Antibodies that immunospecifically bind to trail receptors

• US 7,064,189 B2
• Filed: 05/07/2002
• Issued: 06/20/2006
• Est. Priority Date: 05/25/2001
• Status: Expired due to Term

• Current Assignee: Human Genome Sciences Incorporated (GSK plc)
• Original Assignee: Human Genome Sciences Incorporated (GSK plc)
• Inventors: Salcedo, Theodora, Vaughan, Tristan, Rosen, Craig A., Ruben, Steven M., Albert, Vivian R., Dobson, Claire
• Primary Examiner(s): HELMS, LARRY RONALD
• Application Number:
  US10/139,785
• Publication Number:
  US 20030190685A1
• Time in Patent Office: 1,505 Days
• Field of Search: 530/387.1, 530/387.3, 530/388.22, 530/388.85, 530/391.3, 530/391.1, 424/130.1, 424/133.1, 424/156.1, 424/135.1, 424/180.1, 424/178.1
• US Class Current: 530/388.85
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/00 Medicinal preparations containing organic active ingredients
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)

Abstract

The present invention relates to antibodies and related molecules that immunospecifically bind to TRAIL receptor, TR4. Such antibodies have uses, for example, in the prevention and treatment of cancers and other proliferative disorders. The invention also relates to nucleic acid molecules encoding anti-TR4 antibodies, vectors and host cells containing these nucleic acids, and methods for producing the same. The present invention relates to methods and compositions for preventing, detecting, diagnosing, treating or ameliorating a disease or disorder, especially cancer and other hyperproliferative disorders, comprising administering to an animal, preferably a human, an effective amount of one or more antibodies or fragments or variants thereof, or related molecules, that immunospecifically bind to TRAIL receptor TR4.

Claims

1. An isolated antibody or fragment thereof comprising amino acid residues 26–

35, 50–

66, 99–

107, 157–

170, 186–

192, and 225–

234 of SEQ ID NO;

43, wherein said antibody or fragment thereof immunospecifically binds a TR4 protein selected from the group consisting of;

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell.

2. The antibody or fragment thereof of claim 1, that preferentially binds said TR4 protein relative to its ability to bind TR1 (SEQ ID NO:

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

3. The antibody or fragment thereof of claim 1, wherein said TR4 protein is expressed on the surface of a cell.

64. The antibody or fragment thereof of claim 1 wherein said antibody or fragment thereof immunospecifically binds a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO:

1 and a polypeptide having the amino acid sequence of amino acid residues 109 to 240 of SEQ ID NO;

1.

65. The antibody or fragment thereof of claim 1 wherein said antibody or fragment thereof immunospecifically binds a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO:

1 and a multimer of a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO;

1 expressed on the surface of a cell.

4. An isolated antibody or fragment thereof comprising ammo acid residues 1–

118 and 135–

245 of SEQ ID NO;

43, wherein said antibody or fragment thereof immunospecifically binds a TR4 protein selected from the group consisting of;

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell.

6. The antibody or fragment thereof of claim 4, that preferentially binds said TR4 protein relative to its ability to bind TR1 (SEQ ID NO:

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

7. The antibody or fragment thereof of claim 4, wherein said TR4 protein is expressed on the surface of a cell.

8. The antibody or fragment thereof of claim 4 wherein the antibody is a whole immunoglobulin molecule.

9. The antibody or fragment thereof of claim 4 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:

(a) a human IgM constant domain;

(b) a human IgG1 constant domain;

(c) a human IgG2 constant domain;

(d) a human IgG3 constant domain;

(e) a human IgG4 constant domain; and

(f) a human IgA constant domain.

10. The antibody or fragment thereof of claim 4 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:

(a) a human Ig kappa constant domain; and

(b) a human Ig lambda constant domain.

11. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof has a dissociation constant (K_D) selected from the group consisting of:

(a) a dissociation constant (K_D) between 10<sup>−

7</sup> M (inclusive) and 10<sup>−

8</sup> M; and

(b) a dissociation constant (K_D) between 10<sup>−

8</sup> M (inclusive) and 10<sup>−

9</sup> M.

12. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof has a dissociation constant (K_D) of less than or equal to 10⁻

9 M.

13. The antibody or fragment thereof of claim 12 wherein the antibody or fragment thereof has a K_D between 10⁻

9 M and 10<sup>−

10</sup> M.

14. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof is conjugated to a detectable label.

15. The antibody or fragment thereof of claim 14, wherein the detectable label is a radiolabel.

16. The antibody or fragment thereof of claim 15, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

17. The antibody or fragment thereof of claim 14, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

18. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof is biotinylated.

19. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof is conjugated to a therapeutic or cytotoxic agent.

20. The antibody or fragment thereof of claim 19, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(e) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

21. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof is attached to a solid support.

22. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in a Western blot.

23. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in an ELISA.

24. An isolated cell that produces the antibody or fragment thereof of claim 4.

25. The antibody or fragment thereof of claim 4 that does not inhibit the ability of TRAIL to bind said TR4 protein.

26. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof is an agonist of said TR4 protein.

27. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof stimulates apoptosis of a cell expressing said TR4 protein.

28. The antibody or fragment thereof of claim 27 wherein the antibody or fragment thereof stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

29. The antibody or fragment thereof of claim 27 wherein the antibody or fragment thereof stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

30. The antibody or fragment thereof of claim 27 wherein the antibody or fragment thereof is not hepatotoxic.

31. The antibody or fragment thereof of claim 4 wherein the antibody or fragment thereof inhibits TRAIL binding to said TR4 protein.

32. The antibody or fragment thereof of claim 4 in a pharmaceutically acceptable carrier.

33. A kit comprising the antibody or fragment thereof of claim 4.

34. The kit of claim 33 comprising a control antibody.

35. The kit of claim 33, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.

59. The antibody or fragment thereof of claim 4 which is monoclonal.

60. The antibody or fragment thereof of claim 4 which is human.

61. The antibody or fragment thereof of claim 4 which is chimeric.

62. The antibody or fragment thereof of claim 4 which is humanized.

63. The antibody or fragment thereof of claim 4 wherein the fragment thereof is selected from the group consisting of:

(a) an scFv;

(b) a Fab fragment;

(c) an Fab′

fragment;

(d) an F(ab′

)2;

(e) an Fv; and

(f) a disulfide linked Fv.

66. The antibody or fragment thereof of claim 4 wherein said antibody or fragment thereof immunospecifically binds a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO:

1 and a polypeptide having the amino acid sequence of amino acid residues 109 to 240 of SEQ ID NO;

1.

67. The antibody or fragment thereof of claim 4 wherein said antibody or fragment thereof immunospecifically binds a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO:

1 and a multimer of a polypeptide having the amino acid sequence of amino acid residues 24 to 238 of SEQ ID NO;

1 expressed on the surface of a cell.

5. An isolated scFv comprising amino acid residues 1 to 245 of SEQ ID NO:

43.

36. The antibody expressed by the cell line of ATCC Deposit PTA-3570.

37. The antibody of claim 36, that preferentially binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

relative to its ability to bind TR1 (SEQ ID NO;

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

38. The antibody of claim 36 wherein the antibody is conjugated to a detectable label.

39. The antibody of claim 38, wherein the detectable label is a radiolabel.

40. The antibody of claim 39, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

41. The antibody of claim 38, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

42. The antibody of claim 36 wherein the antibody as biotinylated.

43. The antibody of claim 36 wherein the antibody is conjugated to a therapeutic or cytotoxic agent.

44. The antibody of claim 43, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(c) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

45. The antibody of claim 36 wherein the antibody is attached to a solid support.

46. The antibody of claim 36 wherein the antibody immunospecifically binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

in a Western blot.

47. The antibody of claim 36 wherein the antibody immunospecifically binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

in an ELISA.

48. The antibody of claim 36 wherein the antibody is an agonist of a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell.

49. The antibody of claim 36 wherein the antibody stimulates apoptosis of a cell expressing a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1; and

(b) a multimer of the polypeptide of (a),

wherein said TR4 protein is expressed on the surface of said cell.

