ANTI HUMAN INTERLEUKIN-1 RECEPTOR ACCESSORY PROTEIN (IL1 RAP) ANTIBODIES AND USES THEREOF

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof with binding specificity for human interleukin-1 receptor accessory protein (IL1RAP) wherein the antibody or antigen-binding fragment is capable of inhibiting the binding of antibody ‘CAN04’ to human IL1RAP. The invention further provides the use of such antibodies or an antigen-binding fragments in the treatment and/or diagnosis of IL-1 associated diseases and conditions, including cancers such as acute myeloid leukemia and melanoma.

Claims

1. An antibody or an antigen-binding fragment thereof with binding specificity for human interleukin-1 receptor accessory protein (IL1RAP) wherein the antibody or antigen-binding fragment is capable of inhibiting the binding of reference antibody ‘CAN04’ to human IL1RAP.

2. An antibody or antigen-binding fragment thereof according to claim 1 wherein the antibody or antigen-binding fragment exhibits one or more of the following properties: a) a binding affinity (K.sub.D) for human IL1RAP of 200 pM or greater; b) cross-reactivity with IL1RAP from Macaca fascicularis; c) an inhibitory action on IL1 signalling; d) capability of inducing ADCC in one or more cancer cell lines (such as a CML, AML and/or melanoma cell line); and/or e) capability of internalisation upon binding to one or more cancer cell lines (such as a CML, AML and/or melanoma cell line).

3. An antibody or antigen-binding fragment thereof according to claim 2 wherein the antibody or antigen-binding fragment exhibits all of the following properties: a) a binding affinity (K.sub.D) for human IL1RAP of 200 pM or greater; b) cross-reactivity with IL1RAP from Macaca fascicularis; c) an inhibitory action on IL1 signalling; d) capability of inducing ADCC in one or more cancer cell lines (such as a CML, AML and/or melanoma cell line); and e) capability of internalisation upon binding to one or more cancer cell lines (such as a CML, AML and/or melanoma cell line).

4. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody or antigen-binding fragment is capable of inducing ADCC of cells expressing IL1RAP.

5. An antibody or antigen-binding fragment thereof according to claim 1 or 2 wherein the antibody or antigen-binding fragment is not capable of inducing ADCC of cells expressing IL1RAP.

6. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody or antigen-binding fragment is capable of binding to an epitope on the extracellular domain of IL1RAP which overlaps, at least in part, with the epitope on IL1RAP to which antibody CAN04 is capable of binding.

7. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the epitope is located at or within amino acids 135 to 234 of IL1RAP.

8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising or consisting of an intact antibody.

9. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 comprising or consisting of an antigen-binding fragment selected from the group consisting of Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab).sub.2 fragments) and domain antibodies (e.g. single V.sub.H variable domains or V.sub.L variable domains).

10. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a heavy chain variable region comprising the following CDRs: a) GYAFSSS [SEQ ID NO: 3] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; b) YPGDGN [SEQ ID NO: 4] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; and c) GYLDPMDY [SEQ ID NO: 5] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity.

11. An antibody or antigen-binding fragment thereof according to claim 10 comprising a heavy chain variable region comprising the CDRs of SEQ ID NOs 3, 4 and 5.

12. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a heavy chain variable region comprising the following CDRs: a) GYAFSSSWMN [SEQ ID NO: 6] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; b) RIYPGDGNTHYSGKFKG [SEQ ID NO: 7] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; and c) GYLDPMDY [SEQ ID NO: 5] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity.

13. An antibody or antigen-binding fragment thereof according to claim 12 comprising a heavy chain variable region comprising the CDRs of SEQ ID NOs 6, 7 and 5.

14. An antibody or antigen-binding fragment thereof according to claim 13 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1.

15. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 comprising a heavy chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8 to 11 or an amino acid sequence having at least 90% sequence identity therewith: TABLE-US-00010 a) [SEQ ID NO: 8] Q V Q L V Q S G A E V K K P G S S V K V S C K A S G Y A F S S S W M N W V R Q A P G Q G L E W M G R I Y P G D G N T H Y A Q K F Q G R V T L T A D K S T S T A Y M E L S S L R S E D T A V Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; b) [SEQ ID NO: 9] Q V Q L V Q S G A E V K K P G S S V K V S C K A S G Y A F T S S W M N W V R Q A P G Q G L E W M G R I Y P G D G N T H Y A Q K F Q G R V T L T A D K S T S T A Y M E L S S L R S E D T A V Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; c) [SEQ ID NO: 10] Q V Q L V Q S G A E V K K P G S S V K V S C K A S G Y T F T S S W M N W V R Q A P G K G L E W M G R I Y P G D G Q T H Y A Q K F Q G R V T L T A D K S T S T A Y M E L S S L R S E D T A V Y Y C G E G Y L D P M D Y W G Q G T L V T V S S; or d) [SEQ ID NO: 11] Q V Q L V Q S G A E V K K P G S S V K V S C K A S G Y T F T S S W M N W V R Q A P G K G L E W M G R I Y P G D G Q T H Y A Q K F Q G R V T I T A D K S T S T A Y M E L S S L R S E D T A V Y Y C G E G Y L D P M D Y W G Q G T L V T V S S.

16. An antibody or antigen-binding fragment thereof according to claim 15 comprising a heavy chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8 to 11.

17. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a light chain variable region comprising the following CDRs: a) SASQGINNYLN [SEQ ID NO: 12] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; b) YTSGLHA [SEQ ID NO: 13] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity; and c) QQYSILPWT [SEQ ID NO: 14] or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, or 90% sequence identity.

18. An antibody or antigen-binding fragment thereof according to claim 17 comprising a light chain variable region comprising the CDRs of SEQ ID NOs 12, 13 and 14.

19. An antibody or antigen-binding fragment thereof according to claim 18 comprising a light chain variable region having the amino acid sequence of SEQ ID NO:2.

20. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 comprising a light chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 15 to 17 or an amino acid sequence having at least 90% sequence identity therewith: TABLE-US-00011 a) [SEQ ID NO: 15] D I Q M T Q S P S S L S A S V G D R V T I T C S A S Q G I N N Y L N W Y Q Q K P G K A P K L L I H Y T S G L H A G V P S R F S G S G S G T D Y T L T I S S L Q P E D V A T Y Y C Q Q Y S I L P W T F G G G T K V E I K R; b) [SEQ ID NO: 16] D I Q M T Q S P S S L S A S V G D R V T I T C Q A S Q G I N N Y L N W Y Q Q K P G K A P K L L I H Y T S G L H A G V P S R F S G S G S G T D Y T L T I S S L E P E D V A T Y Y C Q Q Y S I L P W T F G G G T K V E I K R; or c) [SEQ ID NO: 17] D I Q M T Q S P S S L S A S V G D R V T I T C Q A S Q G I N N Y L N W Y Q Q K P G K A P K L L I H Y T S G L H A G V P S R F S G S G S G T D F T L T I S S L E P E D V A T Y Y C Q Q Y S I L P W T F G G G T K V E I K R.

21. An antibody or antigen-binding fragment thereof according to claim 20 comprising a light chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 15 to 17.

22. An antibody or antigen-binding fragment thereof according to claim 14 or 19 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1 and a light chain variable region having the amino acid sequence of SEQ ID NO:2.

23. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a heavy chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8 to 11 and a light chain variable region which comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 15 to 17.

24. An antibody or antigen-binding fragment thereof according to claim 23 comprising: a) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15; b) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15; c) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15; d) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 15; e) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16; f) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16; g) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16; h) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 16; i) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 8 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17; j) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 9 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17; k) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17; or l) a heavy chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 11 and a light chain variable region which comprises or consists of the amino acid sequence of SEQ ID NO: 17.

25. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a heavy chain constant region, or part thereof.

26. An antibody or antigen-binding fragment thereof according to claim 25 wherein the heavy chain constant region is of an immunoglobulin subtype selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

27. An antibody or antigen-binding fragment thereof according to claim 26 wherein the heavy chain constant region is of an immunoglobulin subtype IgG1.

28. An antibody or antigen-binding fragment thereof according to claim 27 wherein the heavy chain constant region comprises or consists of an amino acid sequence of SEQ ID NO: 19.

29. An antibody or antigen-binding fragment thereof according to any one of the preceding claims comprising a light chain constant region, or part thereof.

30. An antibody or antigen-binding fragment thereof according to claim 29 wherein the light chain constant region is of a kappa or lambda light chain.

31. An antibody or antigen-binding fragment thereof according to claim 30 wherein the light chain constant region is of a kappa light chain.

32. An antibody or antigen-binding fragment thereof according to claim 31 wherein the light chain constant region comprises or consists of an amino acid sequence of SEQ ID NO: 18.

33. An antibody or antigen-binding fragment thereof according to any one of the preceding claim comprising an Fc region.

34. An antibody or antigen-binding fragment thereof according to claim 33 wherein the Fc region is non-naturally occurring.

35. An antibody or antigen-binding fragment thereof according to claim 34 wherein the Fc region comprises one or more of the mutations identified in Table 1.

36. An antibody or antigen-binding fragment thereof according to any one of the preceding claims lacking or low in fucose residues in the Fc region.

37. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody or antigen-binding fragment thereof is capable of inhibiting IL1 signalling.

38. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody or antigen-binding fragment thereof is capable of inhibiting IL33 signalling.

39. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody or antigen-binding fragment thereof is capable of inhibiting IL36 signalling.

40. An antibody or antigen-binding fragment thereof according to any one of the preceding claim further comprising a moiety for increasing the in vivo half-life of the agent.

41. An antibody or antigen-binding fragment thereof according to claim 40 wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran.

42. An antibody or antigen-binding fragment thereof according to claim 41 wherein the antibody or antigen-binding fragment thereof is PEGylated.

43. An antibody or antigen-binding fragment thereof according to any one of the preceding claim further comprising a cytotoxic moiety.

44. An antibody or antigen-binding fragment thereof according to claim 43 wherein the cytotoxic moiety comprises or consists of a radioisotope.

45. An antibody or antigen-binding fragment thereof according to claim 44 wherein the radioisotope is selected from the group consisting of beta-emitters, auger-emitters, conversion electron-emitters, alpha-emitters, and low photon energy-emitters.

46. An antibody or antigen-binding fragment thereof according to claim 44 wherein the radioisotope has an emission pattern of locally absorbed energy that creates a high dose absorbance in the vicinity of the agent.

