Apoptosis-inducing factor

• US 20030211514A1
• Filed: 12/02/2002
• Published: 11/13/2003
• Est. Priority Date: 11/23/1998
• Status: Active Grant

• Current Assignee: Bryan E.L. Snow, David Peter Siderovski, Guido P. Kroemer, Josef M. Penninger, Naoufal Zamzami, Santos A. Susin
• Original Assignee: Bryan E.L. Snow, David Peter Siderovski, Guido P. Kroemer, Josef M. Penninger, Naoufal Zamzami, Santos A. Susin
• Inventors: Snow, Bryan E.L., Penninger, Josef M., Susin, Santos A., Zamzami, Naoufal, Siderovski, David Peter, Kroemer, Guido P.
• Granted Patent: US 7,371,834 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins

Abstract

The present invention related generally novel mammalian apoptosis-inducing factors, polynucleotides encoding such factors and methods related thereto.

Claims

1. An isolated polynucleotide encoding a mammalian apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof.

2. The isolated polynucleotide according to claim 1, wherein said polynucleotide is selected from the group consisting of cDNA, genomic DNA, and chemically synthesized DNA.

3. The isolated polynucleotide according to claim 2, wherein the mammalian apoptosis-inducing factor is murine apoptosis-inducing factor, and wherein the murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

2 or SEQ ID NO;

3.

4. The isolated polynucleotide according to claim 2, wherein the mammalian apoptosis-inducing factor is an isoform of murine apoptosis-inducing factor, and wherein said isoform of murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

5 or SEQ ID NO;

6.

5. The isolated polynucleotide according to claim 2, wherein the mammalian apoptosis-inducing factor is human apoptosis-inducing factor, and wherein the human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

8 or SEQ ID NO;

9.

6. The isolated polynucleotide according to claim 2, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor, and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

11 or SEQ ID NO;

12.

7. The isolated polynucleotide according to claim 2, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor, and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

14 or SEQ ID NO;

15.

8. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 70 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

9. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 75 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

10. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 80 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

11. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 85 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

12. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 90 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

13. An isolated polynucleotide according to claim 1, wherein the polynucleotide encodes a polypeptide having at least 95 percent identity to the polypeptides comprising the amino acid sequences set forth in SEQ ID NOS:

2, 3, 5, 6, 8, 9, 11, 12, 14, and 15.

15. A detectably labeled nucleic acid hybridizable to a polynucleotide according to any of claims 1-14.

16. A cloning vector which comprises a polynucleotide according to any of claims 1-14.

17. An expression vector which comprises a polynucleotide according to any of claims 1-14.

18. The expression vector which comprises a polynucleotide according to any of claims 1-14, operatively associated with an expression control sequence.

19. The expression vector of claim 18 wherein said expression control sequence is selected from the group consisting of the immediate early promoters of human cytomegalovirus (hCMV), early promoters of SV-40, early promoters of adenovirus, early promoters of polyoma virus, late promoters of SV-40, late promoters of vaccinia virus, late promoters of polyoma virus, retroviral LTR, inducible promoters, promoters of the lac system, promoters of the trp system, promoters of the TAC system, promoters of the TRC system, the major operators and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, promoters of yeast α

mating factor.

20. A unicellular host transformed with a polynucleotide according to any of claims 1-14.

21. The unicellular host of claim 20 wherein the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomvces, yeast, CHO, R1.1, B-W, LM, C051, C057, BSC1, BSC40, BMT10 and SF9 cells.

22. The unicellular host according to claim 21 wherein the unicellular host is a yeast selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula, and Torulopsis.

25. A method for producing a apoptosis-inducing factor polypeptide, the method comprising the steps of:

(a) culturing a host cell according to claims 20 or 23 under conditions suitable for the expression of the apoptosis-inducing factor polypeptide; and

(b) recovering the expressed apoptosis-inducing factor polypeptide.

