascularization of the wound tissue is also induced. Further, it has also been found that IGFs have an erythropoietin-like activity in that they induce hematopoiesis. Recent studies have also demonstrated that IGFs produced by certain cancer cells, e.g. breast and kidney cancer cells, auto-stimulate the proliferation of cancer cells and the vascular and fibrous tissues required to support the growth of cancer tissues. In addition to this, both IGFs show a spectrum of metabolic activities similar to those of insulin, in that they stimulate, in particular, the transport and metabolism of glucose. The biological effects of IGFs and insulin are mediated through their binding to specific receptors. In particular, both IGFs have the ability to bind to the insulin receptor with approximately 100-fold lower affinity than does insulin. Both IGFs have a concentration in blood approximately a hundred-fold higher than that of insulin. Hypoglycemia is prevented by a regulatory mechanism which involves carrier proteins present in blood and able to form complexes with IGFs. Thus, IGFs circulate in the blood in the form of a complex which has no insulin-like activity. Through their association with carrier proteins (hereinafter referred to as IGF binding proteins or ICFBPs), binding of IGFs to cell surface receptors is inhibited. It has also been demonstrated that another function of the IGF binding proteins is to increase the short half-life of IGFs, which are subjected to rapid proteolytic degradation when present in the free form in blood. In accordance with the foregoing, IGFs may be useful in vitro to stimulate a) the growth of animals and humans with growth hormone deficiency, b) tissue regeneration, such as erythropoiesis and chondrogenesis, c) wound healing and d) the functions of various organs e.g. liver or kidney. As a result of their chondrogenesis stimulating activity, IGFs are of particularly suitable use for bone formation, e.g. in the treatment of osteoporosis. IGFs for use in the above-referred treatments are advantageously administered to a subject in association with at least one IGF binding protein. Through their association with carrier proteins (hereinafter referred to as IGF binding proteins or IGFBPs), binding of IGFs to cell surface receptors is inhibited. It has also been demonstrated that another function of the IGF binding proteins is to increase the short half-life of IGFs, which are subjected to rapid proteolytic degradation when present in the free form in blood. Administration of the combination of IGF and an IGF binding protein, rather than IGF alone, has beneficial effects including the prevention of hypoglycemia and possible mitogenic effects at injection sites and the prolongation of IGF half-life. Furthermore, it has been found that binding proteins are also useful for potentiating the erythropoietin like-effect of IGF-I. The binding proteins may also be useful for targeting IGFs to specific tissues. When administered alone, i.e., without any IGF, the binding proteins may also be therapeutically useful for blocking the adverse effects of IGFs, such as those which occur when IGFs are produced in excess, e.g. free IGFs secreted by certain cancer cells e.g. hormone-producing cancer cells such as breast or kidney cancer cells. IGF binding protein therapy may also prevent blindness as a secondary effect of diabetic proliferation retinopathy. Indeed it has been shown that IGFs may be one of the factors stimulating endothelial and fibroblast proliferation in diabetic retinopathy. Another therapeutic use of IGFBPs is the control of excessive growth in IGF binding protein-deficient subjects, since it is very likely that high IGF levels combined with abnormally low levels of binding protein are responsible for excessive growth. Known forms of IGFBPs include IGFBP-1, having a molecular weight of approximately 30-40 kd in humans. See, e.g., Povoa, G. et al., Eur. J. Biochem (1984) 144:199-204, relates to IGFBP-1, isolated and purified from amniotic fluid; Koistinen, R. et al., Endocrinology (1986) 118:1375-378, relates to IGFBP-1 isolated and purified from: human placenta; Powell, D. R. et al., J. Chromatogr. (1987) 420:163-170, relates to a 30-40 kd IGFBP-1 isolated and purified from conditioned medium of hepatoma G2 (Hep-G2) cells; Lee, Y. L. et al., Mol. Endocrinol. (1988) 2:404-411, relates to an amino acid sequence of IGFBP-1 isolated from Hep-G2 cells; Brinkman, A. et al., The EMBO Journal (1988) 7: 2417-2423, relates to an IGFBP-1 placental cDNA library; Brewer, M. T. et al., Bioch. Biophys. Res. Com. (1988) 152:1289-1297, pertains to nucleotide and amino acid sequences for IGFBP-1 cloned from a human uterine decidua library; WO89/09792, published Oct. 19, 1990, Clemmons, D. R., et al., pertains to cDNA sequences and cloning vectors for IGFBP-1 and IGFBP-2; WO89/08667, published Sept. 21, 1989, Drop, L. S., et al., relates to an amino acid sequence of insulin-like-growth factor binding protein 1 (IGFBP-1); WO89/09268, published Oct. 5, 1989, Baxter, R. C., relates to a cDNA sequence of IGFBP-1 and methods of expression for IGFBP-1. IGFBP-2 has a molecular weight of approximately 33-36 kd. See, e.g., Binkert, C. et al., The EMBO Journal (1989) 8:2497-2502, relates to a nucleotide and deduced amino acid sequence for IGFBP-2. IGFBP-3 