47. An antibody or antigen-binding fragment thereof according to any one of claims 44 to 44 wherein the radioisotope is selected from the group consisting of long-range beta-emitters, such as .sup.90Y, .sup.32P, .sup.186Re/.sup.186Re; .sup.166Ho, .sup.76As/.sup.77As, .sup.153Sm; medium range beta-emitters, such as .sup.131I, .sup.177Lu, .sup.67Cu, .sup.161Tb; low-energy beta-emitters, such as .sup.45Ca, .sup.35S or .sup.14C; conversion or auger-emitters, such as .sup.51Cr, .sup.67Ga, .sup.99Tc.sup.m, .sup.111In, .sup.123I, .sup.125I, .sup.201Tl; and alpha-emitters, such as .sup.212Bi, .sup.213Bi, .sup.223Ac, and .sup.221At.

48. An antibody or antigen-binding fragment thereof according to claim 47 wherein the radioisotope is .sup.177Lu.

49. An antibody or antigen-binding fragment thereof according to claim 43 wherein the cytotoxic moiety comprises or consists of a cytotoxic drug.

50. An antibody or antigen-binding fragment thereof according to claim 49 wherein the cytotoxic drug is selected from the group consisting of a cytostatic drug; an anti-androgen drug; cortisone and derivatives thereof; a phosphonate; a testosterone-5-α-reductase inhibitor; a boron addend; a cytokine; thapsigargin and its metabolites; a toxin (such as saporin or calicheamicin); a chemotherapeutic agent (such as an antimetabolite); or any other cytotoxic drug useful in the treatment of neoplastic disorders.

51. An antibody or antigen-binding fragment thereof according to claim 50 wherein the cytotoxic drug is suitable for use in activation therapy, such as photon activation therapy, neutron activation therapy, neutron induced Auger electron therapy, synchrotron irradiation therapy, or low energy X-ray photon activation therapy.

52. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody polypeptide further comprises a detectable moiety.

53. An antibody or antigen-binding fragment thereof according to claim 52 wherein the detectable moiety comprises or consists of a radioisotope.

54. An antibody or antigen-binding fragment thereof according to claim 53 wherein the radioisotope is selected from the group consisting of .sup.99mTc, .sup.111In, .sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, .sup.123I and .sup.201Tl.

55. An antibody or antigen-binding fragment thereof according to claim 54 wherein the radioisotope is .sup.89Zr.

56. An antibody or antigen-binding fragment thereof according to any one of the preceding claims wherein the antibody polypeptide comprises a pair of detectable and cytotoxic radionuclides, such as .sup.86Y/.sup.90Y or .sup.124I/.sup.211At.

57. An antibody or antigen-binding fragment thereof according to claim 56 wherein the radioisotope is capable of simultaneously acting in a multi-modal manner as a detectable moiety and also as a cytotoxic moiety.

58. An antibody or antigen-binding fragment thereof according to claim 57 wherein the detectable moiety comprises or consists of a paramagnetic isotope.

59. An antibody or antigen-binding fragment thereof according to claim 58 wherein the paramagnetic isotope is selected from the group consisting of .sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Cr and .sup.56Fe.

60. An antibody or antigen-binding fragment thereof according to any of claims 52 to 59 wherein the detectable moiety is detectable by an imaging technique such as SPECT, PET, MRI, optical or ultrasound imaging.

61. An antibody or antigen-binding fragment thereof according to any of claims 43 to 60 wherein the cytotoxic moiety and/or detectable moiety is joined to the antibody or antigen-binding fragment thereof indirectly, via a linking moiety.

62. An antibody or antigen-binding fragment thereof according to claim 61 wherein the linking moiety is a chelator.

63. An antibody or antigen-binding fragment thereof according to claim 62 wherein the chelator is selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives of S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and derivatives of 1,4,8,11-tetraazacyclodocedan-1,4,8,11-tetraacetic acid (TETA).

64. An antibody or antigen-binding fragment thereof according to any of claims 1 to 42 wherein the antibody or antigen-binding fragment does not comprise a cytotoxic moiety.

65. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of the preceding claims or a component polypeptide chain thereof.

66. A nucleic acid molecule according to claim 65 wherein the molecule is a cDNA molecule.

67. A nucleic acid molecule according to claim 65 or 66 encoding an antibody heavy chain or variable region thereof.

68. A nucleic acid molecule according to any one of claims 65 to 67 encoding an antibody light chain or variable region thereof.

69. A vector comprising a nucleic acid molecule according to any one of claims 65 to 68.

70. A vector according to claim 69 wherein the vector is an expression vector.

71. A recombinant host cell comprising a nucleic acid molecule according to any one of claims 55 to 58 or a vector according to claim 69 or 70.

72. A host cell according to claim 71 wherein the host cell is a bacterial cell.

73. A host cell according to claim 72 wherein the host cell is a mammalian cell.

74. A host cell according to claim 73 wherein the host cell is a human cell.

75. A method for producing an antibody or antigen-binding fragment according to any one of the claims 1 to 64, the method comprising culturing a host cell as defined in any of claims 72 to 74 under conditions which permit expression of the encoded antibody or antigen-binding fragment thereof.

76. A pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 and a pharmaceutically-acceptable diluent, carrier or excipient.

77. A pharmaceutical composition according to claim 76 adapted for parenteral delivery.

78. A pharmaceutical composition according to claim 77 adapted for intravenous delivery.

79. A pharmaceutical composition according to claim 77 adapted for topical delivery.

80. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 for use in medicine.

81. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 for use in inducing cell death and/or inhibiting the growth and/or proliferation of pathological cells associated with a neoplastic disorder in a subject, or stem cells or progenitor cells thereof, wherein the cells express IL1RAP.

82. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 for use in the treatment of a neoplastic disorder in a subject, wherein the neoplastic disorder is associated with cells expressing IL1RAP.

83. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 82 wherein the neoplastic disorder is a neoplastic hematologic disorder.

84. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 83 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

85. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 84 wherein the neoplastic hematologic disorder is CML.

86. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 84 wherein the neoplastic hematologic disorder is ALL.

87. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 82 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.

88. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 87 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas.

89. An antibody or antigen-binding fragment thereof for use in the treatment of a neoplastic disorder according to claim 88 wherein the solid tumour is a melanoma.

90. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 in the preparation of a medicament for the treatment or diagnosis of a neoplastic disorder in a subject, wherein the neoplastic disorder is associated with cells expressing IL1RAP.

91. The use according to claim 90 wherein the neoplastic disorder is a neoplastic hematologic disorder.

92. The use according to claim 91 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

93. The use according to claim 92 wherein the neoplastic hematologic disorder is CML.

94. The use according to claim 92 wherein the neoplastic hematologic disorder is ALL.

95. The use according to claim 90 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.

96. The use according to claim 95 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas.

97. The use according to claim 96 wherein the solid tumour is a melanoma.

98. A method for the treatment or diagnosis of a neoplastic disorder in a subject, comprising the step of administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64, wherein the neoplastic disorder is associated with cells expressing IL1RAP.

99. A method according to claim 98 wherein the neoplastic disorder is a neoplastic hematologic disorder.

100. A method according to claim 99 wherein the neoplastic hematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

101. A method according to claim 100 wherein the neoplastic hematologic disorder is CML.

102. A method according to claim 100 wherein the neoplastic hematologic disorder is ALL.

103. A method according to claim 99 wherein the neoplastic disorder is associated with the formation of solid tumours within the subject's body.

104. A method according to claim 103 wherein the solid tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas.

105. A method according to claim 104 wherein the solid tumour is a melanoma.

106. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 for use in the treatment of a disease or condition susceptible to treatment with an inhibitor of IL-1 signalling.

107. An antibody or antigen-binding fragment thereof according to claim 106 wherein the disease or condition susceptible to treatment with an inhibitor of IL-1 signalling is selected from the group consisting of rheumatoid arthritis, all types of juvenile arthritis including systemic onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells disease, neonatal onset multi-system inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult onset Still's disease, hyper IgD syndrome, type 2 diabetes mellitus, macrophage activation syndrome, TNF receptor-associated periodic syndrome, Blau disease, ankylosing spondylitis, Sweets disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, atherosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes mellitus, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), Hepatitis C, ischaemia-reperfusion injury, multiple sclerosis, Neisserial or pneumococcal meningitis, tuberculosis, Bechet's syndrome, septic shock, graft versus host disease, asthma, type I diabetes, Alzheimer's disease, atherosclerosis, adult T cell leukaemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (for example, metabolic syndrome, cardiomegaly, congestive heart failure, myocardial infarction, varicose veins, polycystic ovarian syndrome, gastroesophageal reflux disease (GERD), fatty liver disease, colorectal cancer, breast cancer, uterine cancer, chronic renal failure, stroke and hyperuricemia), intervertebral disc disease, irritable bowel syndrome, Schnitzler syndrome, allergy/atopic dermatitis and gout.

108. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 in the preparation of a medicament for the treatment of a disease or condition susceptible to treatment with an inhibitor of IL-1 signalling.

109. The use of an antibody or antigen-binding fragment thereof according to claim 108 wherein the disease or condition susceptible to treatment with an inhibitor of IL-1 signalling is selected from the group consisting of rheumatoid arthritis, all types of juvenile arthritis including systemic onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells disease, neonatal onset multi-system inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult onset Still's disease, hyper IgD syndrome, type 2 diabetes mellitus, macrophage activation syndrome, TNF receptor-associated periodic syndrome, Blau disease, ankylosing spondylitis, Sweets disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, atherosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes mellitus, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), Hepatitis C, ischaemia-reperfusion injury, multiple sclerosis, Neisserial or pneumococcal meningitis, tuberculosis, Bechet's syndrome, septic shock, graft versus host disease, asthma, type I diabetes, Alzheimer's disease, atherosclerosis, adult T cell leukaemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (for example, metabolic syndrome, cardiomegaly, congestive heart failure, myocardial infarction, varicose veins, polycystic ovarian syndrome, gastroesophageal reflux disease (GERD), fatty liver disease, colorectal cancer, breast cancer, uterine cancer, chronic renal failure, stroke and hyperuricemia), intervertebral disc disease, irritable bowel syndrome, Schnitzler syndrome, allergy/atopic dermatitis and gout.

110. A method for the treatment of a disease or condition susceptible to treatment with an inhibitor of IL-1 signalling in a subject, comprising the step of administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64.