50. The antibody of claim 49 wherein the antibody stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

51. The antibody of claim 49 wherein the antibody stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

52. The antibody of claim 49 wherein the antibody is not hepatotoxic.

53. An isolated cell line that produces the antibody of claim 36.

54. The isolated cell line of claim 53 wherein the cell line is contained in ATCC Deposit PTA-3570.

55. The antibody or fragment thereof of claim 36 in a pharmaceutically acceptable carrier.

56. A kit comprising the antibody or fragment thereof of claim 36.

57. The kit of claim 56 comprising a control antibody.

58. The kit of claim 56, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.

68. An isolated antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of:

(a) an amino acid sequence that is at least 80% identical to the VH domain of SEQ ID NO;

43;

(b) an amino acid sequence that is at least 80% identical to the VL domain of SEQ ID NO;

43; and

(c) the amino acid sequence of both (a) and (b);

wherein said antibody or fragment thereof immunospecifically binds a TR4 protein selected from the group consisting of;

(i) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(ii) a multimer of the polypeptide of (i);

(iii) the polypeptide of (i) expressed on the surface of a cell; and

(iv) the multimer of (ii) expressed on the surface of a cell.

69. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 85% identical to the VH domain of SEQ ID NO:

43.

70. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 85% identical to the VL domain of SEQ ID NO:

43.

71. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 90% identical to the VH domain of SEQ ID NO:

43.

72. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 90% identical to the VL domain of SEQ ID NO:

43.

73. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 95% identical to the VH domain of SEQ ID NO:

43.

74. The antibody or fragment thereof of claim 68 comprising an amino acid sequence that is at least 95% identical to the VL domain of SEQ ID NO:

43.

75. The antibody or fragment thereof of claim 68, that preferentially binds said TR4 protein relative to its ability to bind TR1 (SEQ ID NO:

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

76. The antibody or fragment thereof of claim 68, wherein said TR4 protein is expressed on the surface of a cell.

77. The antibody or fragment thereof of claim 68 wherein the antibody is a whole immunoglobulin molecule.

78. The antibody or fragment thereof of claim 68 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:

(a) a human IgM constant domain;

(b) a human IgG1 constant domain;

(c) a human IgG2 constant domain;

(d) a human IgG3 constant domain;

(e) a human IgG4 constant domain; and

(f) a human IgA constant domain.

79. The antibody or fragment thereof of claim 68 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:

(a) a human Ig kappa constant domain; and

(b) a human Ig lambda constant domain.

80. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof has a dissociation constant (K_D) selected from the group consisting of:

(a) a dissociation constant (K_D) between 10<sup>−

7</sup> M (inclusive) and 10<sup>−

8</sup> M; and

(b) a dissociation constant (K_D) between 10<sup>−

8</sup> M (inclusive) and 10<sup>−

9</sup> M.

81. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof has a dissociation constant (K_D) of less than or equal to 10⁻

9 M.

82. The antibody or fragment thereof of claim 81 wherein the antibody or fragment thereof has a K_D between 10⁻

9 M and 10<sup>−

10</sup> M.

83. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof is conjugated to a detectable label.

84. The antibody or fragment thereof of claim 83, wherein the detectable label is a radiolabel.

85. The antibody or fragment thereof of claim 84, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

86. The antibody or fragment thereof of claim 83, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

87. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof is biotinylated.

88. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof is conjugated to a therapeutic or cytotoxic agent.

89. The antibody or fragment thereof of claim 88, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(e) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

90. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof is attached to a solid support.

91. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in a Western blot.

92. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in an ELISA.

93. An isolated cell that produces the antibody or fragment thereof of claim 68.

94. The antibody or fragment thereof of claim 68 that does not inhibit the ability of TRAIL to bind said TR4 protein.

95. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof is an agonist of said TR4 protein.

96. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof stimulates apoptosis of a cell expressing said TR4 protein.

97. The antibody or fragment thereof of claim 96 wherein the antibody or fragment thereof stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

98. The antibody or fragment thereof of claim 96 wherein the antibody or fragment thereof stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

99. The antibody or fragment thereof of claim 96 wherein the antibody or fragment thereof is not hepatotoxic.

100. The antibody or fragment thereof of claim 68 wherein the antibody or fragment thereof inhibits TRAIL binding to said TR4 protein.