14. An isolated polynucleotide encoding a mammalian apoptosis-inducing factor the polynucleotide being selected from the group consisting of:

(a) the DNA molecules set forth in SEQ ID NOS;

1, 4, 7, 10, 13, DNA molecules encoding variants including conserved variants, allelic variants, analogs, and fragments thereof;

(b) DNA molecules which hybridize, under high stringency conditions, to the DNA molecules defined in a) or hybridizable fragments thereof; and

(c) DNA molecules that code an expression for the amino acids encoded by any of the foregoing DNA molecules.

23. A mammalian cell containing a mammalian apoptosis-inducing factor encoding DNA modified so as to permit higher expression of the apoptosis-inducing factor by means of a homologous recombinational event consisting of inserting an expression regulatory sequence in functional proximity to the apoptosis-inducing factor encoding DNA.

24. A mammalian cell according to claim 23 wherein the inserted expression regulatory sequence is not a native apoptosis-inducing factor expression regulatory sequence.

26. An isolated nucleic acid encoding an analog of a mammalian apoptosis-inducing factor comprising the amino acid sequence set forth in SEQ ID.:

3 or SEQ ID NO.;

9, wherein one or more amino acids selected from the group consisting of amino acids

27. An isolated purified mammalian apoptosis-inducing factor and biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 108, 109, 110, 111, 112, 113, 115, 116, 117, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 140, 141, 143, 144, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 174, 175, 176, 177, 178, 179, 180, 181, 182, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 225, 226, 227, 228, 229, 230, 232, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 306, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 411, 412, 413, 414, 415, 416, 417, 418, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 451, 452, 453, 455, 456, 457, 458, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, and 612 is substituted with another amino acid.

28. The isolated purified mammalian apoptosis-inducing factor according to claim 27, wherein the mammalian apoptosis-inducing factor is murine apoptosis-inducing factor. and wherein the murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

2 or SEQ ID NO;

3.

29. The isolated purified mammalian apoptosis-inducing factor according to claim 27, wherein the mammalian apoptosis-inducing factor is an isoform of murine apoptosis-inducing factor, and wherein said isoform of murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

5 or SEQ ID NO;

6.

30. The isolated purified mammalian apoptosis-inducing factor according to claim 27, wherein the mammalian apoptosis-inducing factor is human apoptosis-inducing factor, and wherein the human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

8 or SEQ ID NO;

9.

31. The isolated purified mammalian apoptosis-inducing factor according to claim 27, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor, and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

11 or SEQ ID NO;

12.

32. The isolated purified mammalian apoptosis-inducing factor according to claim 27, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor, and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

14 or SEQ ID NO;

15.

58. A method of inhibiting cell proliferation via the administration of the isolated purified mammalian apoptosis-inducing factor of any of claims 27-33.

33. An isolated purified mammalian apoptosis-inducing factor comprising the amino acid sequence set forth in SEQ ID NO.:

9, wherein one or more amino acids from positions 1-612 is substituted for the amino acid at aligned positions 1-612 of SEQ ID NO.;

3.

34. An antibody which specifically binds human apoptosis-inducing factor.

36. The antibody of claim 34 that is a monoclonal antibody.

35. An antibody which specifically binds murine apoptosis-inducing factor.

37. The antibody of claim 35 that is a monoclonal antibody.

38. A derivative of mammalian apoptosis-inducing factor having one or more chemical moieties attached thereto optionally in an acceptable carrier wherein the polypeptide is selected from the group consisting of mammalian apoptosis-inducing factor and biologically active conserved variants, allelic variants, analogs, and fragments thereof.

39. The derivative according to claim 38 wherein said one or more chemical moieties is a water soluble polymers.

40. The derivative of claim 39, wherein the water soluble is selected from the group consisting of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymer, polyethylated polyols, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6,-trioxane, ethylene/maleic anhydride copolymers, homopolymers of polyamino acids, random copolymers of polyamino acids, poly(n-vinyl pyrrolidone)-polyethylene glycol, and polyvinyl alcohol.