111. A method according to claim 110 wherein the disease or condition susceptible to treatment with an inhibitor of IL-1 signalling is selected from the group consisting of rheumatoid arthritis, all types of juvenile arthritis including systemic onset juvenile idiopathic arthritis (SOJIA), osteoarthritis, familial cold auto-inflammatory syndrome (FCAS), Muckle-Wells disease, neonatal onset multi-system inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis pyoderma gangrenosum and acne (PAPA) syndrome, adult onset Still's disease, hyper IgD syndrome, type 2 diabetes mellitus, macrophage activation syndrome, TNF receptor-associated periodic syndrome, Blau disease, ankylosing spondylitis, Sweets disease, lupus arthritis, Alzheimer's disease, psoriasis, asthma, atherosclerosis, sarcoidosis, atopic dermatitis, systemic lupus erythematosus, bullous pemphigoid, type I diabetes mellitus, chronic obstructive pulmonary disease, Helicobacter pylori gastritis, inflammatory bowel disease (including ulcerative colitis and Crohn's disease), Hepatitis C, ischaemia-reperfusion injury, multiple sclerosis, Neisserial or pneumococcal meningitis, tuberculosis, Bechet's syndrome, septic shock, graft versus host disease, asthma, type I diabetes, Alzheimer's disease, atherosclerosis, adult T cell leukaemia, multiple myeloma, periodontitis, obesity and obesity-related diseases (for example, metabolic syndrome, cardiomegaly, congestive heart failure, myocardial infarction, varicose veins, polycystic ovarian syndrome, gastroesophageal reflux disease (GERD), fatty liver disease, colorectal cancer, breast cancer, uterine cancer, chronic renal failure, stroke and hyperuricemia), intervertebral disc disease, irritable bowel syndrome, Schnitzler syndrome, allergy/atopic dermatitis and gout.

112. An in vitro method for the detection of cancer cells in a subject, the method comprising: (a) providing a sample of cells (e.g. white blood stem/progenitor cells or biopsy tissue) from a subject to be tested; (b) optionally, extracting and/or purifying the cells present in the sample; (c) contacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 64 with cells present in the sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cells wherein the binding of the antibody or antigen-binding fragment thereof to the cells is indicative of the presence of cancer cells in the tissue of a subject.

113. An in vitro method for identifying a patient with cancer who would benefit from treatment with an antibody or antigen-binding fragment thereof according to any one of claims 43 to 64, the method comprising: (a) providing a sample of cancer cells (e.g. white blood stem/progenitor cells or biopsy tissue) from a patient to be tested; (b) optionally, extracting and/or purifying the cells present in the sample; (c) contacting an antibody or antigen-binding fragment thereof according to any one of claims 1 to 62 with cells present in the sample; (d) determining whether the antibody or antigen-binding fragment thereof binds to the cells wherein the binding of the antibody or antigen-binding fragment thereof to the cancer cells is indicative of a patient who would benefit from treatment with an antibody or antigen-binding fragment thereof according to any one of claims 43 to 64.

114. A method for treating a patient with cancer, the method comprising administering to a subject identified as having cancer using a method according to claim 112 or 113 a therapeutic agent effective in the treatment of said cancer.

115. A method for treating a patient with cancer according to claim 114 wherein the therapeutic agent is an antibody or antigen-binding fragment thereof according to any one of claims 1 to 4.

116. A method for treating a patient with cancer comprising: (a) arranging for a sample of cells (e.g. white blood stem/progenitor cells or biopsy tissue) from a subject to be tested for the presence of cancer cells expressing IL1RAP above a threshold criteria using a method according to claim 112 or 113; (b) selecting for treatment subjects whose sample of cells tested in step (a) contains cancer cells with IL1RAP expression above a threshold criteria; and (c) administering to the subject selected in step (b) a therapeutic agent effective in the treatment of said cancer.

117. A method for treating a patient with cancer according to claim 116 comprising (a) obtaining a sample of cells (e.g. white blood stem/progenitor cells or biopsy tissue) from a subject (b) testing said cells for the presence of cancer cells expressing IL1RAP above a threshold criteria using a method according to claims 112 or 113; (c) selecting for treatment subjects whose sample of cells tested in step (b) contains cancer cells with IL1RAP expression above a threshold criteria; and (d) administering to the subject selected in step (c) a therapeutic agent effective in the treatment of said cancer.

118. An antibody or antigen-binding fragment thereof, or use of the same, substantially as herein described with reference to the description.

Description

[0308] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0309] FIG. 1. Binding of the exemplary antibody in an indirect ELISA to human IL-1RAP. The exemplary antibody of the invention, CAN04, was found to possess the highest affinity for human IL-1RAP.

[0310] FIGS. 2A-C. Binding of exemplary antibody of the invention (CAN04) to human CML and AML cells. (2A) The graph shows the MFI value for KU812 cells stained with IL1RAP-targeting monoclonal antibodies at a concentration of 0.1 μg/lmL, and reveals that CAN04 has the highest MFI of the compared antibodies. (2B) CAN04 shows specific binding to five primary AML samples. (2C) In three primary CML samples, CAN04 shows specific binding.

[0311] FIG. 3. Exemplary humanized variants of antibody CAN04 all bind to IL1RAP-expressing BV173 cells, with Variant 6 showing the highest mean fluorescent intensity.

[0312] FIGS. 4A-C. Ability of exemplary antibody CAN04 to block (4A) IL-1β (4B) IL-1α, and (4C) IL-33 signalling.