101. The antibody or fragment thereof of claim 68 in a pharmaceutically acceptable carrier.

102. A kit comprising the antibody or fragment thereof of claim 68.

103. The kit of claim 102 comprising a control antibody.

104. The kit of claim 102, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.

105. The antibody or fragment thereof of claim 68 which is monoclonal.

106. The antibody or fragment thereof of claim 68 which is human.

107. The antibody or fragment thereof of claim 68 which is chimeric.

108. The antibody or fragment thereof of claim 68 which is humanized.

109. The antibody or fragment thereof of claim 68 wherein the fragment thereof is selected from the group consisting of:

(a) an scFv;

(b) a Fab fragment;

(c) an Fab′

fragment;

(d) an F(ab′

)2;

(e) an Fv; and

(f) a disulfide linked Fv.

110. An isolated antibody or fragment thereof comprising amino acid residues 1–

118 and 135–

245 of SEQ ID NO;

42, wherein said antibody or fragment thereof immunospecifically binds a TR4 protein selected from the group consisting of;

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell.

111. The antibody or fragment thereof of claim 110, that preferentially binds said TR4 protein relative to its ability to bind TR1 (SEQ ID NO:

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

112. The antibody or fragment thereof of claim 110, wherein said TR4 protein is expressed on the surface of a cell.

113. The antibody or fragment thereof of claim 110 wherein the antibody is a whole immunoglobulin molecule.

114. The antibody or fragment thereof of claim 110 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:

(a) a human IgM constant domain;

(b) a human IgG1 constant domain;

(c) a human IgG2 constant domain;

(d) a human IgG3 constant domain;

(e) a human IgG4 constant domain; and

(f) a human IgA constant domain.

115. The antibody or fragment thereof of claim 110 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:

(a) a human Ig kappa constant domain; and

(b) a human Ig lambda constant domain.

116. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof has a dissociation constant (K_D) selected from the group consisting of:

(a) a dissociation constant (K_D) between 10<sup>−

7</sup> M (inclusive) and 10<sup>−

8</sup> M; and

(b) a dissociation constant (K_D) between 10<sup>−

8</sup> M (inclusive) and 10<sup>−

9</sup> M.

117. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof has a dissociation constant (K_D) of less than or equal to 10⁻

9 M.

118. The antibody or fragment thereof of claim 117 wherein the antibody or fragment thereof has a K_D between 10⁻

9 M and 10<sup>−

10</sup> M.

119. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof is conjugated to a detectable label.

120. The antibody or fragment thereof of claim 119, wherein the detectable label is a radiolabel.

121. The antibody or fragment thereof of claim 120, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ²¹⁹Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

122. The antibody or fragment thereof of claim 119, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

123. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof is biotinylated.

124. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof is conjugated to a therapeutic or cytotoxic agent.

125. The antibody or fragment thereof of claim 124, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(e) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

126. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof is attached to a solid support.

127. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in a Western blot.

128. The antibody or fragment thereof of claim 112 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in an ELISA.

129. An isolated cell that produces the antibody or fragment thereof of claim 110.

130. The antibody or fragment thereof of claim 110 that does not inhibit the ability of TRAIL to bind said TR4 protein.

131. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof is an agonist of said TR4 protein.

132. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof stimulates apoptosis of a cell expressing said TR4 protein.

133. The antibody or fragment thereof of claim 132 wherein the antibody or fragment thereof stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

134. The antibody or fragment thereof of claim 132 wherein the antibody or fragment thereof stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

135. The antibody or fragment thereof of claim 132 wherein the antibody or fragment thereof is not hepatotoxic.

136. The antibody or fragment thereof of claim 110 wherein the antibody or fragment thereof inhibits TRAIL binding to said TR4 protein.

