B7-H1, a novel immunoregulatory molecule
View Patent ↗The invention provides novel polypeptides useful for co-stimulating T cells, isolated nucleic acid molecules encoding them, vectors containing the nucleic acid molecules, and cells containing the vectors. Also included are methods of making and using these co-stimulatory polypeptides.
1. A polynucleotide which is:
(a) a cDNA that encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1;
(b) a cDNA comprising nucleotides having the sequence set forth in SEQ ID NO:2;
(c) a cDNA which hybridizes to the complement of a polynucleotide over its full length under conditions of incubation at 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C., wherein the second polynucleotide encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1; or
(d) a cDNA which is complementary to the cDNA of at least one of (a)-(c).
2. The polynucleotide according to claim 1 (a) which is a cDNA that encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1.
3. The polynucleotide according to claim 1 (b), which is a cDNA that comprises nucleotides having the sequence set forth in SEQ ID NO:2.
4. An isolated polypeptide comprising:
(a) amino acids having the sequence set forth in SEQ ID NO:1; or
(b) amino acids encoded by a first polynucleotide which hybridizes to the complement of a second polynucleotide over its full length under conditions of incubation at 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C., wherein the second polynucleotide encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1,
wherein said isolated polypeptide is not membrane bound and is expressed in non-human cells.
5. An expression vector comprising
(a) a polynucleotide that encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1;
(b) a polynucleotide comprising nucleotides having the sequence set forth in SEQ ID NO:2;
(c) a first polynucleotide which hybridizes to the complement of a second polynucleotide over its full length under conditions of incubation at 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C., wherein the second polynucleotide encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1; or
(d) a polynucleotide which is complementary to the polynucleotide of at least one of (a)-(c),
said polynucleotide being operably linked to a heterologous regulatory element which allows expression of said polynucleotide in a cell.
6. A cell comprising the vector of claim 5 .
7. A method of producing a polypeptide that co-stimulates a T cell, comprising:
culturing a non-human cell having a vector comprising a polynucleotide which encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1,
wherein the polynucleotide is operably linked to a regulatory element which allows expression of said polynucleotide in said cell; and
purifying the polypeptide from the culture.
8. A fusion protein comprising a first domain joined to at least one unrelated additional domain, wherein the first domain comprises a polypeptide encoded by:
(a) a polynucleotide comprising nucleotides having the sequence set forth in SEQ ID NO:2,
(b) a first polynucleotide which hybridizes to the complement of a second polynucleotide over its full length under conditions of incubation at 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C., wherein the second polynucleotide encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1, or
(c) a polynucleotide which is complementary to any one of the polynucleotides of (a)-(c).
9. A polynucleotide encoding a fusion protein comprising a first domain joined to at least one additional domain, wherein the first domain comprises a polypeptide which is encoded by the polynucleotide according to part (a) or (b),
(a) a polynucleotide that encodes a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1;
(b) a polynucleotide comprising nucleotides having the sequence set forth in SEQ ID NO:2.
10. A vector comprising the polynucleotide of claim 9 .
11. The vector of claim 10 , wherein the polynucleotide is operably linked to a regulatory element which allows expression of the polynucleotide in a cell.
12. A cell comprising the vector of claim 11 .
13. A method of producing a fusion protein, comprising culturing the cell of claim 12 and purifying the fusion protein from the culture.
14. A cDNA comprising nucleotides having a sequence that encodes
a polypeptide comprising amino acids having the sequence of amino acid residue 23 to amino acid residue 290 of SEQ ID NO:1; or
a B7-H1 polypeptide comprising amino acids having a sequence comprising one to 17 conservative substitutions in SEQ ID NO:1.
15. The cDNA according to claim 14 , which encodes
a polypeptide comprising amino acids having a sequence of about amino acid residue 20 to amino acid residue 290 of SEQ ID NO:1.
16. The cDNA according to claim 14 , which encodes a polypeptide comprising amino acids having a sequence from amino acid residue 20 to amino acid residue 290 of SEQ ID NO:1.