[0313] FIG. 5. The total cell expansion of primary primitive CML cells in presence of IL1b is significantly reduced with exemplary antibody CAN04 in comparison with both control and a reference antibody. The graph shows the combined result from three individual patient samples normalised to the control consisting of culturing without antibody or with isotype control antibody.

[0314] FIGS. 6A-D. In vitro ADCC assay shows that exemplary antibody CAN04 induces specific cell killing of CML cells. (6A) KU812, LAMA84, and BV173 cells were specifically killed by addition of 0.1 μg/mL CAN04. (6B) The cell killing mediated by exemplary antibody CAN04 is dose dependant as shown on BV173 target cells. (6C) Cell killing of primary cells from two CML blast crisis patients was induced by 1 μg/mL CAN04. (6D) Cells from a third CML blast crisis patient carrying the T315I mutation were sensitive to the ADCC effect mediated by CAN04. Each experiment was performed at least twice with NK cells from different donors, and the presented data shows one representative experiment from each.

[0315] FIG. 7. In vitro ADCC assay showing that the exemplary CAN04 antibody is efficient in inducing specific cell killing of melanoma cells (SKMEL5 cell line). At all concentrations tested, as low as 0.1 μg/mL CAN04 shows a high specific killing. The experiment was performed at least twice with NK cells from different donors, and the presented data shows one representative experiment.

[0316] FIGS. 8A-B. (8A) Confocal images (one optical section, about 0.9 μm thick) showing LAMA cells incubated for 2 hours with CAN04-AF488 conjugated antibodies (green) on ice, or at 37° C. for 2 hours, or been incubated with CAN04-AF488 conjugated antibodies (green) for 16 hours at 37 C.°. A clearly defined antibody binding to the cell membrane of the majority of cells can be observed after 2 hours incubation on ice (“Ice 2h)”. After incubation for 2 hours with CAN04-AF488 conjugated antibodies (green) at 37° C., in addition to membrane binding, antibodies have started to enter the cells (internalization) and are now localized also in the cytosol. After 16 hours of incubation at 37° C., the cell membrane binding is still present and the antibody internalization has produced accumulation of CAN04-AF488 antibodies in the majority of cells. Scale bar (right image) represents 20 μm in all images. (8B) Control for the CAN04-specific binding and internalization: The confocal images (one optical section, about 0.9 μm thick) show LAMA cells incubated with AF488 conjugated isotype control antibody on ice or at 37° C. for 2 hours, or at 37 C.° for 16 hours. The isotype control antibody showed no specific binding at any of these conditions. Minor binding to cellular debris and necrotic cells (weak green) was noted. Scale bar (right image) represents 20 μm in all images.

[0317] FIGS. 9A-D. Treatment with CAN04 significantly reduces the leukemia burden. (9A) The frequency of leukemic cells in peripheral blood was lower in mice treated with exemplary antibody CAN04 compared to isotype control at day 36 after transplantation (1.2% vs. 22.7%, p<0.0001). (9B) The platelet (PLT) count remained at normal levels with CAN04 (p=0.0001). (9C) At time of sacrifice the frequency of leukemic cells in the bone marrow was reduced with CAN04 (38.7% vs. 91.5%; p<0.0001). (9D) The frequency of leukemic cells in spleen was lower in mice treated with CAN04 compared to isotype control (27.6% vs. 70.4%; p=0.0063).

[0318] FIGS. 10A-C. The effect of low fucose and Fc mutations on ADCC activity, as determined using SKMEL5 cells. (10A) ADCC activity in SKMEL-5 of CAN04 (Fc-1) normal fucose, CAN04 (Fc-1) low fucose, Fc-4 and Fc-7. (10B) ADCC activity in SKMEL-5 of CAN04 (Fc-1) normal fucose, CAN04 (Fc-1) low fucose, Fc-2 and Fc-3. (10C) ADCC activity in SKMEL-5 of CAN04 (Fc-1) normal fucose, CAN04 (Fc-1) low fucose, Fc-5, Fc-6 and Fc-8.

EXAMPLES

A. Binding Affinity of Exemplary Antibodies of the Invention for IL1-RAP Protein

(i) Biacore Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

[0319] Goat anti-mouse IgG was immobilized on a CM5 chip according to the technical manual of capture kit and standard operation principle of BIAcore T200 (Biacore Life Sciences, GE Healthcare Europe GmbH, Uppsala, Sweden).

[0320] The binding analysis cycle consisted of three steps: (i) capture of the ligand on the chip surface by immobilized anti-mouse antibody; (ii) binding of the analyte to the captured ligand; and (iii) dissociation of bound analyte.

[0321] The capture molecule surface was regenerated after each binding cycle using the manufacturer's recommended conditions.

[0322] All binding cycles were run at 25° C.

[0323] After five cycles of start-up, each antibody (100 nM) was injected at a flow rate of 30 μl/min, for 120 s, at the start of the cycle; then the analyte (100 nM) was injected at a flow rate of 30 μl/min, for 120 s, followed by monitoring the dissociation phase for 300 s.

[0324] One exemplary antibody of the invention (CAN04) was tested along with two comparator anti-IL1RAP antibodies (CAN01 and CAN03).