has a molecular weight of 150 kd. See, e.g., Baxter, R. C. et al., Bioch. Biopys. Res. Com. (1986) 139:1256-1261, pertains to a 53 kd subunit of IGFBP-3 that was purified from human serum; Wood, W. I. et al., Mol. Endocrinol. (1988) 2:1176-1185, relates to a full length amino acid sequence for IGFBP-3 and cellular expression of the cloned IGFBP-3 cDNA in mammalian tissue culture cells; WO90/00569, published Jan. 25, 1990, Baxter, R. C., relates to isolating from human plasma an acid-labile subunit (ALS) of (IGFBP) complex and, the particular amino acid sequence for ALS pertains to a subunit of IGFBP-3. For nonhuman forms, see, e.g., Mottola, C. et al., Journ. of Biol. Chem. (1986) 261: 11180-11188, relates to a non-human form of IGFBP that was isolated in conditioned medium from rat liver BRL-3A cells and has a molecular weight of approximately 33-36 kd; Lyons, R. M. et al., Mol. Cell. Endocrinol. (1986) 45: 263-270, relates to a 34 kd cloned BRL-3A rat liver cell protein designated MCP; EPO Publ. No. 369 943, published May 23 1990, Binkert, C., et al., relates to a cDNA sequence of the rat BRL-3A binding protein and uses this sequence to screen three human cDNA libraries. Mohan, S. et al., Proc. Natl. Acad. Scii. (1989) 86:8338-8342, relates to an N-terminal amino acid sequence for an IGFBP (designated therein as IGFBP-4 but, using Applicants' terminology as defined in the applications listed below, actually corresponding to IGFBP-5) isolated from medium conditioned by human osteosarcoma cells and Shimasaki, S. et al., Mol. Endocrinology (1990) 4:1451-1458, pertains to IGFBP cDNAs encoding an IGFBP (designated therein as IGFBP-4 but, using Applicants' terminology, actually corresponding to IGFBP-5) from rat and human. Copending application Ser. No. 07/574,613, filed Aug. 28, 1990, which is co-owned by the present assignee, relates to IGFBP-6 and IGFBP-4 genetic material and amino acid sequences; copending application Ser. No. 07/576,648, filed Aug. 31, 1990, which is co-owned by the present assignee, relates to IGFBP-6 amino acid sequences; copending application Ser. No. 07/576,629, filed Aug. 31, 1990, which is co-owned by the present assignee, relates to genetic material encoding IGFBP-6; copending application Ser. No. 07/577,391, filed Aug. 31, 1990, which is co-owned by the present assignee, relates to IGFBP-4 amino acid sequences; copending application Ser. No. 07/577,392, filed Aug. 31, 1990, which is co-owned by the present assignee, relates to genetic material encoding IGFBP-4. Zapf, J. et al., J. of Biol. Chem. (1990) 265:14892-14898, pertains to four IGFBP's (IGFBP-2, IGFBP-3, a truncated form of IGFBP-3, and IGFBP-4) isolated from adult human serum by insulin-like growth factor (IGF) affinity chromatography and high performance liquid chromatography. The existence of a number of different IGF-binding proteins indicates that these proteins may have different functions. Because it is possible to diagnose disease states and to modify in various different ways the biological activity of IGFs using the currently known binding proteins, there is significant interest, in the discovery of new IGF-binding proteins having the same or different biological properties. SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide an IGF binding protein that differs from IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-6. It is further an object of the present invention to provide a new IGF binding protein using recombinant DNA molecules capable of expressing the new IGF binding protein (designated herein as IGFBP-5) in order to produce the binding protein. These and other objects of the invention have been accomplished by providing a purified IGFBP selected from a group consisting of an IGFBP having an amino acid sequence which is at least 60%, preferably 70% and more preferably 85%, and most preferably 90%, homologous to the amino acid sequence of FIG. 1 and fragments thereof wherein the fragments are of a sufficient length to be unique to this binding protein (e.g., 10, 15, 20, or 25 consecutive amino acids of said sequence), and further wherein the purified binding protein is capable of binding to an antibody specific for IGFBP-5 or an insulin-like growth factor. Recombinantly produced binding protein molecules and antibodies that recognize the new binding protein are also part of the invention. A significant advantage of producing IGFIBP-5 by recombinant DNA techniques rather than by isolating IGFBP-5 from natural sources is that equivalent quantities of IGFBP-5 can be produced by using less starting material than would be required for isolating the binding protein from a natural source. Producing IGFBP-5 by recombinant techniques also permits IGFBP-5 to be isolated in the absence of some molecules normally present in cells that naturally produce IGFBP-5. Indeed, IGFBP compositions entirely free of any trace of human protein contaminants can readily be produced since the only human protein produced by the recombinant non-human host is the recombinant IGFBP. Potential viral agents from natural sources are also avoided. It is also apparent that recombinant DNA techniques can be used to produce IGFBP-5 polypeptide derivatives that are not found in nature, such as the variations described above |
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