17. A cDNA which
(a) comprises nucleotides having sequence that encodes a polypeptide comprising at least 95% amino acid sequence identity to SEQ ID NO:1; or
(b) is complementary to the nucleic acid according to (a);
wherein the nucleic acid of (a) encodes a co-stimulatory B7-H1 polypeptide.
18. A cDNA comprising nucleotides having a sequence encoding amino acids 23-239 of SEQ ID NO:1.
19. A polynucleotide having a first part and a second part, which polynucleotide encodes a fusion protein,
wherein the first part (a) comprises nucleotides having a sequence that encodes a co-stimulatory B7-H1 polypeptide comprising at least 95% amino acid sequence identity to SEQ ID NO:1; or (b) is complementary to the nucleic acid according to (a); and
wherein the second part comprises nucleotides having a sequence that encodes a heterologous polypeptide.
20. A vector comprising the cDNA of claim 17 .
21. The vector of claim 20 , which is an expression vector.
22. A host cell transfected with the expression vector of claim 21 .
23. A method of producing a polypeptide encoded by a polynucleotide comprising nucleotides having the sequence set forth in SEQ ID NO:2, comprising culturing a host cell transfected with an expression vector comprising a cDNA according to claim 17 (a) in an appropriate culture medium, to thereby produce the polypeptide.
24. A method of producing a polypeptide comprising amino acids having the sequence set forth in SEQ ID NO:1, comprising culturing the host cell of claim 22 in an appropriate culture medium, to thereby produce the polypeptide.
25. A cDNA which
(a) comprises nucleotides having a sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:2 or a complement of said nucleic acid;
(b) encodes a polypeptide comprising amino acids having the sequence of SEQ ID NO:1; or
(c) encodes a fragment of a polypeptide comprising amino acids having the sequence of SEQ ID NO:1 wherein the fragment comprises at least 10 consecutive amino acid residues of SEQ ID NO:1.
26. The cDNA according to claim 25 , which is
(a) a polynucleotide comprising nucleotides having the sequence of SEQ ID NO:2, or a complement thereof; or
(b) a polynucleotide which encodes a polypeptide comprising amino acids having the sequence of SEQ ID NO:1.
27. The cDNA of claim 25 , further comprising a nucleic acid sequence of a vector.
28. A non-human mammalian host cell containing the cDNA of claim 25 .
29. A kit comprising a cDNA according to claim 25 and instructions for use.
30. A polynucleotide which comprises said cDNA (c) according to claim 25 .
31. A method for detecting the presence of the cDNA of claim 25 in a sample, comprising
(a) contacting the sample with a nucleic acid probe or primer which hybridizes with the cDNA; and
(b) determining whether the nucleic acid probe or primer binds with the cDNA in the sample under conditions of incubation at 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.
32. The polynucleotide according claim 1 , wherein each of (a) to (c) encodes a co-stimulatory B7-H1 polypeptide having the ability to co-stimulate a T cell.
33. The cDNA according to claim 14 which encodes a B7-H1 polypeptide comprising amino acids having a sequence comprising 1 to 14 conservative substitutions in SEQ ID NO:1.
34. A cDNA which encodes a soluble B7-H1 polypeptide which is not membrane-bound, wherein said soluble B7-H1 polypeptide comprises at least one amino acid domain(s) selected from the group consisting of Ig-V-like and Ig-C-like domains having the sequence(s) shown in FIG. 2A (SEQ ID NO:1).
35. A cDNA comprising:
(a) a cDNA sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the cDNA sequence hybridizes under highly stringent conditions to the complement of a nucleotide sequence that encodes a polypeptide with an amino acid sequence with SEQ ID NO:1; or
(b) the complement of the cDNA sequence,
wherein the highly stringent conditions of (a) are defined as hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.
36. A vector comprising the cDNA of claim 25 .
37. The vector of claim 36 , wherein the nucleic acid sequence is operably linked to a regulatory element which allows expression of said nucleic acid cDNA sequence in a cell.