Results & Conclusions

[0325] Results are shown in Table 2 below:

TABLE-US-00006 TABLE 2 Measurement of K.sub.on, K.sub.off and K.sub.D Antibody ka (1/M .Math. s) kd (1/s) KD (M) CAN01 2.34E+05 3.35E−04 1.43E−09 CAN03 2.26E+05 7.25E−05 3.21E−10 CAN04 4.27E+05 4.72E−05 1.10E−10

[0326] The exemplary antibody of the invention, CAN04, exhibited the highest affinity for human IL1RAP.

(ii) ELISA Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

[0327] An indirect ELISA assay was performed. All samples were analysed in duplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ng of recombinant hIL1RAP 21-367 (100 μl/well) diluted in 0.01M PBS, pH 7.4, and incubated overnight at 4° C. Plates were washed with ELISA washing buffer (0.01M PBS, 0.05% Tween 20, pH 7.4) followed by a blocking step using 150 μl/well of ELISA blocking solution (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4). After 1 h incubation at room temperature (RT) on agitation the plates were washed again using ELISA washing buffer. Samples were diluted in three fold serial dilution (ranging from 1000 ng/ml to 0.5 ng/ml) in ELISA blocking solution and then transferred to the ELISA plate, 100 μl/well. Plates were incubated at RT for 1 h on agitation and then washed with ELISA washing solution. 100 μl/well of rabbit anti-mouse IgG conjugated to Alkaline Phosohatase (DAKO, 1:1000) was added and incubated 1 hour at RT on agitation. The plates were washed followed by addition of substrate (4-Nitrophenyl phosphatise disodium salt hexahydrate, SIGMA, 1 mg/ml), 100 μl/well. The plates were thereafter incubated at RT on agitation and absorbance at 405 nm measured consecutively for 30 min. Absorbance at 0 min was taken as background signal.

Results & Conclusions

[0328] Results are shown in FIG. 1

[0329] The exemplary antibody of the invention, CAN04, was found to possess the highest binding signal for human IL1RAP.

(iii) ELISA Study—Humanised Versions of the Exemplary ‘CAN04’ Antibody

Materials & Methods

[0330] Heavy and light chain variable domains of sixteen humanised variants of CAN04 (hCAN04) were sub-cloned into vectors containing human constant domains: [0331] Kappa constant domain for VL domain [SEQ ID NO:18] [0332] IgG1za heavy chain constant for VH domain [SEQ ID NO:19]

[0333] Antibodies were transiently expressed in CHOK1SV cells in a volume of 200 ml (shaker flask).

[0334] Antibodies were purified using Protein A affinity chromatography.

[0335] Purified antibodies were analysed by SDS-PAGE and SE-HPLC.

[0336] An indirect ELISA assay was performed as described above with the alteration that the samples were diluted in a three-fold serial dilution starting at 3.5 nM and run in replicates of four.

Results & Conclusions

[0337] Results are shown in Table 3.

TABLE-US-00007 TABLE 3 Affinity for hIL1RAP as determined by ELISA Antibody Heavy chain* Light Chain** K.sub.D (pM) CAN04 (murine) SEQ ID NO: 1 SEQ ID NO: 2  88 ± 2 hCAN04 Variant 1 SEQ ID NO: 8 SEQ ID NO: 15 125 ± 2 hCAN04 Variant 2 SEQ ID NO: 9 SEQ ID NO: 15 171 ± 4 hCAN04 Variant 3 SEQ ID NO: 10 SEQ ID NO: 15 450 ± 68 hCAN04 Variant 4 SEQ ID NO: 11 SEQ ID NO: 15 470 ± 50 hCAN04 Variant 5 SEQ ID NO: 8 SEQ ID NO: 16 135 ± 2 hCAN04 Variant 6 SEQ ID NO: 9 SEQ ID NO: 16 173 ± 4 hCAN04 Variant 7 SEQ ID NO: 10 SEQ ID NO: 16 526 ± 44 hCAN04 Variant 8 SEQ ID NO: 11 SEQ ID NO: 16 402 ± 64 hCAN04 Variant 9 SEQ ID NO: 8 SEQ ID NO: 17 210 ± 4 hCAN04 Variant 10 SEQ ID NO: 9 SEQ ID NO: 17 301 ± 5 hCAN04 Variant 11 SEQ ID NO: 10 SEQ ID NO: 17 504 ± 26 hCAN04 Variant 12 SEQ ID NO: 11 SEQ ID NO: 17 620 ± 68 *The heavy chain also comprised the IgG1za heavy chain constant domains [SEQ ID NO: 19]. **The light chain also comprised a kappa constant domain [SEQ ID NO: 18].

[0338] Four of the sixteen humanised versions of CAN04 showed minimal or no binding to IL1RAP (data not shown).

B. Binding of Exemplary Antibodies of the Invention to IL1RAP-Expressing Cells

(i) Flow Cytometry Study—Anti-IL1RAP Antibodies of Murine Origin

Materials & Methods

[0339] Chronic myeloid leukemia (CML) cell line KU812 cells were stained with antibodies raised against IL1RAP or a relevant isotype control. For detection, a secondary anti-mIg-APC was used.

[0340] One exemplary antibody of the invention (CAN04) was tested along with seven comparator anti-IL1RAP antibodies (CAN01, CAN02, CAN03, CAN05, CAN07, CAN08 and CAN09). An isotype negative control antibody was also included.