137. The antibody or fragment thereof of claim 110 in a pharmaceutically acceptable carrier.

138. A kit comprising the antibody or fragment thereof of claim 110.

139. The kit of claim 138 comprising a control antibody.

140. The kit of claim 138, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.

141. The antibody or fragment thereof of claim 110 which is monoclonal.

142. The antibody or fragment thereof of claim 110 which is human.

143. The antibody or fragment thereof of claim 110 which is chimeric.

144. The antibody or fragment thereof of claim 110 which is humanized.

145. The antibody or fragment thereof of claim 110 wherein the fragment thereof is selected from the group consisting of:

(a) an scFv;

(b) a Fab fragment;

(c) an Fab′

fragment;

(d) an F(ab′

)2;

(e) an Fv; and

(f) a disulfide linked Fv.

146. An isolated antibody or fragment thereof comprising an amino acid sequence selected from the group consisting of:

(a) the VH domain of any one of SEQ ID NOs;

45, 46, 47, 48, 49, or 51;

(b) the VL domain of any one of SEQ ID NOs;

45, 46, 47, 48, 49, or 51; and

(c) the amino acid sequence of both (a) and (b);

wherein said antibody or fragment thereof immunospecifically binds a TR4 protein selected from the group consisting of;

(i) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(ii) a multimer of the polypeptide of (i);

(iii) the polypeptide of (i) expressed on the surface of a cell; and

(iv) the multimer of (ii) expressed on the surface of a cell.

147. The antibody or fragment thereof of claim 146, that preferentially binds said TR4 protein relative to its ability to bind TR1 (SEQ ID NO:

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

148. The antibody or fragment thereof of claim 146, wherein said TR4 protein is expressed on the surface of a cell.

149. The antibody or fragment thereof of claim 146 wherein the antibody is a whole immunoglobulin molecule.

150. The antibody or fragment thereof of claim 146 which comprises a heavy chain immunoglobulin constant domain selected from the group consisting of:

(a) a human IgM constant domain;

(b) a human IgG1 constant domain;

(c) a human IgG2 constant domain;

(d) a human IgG3 constant domain;

(e) a human IgG4 constant domain; and

(f) a human IgA constant domain.

151. The antibody or fragment thereof of claim 146 which comprises a light chain immunoglobulin constant domain selected from the group consisting of:

(a) a human Ig kappa constant domain; and

(b) a human Ig lambda constant domain.

152. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof has a dissociation constant (K_D) selected from the group consisting of:

(a) a dissociation constant (K_D) between 10<sup>−

7</sup> M (inclusive) and 10<sup>−

8</sup> M; and

(b) a dissociation constant (K_D) between 10<sup>−

8</sup> M (inclusive) and 10<sup>−

9</sup> M.

153. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof has a dissociation constant (K_D) of less than or equal to 10⁻

9 M.

154. The antibody or fragment thereof of claim 153 wherein the antibody or fragment thereof has a K_D between 10⁻

9 M and 10<sup>−

10</sup> M.

155. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof is conjugated to a detectable label.

156. The antibody or fragment thereof of claim 155, wherein the detectable label is a radiolabel.

157. The antibody or fragment thereof of claim 156, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ²⁵⁵Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

158. The antibody or fragment thereof of claim 155, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

159. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof is biotinylated.

160. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof is conjugated to a therapeutic or cytotoxic agent.

161. The antibody or fragment thereof of claim 160, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(e) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

162. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof is attached to a solid support.

163. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in a Western blot.

164. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof immunospecifically binds said TR4 protein in an ELISA.

165. An isolated cell that produces the antibody or fragment thereof of claim 146.

166. The antibody or fragment thereof of claim 146 that does not inhibit the ability of TRAIL to bind said TR4 protein.

167. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof is an agonist of said TR4 protein.

168. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof stimulates apoptosis of a cell expressing said TR4 protein.

169. The antibody or fragment thereof of claim 168 wherein the antibody or fragment thereof stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

170. The antibody or fragment thereof of claim 168 wherein the antibody or fragment thereof stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

171. The antibody or fragment thereof of claim 168 wherein the antibody or fragment thereof is not hepatotoxic.

172. The antibody or fragment thereof of claim 146 wherein the antibody or fragment thereof inhibits TRAIL binding to said TR4 protein.