38. A cell comprising the vector of claim 36 .
39. A cell comprising the vector of claim 37 .
40. A method of producing a polypeptide that co-stimulates a T cell, comprising:
culturing a cell having a vector comprising a DNA comprising a cDNA sequence according to claim 35 part (a), wherein the cDNA sequence is operably linked to a regulatory element which allows expression of said cDNA sequence in said cell; and
purifying the polypeptide from the culture.
41. A nucleic acid molecule encoding a fusion protein comprising a first domain joined to at least one additional domain, wherein the first domain comprises a polypeptide encoded by (a) a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the nucleic acid sequence hybridizes under highly stringent conditions to the complement of a sequence that encodes a polypeptide with an amino acid sequence with SEQ ID NO:1; or
(b) the complement of the nucleic acid sequence,
wherein the highly stringent conditions of (a) are defined as hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.
and the at least one additional domain comprises the constant region of an immunoglobulin heavy chain or a fragment thereof.
42. A vector comprising the nucleic acid molecule of claim 41 .
43. The vector of claim 10 , wherein the nucleic acid molecule is operably linked to a regulatory element which allows expression of the nucleic acid in a cell.
44. A cDNA cell comprising the vector of claim 43 .
45. A method of producing a fusion protein, the method comprising culturing the cell of claim 44 and purifying the fusion protein from the culture.
46. A cDNA comprising: a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 2.
47. A cDNA comprising: a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cells, wherein the amino acid sequence consists of SEQ ID NO:1 but lacking amino acid residues 1-22 of SEQ ID NO:1.
48. A cDNA comprising:
(a) a cDNA sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the polypeptide is an amino acid sequence consisting of SEQ ID NO:1; or
(b) the complement of the cDNA sequence.
49. The cDNA of claim 48 , wherein the nucleic acid sequence is a nucleotide sequence consisting of SEQ ID NO:2.
50. A vector comprising the cDNA of claim 48 .
51. The vector of claim 50 , wherein the cDNA sequence is operably linked to a regulatory element which allows expression of said cDNA sequence in a cell.
52. A cell comprising the vector of claim 50 .
53. A cell comprising the vector of claim 51 .
54. A method of producing a polypeptide that co-stimulates a T cell, comprising:
culturing a non-human cell having a vector comprising a cDNA that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the polypeptide is an amino acid sequence consisting of SEQ ID NO:1, wherein the nucleic acid sequence is operably linked to a regulatory element which allows expression of said nucleic acid sequence in said cell; and
purifying the polypeptide from the culture.
55. A cDNA comprising:
(a) a cDNA sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the cDNA sequence is at least 50 nucleotides long and wherein the polypeptide consists of a functional fragment of an amino acid sequence consisting of SEQ ID NO:1; or
(b) the complement of the cDNA sequence,
wherein the functional fragment has the ability to co-stimulate a T cell.
56. The cDNA of claim 55 , wherein the functional fragment consists of (i) SEQ ID NO:1 but lacking amino acid residues 1-22 of SEQ ID NO:1.
57. A vector comprising
(a) a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the nucleic acid sequence is at least 50 nucleotides long and wherein the polypeptide consists of a functional fragment of an amino acid sequence consisting of SEQ ID NO:1; or
(b) the complement of the nucleic acid sequence,
wherein the functional fragment has the ability to co-stimulate a T cell.
58. The vector of claim 57 , wherein the nucleic acid sequence is operably linked to a regulatory element which allows expression of said nucleic acid sequence in a cell.
59. An isolated cell comprising the vector of claim 57 .
60. An isolated cell comprising the vector of claim 58 .
61. A method of producing a polypeptide that co-stimulates a T cell, comprising:
culturing a non-human cell having a vector comprising a DNA comprising a DNA which encodes a soluble B7-H1 polypeptide which is not membrane-bound, wherein said soluble B7-H1 polypeptide comprises at least one amino acid domain(s) selected from the group consisting of Ig-V-like and Ig-C-like, having the sequence(s) shown in FIG. 2A (SEQ ID NO:1)
wherein the nucleic acid sequence is operably linked to a regulatory element which allows expression of said nucleic acid sequence in said cell; and
purifying the polypeptide from the culture.