41. A method for determining the presence of mammalian apoptosis-inducing factor in a biological sample comprising the steps of:

(a) obtaining a biological sample;

(b) exposing said biological sample to a mammalian apoptosis-inducing factor-specific antibody; and

(c) detecting the binding of mammalian apoptosis-inducing factor-specific antibody in said biological sample.

42. A diagnostic reagent comprising a detectably labeled polynucleotide encoding part or all of human apoptosis inducing factor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

8, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

14, and SEQ ID NO;

15 and conserved variants, allelic variants, fragments, and analogs thereof.

44. The diagnostic reagent of claim 42 or claim 43 wherein said labeled polynucleotide is a DNA.

45. A diagnostic reagent of claim 42 or claim 43 wherein said labeled polynucleotide is a first-strand cDNA.

46. A method for determining the presence of mammalian apoptosis-inducing factor-specific polynucleotide molecule in a biological sample comprising the steps of:

(a) collecting a biological sample;

(b) isolating polynucleotide molecules from said biological sample;

(c) hybridizing to said polynucleotide molecules a diagnostic reagent according to claim 42 or claim 43;

and

(d) detecting the binding of the mammalian apoptosis-inducing factor-specific polynucleotide molecules in said biological samples.

47. A method for determining the presence of mammalian-apoptosis inducing factor-specific polynucleotide molecule in a tissue or cellular sample comprising the steps of:

(a) collecting tissue or cellular sample;

(b) hybridizing said tissue or cellular sample to a diagnostic reagent according to claim 42 or claim 43;

and

(c) detecting the binding of the mammalian apoptosis-inducing factor-specific polynucleotide molecules in the tissue or cellular sample to said diagnostic reagent.

48. The method of claim 46 or claim 47, wherein said polynucleotide molecule is DNA.

49. The method of claim 46 or claim 47, wherein said polynucleotide acid molecule is RNA.

43. A diagnostic reagent comprising a detectably labeled polynucleotide encoding part or all of murine apoptosis inducing factor comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

5, and SEQ ID NO;

6, and conserved variants, allelic variants, fragments, and analogs thereof.

50. A method of identifying a candidate inhibitor of mammalian apoptosis-inducing factor binding to a mammalian apoptosis-inducing factor binding protein comprising the steps of:

(a). exposing mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein under conditions which permit binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein in the presence or absence of a candidate inhibitor;

(b) measuring the binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein in the presence or absence of the candidate inhibitor;

(c) comparing the level of binding observed in step (a); and

(d) identifying the compound as an inhibitor of mammalian apoptosis-inducing factor binding by its ability to prevent binding of mammalian apoptosis-inducing factor to a mammalian apoptosis-inducing factor binding protein.

51. A compositon comprising an isolated purified mammalian apoptosis-inducing factor or biologically active conserved variants, allelic variants, isoforms, analogs, and fragments thereof and an acceptable carrier, diluent and/or adjuvant.

52. The composition according to claim 51, wherein the mammalian apoptosis-inducing factor is murine apoptosis-inducing factor. and wherein the murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

2 or SEQ ID NO;

3.

53. The composition according to claim 51, wherein the mammalian apoptosis-inducing factor is an isoform of murine apoptosis-inducing factor, and wherein said isoform of murine apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

5 or SEQ ID NO;

6.

54. The composition according to claim 51, wherein the mammalian apoptosis-inducing factor is human apoptosis-inducing factor, and wherein the human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

8 or SEQ ID NO;

9.

55. The composition according to claim 51, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

11 or SEQ ID NO;

12.

56. The composition according to claim 51, wherein the mammalian apoptosis-inducing factor is an isoform of human apoptosis-inducing factor, and wherein said isoform of human apoptosis-inducing factor comprises the amino acid sequence set out in either SEQ ID NO:

14 or SEQ ID NO;

15.

57. A method of inhibiting cell proliferation via the administration of any of the compositions of claims 51-56