173. The antibody or fragment thereof of claim 146 in a pharmaceutically acceptable carrier.

174. A kit comprising the antibody or fragment thereof of claim 146.

175. The kit of claim 174 comprising a control antibody.

176. The kit of claim 174, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.

177. The antibody or fragment thereof of claim 146 which is monoclonal.

178. The antibody or fragment thereof of claim 146 which is human.

179. The antibody or fragment thereof of claim 146 which is chimeric.

180. The antibody or fragment thereof of claim 146 which is humanized.

181. The antibody or fragment thereof of claim 146 wherein the fragment thereof is selected from the group consisting of:

(a) an scFv;

(b) a Fab fragment;

(c) an Fab′

fragment;

(d) an F(ab′

)2;

(e) an Fv; and

(f) a disulfide linked Fv.

182. An isolated antibody selected from the group consisting of:

(a) the antibody expressed by the cell line of ATCC Deposit PTA-3571;

or

(b) the antibody expressed by the cell line of ATCC Deposit PTA-3675.

183. The antibody of claim 182, that preferentially binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

relative to its ability to bind TR1 (SEQ ID NO;

5), TR5 (SEQ ID NO;

2), TR7 (SEQ ID NO;

3) and TR10 (SEQ ID NO;

4).

184. The antibody of claim 182 wherein the antibody is conjugated to a detectable label.

185. The antibody of claim 184, wherein the detectable label is a radiolabel.

186. The antibody of claim 185, wherein the radiolabel is ¹²⁵I, ¹³¹I, ¹¹¹In, ⁹⁰Y, ⁹⁹Tc, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

187. The antibody of claim 184, wherein the detectable label is an enzyme, a fluorescent label, a luminescent label, or a bioluminescent label.

188. The antibody of claim 182 wherein the antibody is biotinylated.

189. The antibody of claim 182 wherein the antibody is conjugated to a therapeutic or cytotoxic agent.

190. The antibody of claim 189, wherein the therapeutic or cytotoxic agent is selected from the group consisting of:

(a) an anti-metabolite;

(b) an alkylating agent;

(c) an antibiotic;

(d) a growth factor;

(e) a cytokine;

(f) an anti-angiogenic agent;

(g) an anti-mitotic agent;

(h) an anthracycline;

(i) toxin; and

(j) an apoptotic agent.

191. The antibody of claim 182 wherein the antibody is attached to a solid support.

192. The antibody of claim 182 wherein the antibody immunospecifically binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

in a Western blot.

193. The antibody of claim 182 wherein the antibody immunospecifically binds a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell;

in an ELISA.

194. The antibody of claim 182 wherein the antibody is an agonist of a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1;

(b) a multimer of the polypeptide of (a);

(c) the polypeptide of (a) expressed on the surface of a cell; and

(d) the multimer of (b) expressed on the surface of a cell.

195. The antibody of claim 182 wherein the antibody stimulates apoptosis of a cell expressing a TR4 protein selected from the group consisting of:

(a) a polypeptide having the amino acid sequence of any one of amino acid residues 1 to 468 of SEQ ID NO;

1, amino acid residues 24 to 468 of SEQ ID NO;

1, amino acid residues 24 to 238 of SEQ ID NO;

1, and amino acid residues 109 to 240 of SEQ ID NO;

1; and

(b) a multimer of the polypeptide of (a),

wherein said TR4 protein is expressed on the surface of said cell.

196. The antibody of claim 195 wherein the antibody stimulates apoptosis of said cell better than an equal concentration of TRAIL polypeptide stimulates apoptosis of said cell.

197. The antibody of claim 195 wherein the antibody stimulates apoptosis of said cell equally well in the presence or absence of antibody cross-linking reagents.

198. The antibody of claim 195 wherein the antibody is not hepatotoxic.

199. An isolated cell line that produces the antibody of claim 182.

200. The isolated cell line of claim 199 wherein the cell line is contained in ATCC Deposit PTA-3571.

201. The isolated cell line of claim 199 wherein the cell line is contained in ATCC Deposit PTA-3675.

202. The antibody or fragment thereof of claim 182 in a pharmaceutically acceptable carrier.

203. A kit comprising the antibody or fragment thereof of claim 182.

204. The kit of claim 203 comprising a control antibody.

205. The kit of claim 204, wherein the antibody or fragment thereof is coupled or conjugated to a detectable label.