62. The DNA of claim 55 , wherein the cDNA sequence is a segment of a cDNA sequence consisting of SEQ ID NO:2.
63. An isolated polypeptide encoded by a DNA comprising:
(a) a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the nucleic acid sequence hybridizes under highly stringent conditions to the complement of a sequence that encodes a polypeptide with an amino acid sequence with SEQ ID NO:1; or
(b) the complement of the nucleic acid sequence,
wherein the highly stringent conditions of (a) are defined as hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.,
wherein said isolated polypeptide is not membrane bound and is expressed in non-human cells.
64. The isolated polypeptide of claim 63 , wherein the polypeptide comprises an amino acid sequence of amino acid residue 23 to amino acid residue 290 of SEQ ID NO:1, or said amino acid sequence but differing solely by conservative substitutions.
65. The isolated polypeptide of claim 63 , wherein the polypeptide comprises an amino acid sequence of SEQ ID NO:1, or said amino acid sequence but differing solely by conservative substitutions.
66. An isolated polypeptide encoded by an isolated DNA comprising a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the polypeptide is an amino acid sequence consisting of SEQ ID NO:1 and wherein said isolated polypeptide is not membrane bound and is expressed in non-human cells.
67. A fusion protein comprising a first domain joined to at least one additional domain comprising an unrelated amino acid sequence, wherein the first domain comprises a polypeptide encoded by a DNA comprising:
(a) a nucleic acid sequence that encodes a polypeptide with the ability to co-stimulate a T cell, wherein the nucleic acid sequence hybridizes under highly stringent conditions to the complement of a sequence that encodes a polypeptide with an amino acid sequence with SEQ ID NO:1; or
(b) the complement of the nucleic acid sequence,
wherein the highly stringent conditions of (a) are defined as hybridization in 6× sodium chloride/sodium citrate (SSC) at 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C.
68. The fusion protein of claim 67 , wherein the at least one additional domain comprises the constant region of an immunoglobulin heavy chain or a fragment thereof.
69. A non-human cell comprising the vector of claim 5 .
70. A non-human cell comprising the vector of claim 11 .
71. A non-human host cell transfected with the expression vector of claim 51 .
72. A non-human cell comprising the vector of claim 36 .
73. A non-human cell comprising the vector of claim 37 .
74. A non-human cell comprising the vector of claim 43 .
75. A non-human cell comprising the vector of claim 50 .
76. A non-human cell comprising the vector of claim 51 .
77. A non-human cell comprising the vector of claim 57 .
78. A non-human cell comprising the vector of claim 58 .
79. A mammalian cell transfected with an expression vector comprising a polynucleotide that encodes the Ig-V-like domain and Ig-C-like domain as shown in FIG. 2A , said cell also including heterologous regulatory elements.
80. A method of producing a polypeptide comprising an Ig-V-like domain and an Ig-C-like domain of a B7-H1 polypeptide comprising:
growing cells according to claim 79 ; and
separating the polypeptide from the cells.
81. The cDNA of claim 34 , wherein the at least one amino acid domain is the Ig-V-like domain having the sequence shown in FIG. 2A (SEQ ID NO:1).
82. The polynucleotide of claim 34 , wherein the at least one amino acid domain is the Ig-C-like domain having the sequence shown in FIG. 2A (SEQ ID NO:1).
83. The polynucleotide of claim 34 , wherein the at least one amino acid domain(s) comprises the Ig-V-like domain and the Ig-C-like domain having the sequences shown in FIG. 2A (SEQ ID NO:1).
84. The polynucleotide of claim 34 , wherein the polynucleotide further encodes an additional domain at the 3′-end which encodes a heterologous sequence.