[0341] For analysis of primary leukemic cells, three CD34-enriched CML patient samples and five acute myeloid leukemia (AML) patient samples enriched for mononuclear cells were stained with CAN04-PE at concentrations of 1 μg/mL and 5 μg/mL respectively, or a PE-conjugated isotype control. Cells were analysed using a FACS CANTO flow cytometer (BD).

Results & Conclusions

[0342] Staining of IL1RAP-expressing KU812 leukemia cells reveals a higher mean fluorescence intensity (MFI) for CAN04 compared to the isotype control and other comparator antibodies targeting IL1RAP (FIG. 2 A). Labelling of primary cells from five AML and three CML patients using CAN04 result in staining above the isotype control in flow cytometric analysis (FIG. 2 B-C). The present study shows that CAN04 specifically bind IL1RAP with a higher MFI than other tested monoclonal antibodies on a CML cell line, and that CAN04 also bind to primary CML and AML cells.

(ii) Flow Cytometry Study—Humanised Versions of ‘CAN04’ Antibody

Materials & Methods

[0343] Chronic myeloid leukemia (CML) cell line BV173 cells were stained with 1 μg/mL test antibody or a relevant isotype control. For detection, a secondary anti-hIgG-PE was used. Cells were analysed using a FACS CANTO flow cytometer (BD).

[0344] The test antibodies included one chimeric anti-IL1RAP antibody (‘chimary’) and twelve different humanised versions of the exemplary CAN04 antibody of the invention (‘Var1’ to ‘Var 12’).

Results & Conclusions

[0345] Staining of IL1RAP-expressing BV173 leukemia cells shows that the humanised CAN04 variants stain with different intensity, but all at levels above the isotype control (see FIG. 3. Humanized variants 1, 2, 5, 6, 9 and 10 of CAN04 (see Table 3) exhibit the strongest labelling.

C. Epitope/Domain Mapping of Exemplary Antibodies of the Invention

Materials & Methods

[0346] In order to understand where the different antibody clones bind on the IL1RAP, a structural analysis of the protein was performed revealing that the extracellular part of the receptor could be divided into three distinct domains hereafter referred to as domains 1, 2 and 3 (D1, D2, D3) (see Wang et al., 2010, Nature Immunology, 11:905-912, the disclosures of which are incorporated herein by reference). In order to determine the domain-binding pattern for the different antibody clones, a series of receptor constructs were generated and binding to these tested in an ELISA assay.

[0347] An indirect ELISA assay was performed. All samples were analysed in duplicate. Nunc-MaxiSorp 96 Micro Well™ Plates were coated with 100 ng of Rec hIL1 RAP Domain123 (aa21-367) (positive control), Rec hIL1RAP Domain12 (aa21-234), Domain1 (aa21-134) or Rec hIL1RAP Domain3 (aa235-367) (100 μl/well) diluted in 0.01M PBS, pH 7.4, and incubated overnight at 4° C. Plates were washed with ELISA washing buffer (0.01M PBS, 0.05% Tween 20, pH 7.4) followed by a blocking step using 150 μl/well of ELISA blocking solution (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4). After 1 h incubation at room temperature (RT) on agitation the plates were washed again using ELISA washing buffer. CAN01, CAN03, CAN04, CAN05, CAN07, CAN08 and KMT-1 (positive control) were diluted in three fold serial dilution (ranging from 1000 ng/ml to 0.5 ng/ml) in ELISA blocking solution and then transferred to the ELISA plate, 100 μl/well. Plates were incubated at RT for 1 h on agitation and then washed with ELISA washing solution. 100 μl/well of rabbit anti-mouse IgG conjugated to Alkaline Phosphatase (DAKO, 1:1000) was added and incubated 1 hour at RT on agitation. The plates were washed followed by addition of substrate (4-Nitrophenyl phosphatise disodium salt hexahydrate, SIGMA, 1 mg/ml), 100 l/well. The plates were thereafter incubated at RT on agitation and absorbance at 405 nm measured consecutively for 30 min. Absorbance at 0 min was taken as background signal.

[0348] One exemplary antibody of the invention (CAN04) was tested along with nine comparator anti-IL1RAP monoclonal antibodies (CAN01, CAN02, CAN03, CAN05, CAN07, CAN08, CAN10, and CAN11, together with a polyclonal anti-IL1RAP antibody (KMT-1) as a positive control.

Results & Conclusions

[0349] A majority of anti-IL1RAP antibodies tested for target validation bind to domain 3 (D3). However, the exemplary CAN04 antibody of the invention is distinct in that it binds to domain 2 (D2). The entire domain mapping data can be found summarized in the Table 4 below.

TABLE-US-00008 TABLE 4 Epitope mapping of exemplary anti-IL1RAP antibody clones. Domain123 Domain12 Domain1 Domain3 Suggested Clone (aa21-367) (aa21-234) (aa21-134) (aa235-367) epitope CAN03 + + D3 CAN05 + + + D1 CAN07 + + D3 CAN08 + + D3 CAN04 + + D2 CAN01 + + D3 CAN02 + nd* KMT-1 + + + + polyclonal nd* = not determined as epitope mapping data could not clearly identify specific domain for these constructs, which may be attributed to binding to a structural epitope containing sequence elements from more than one domain, e.g. D2-D3 junction.

D. Specificity/Cross-Reactivity of Exemplary Antibodies of the Invention

Materials & Methods

[0350] An important feature of a good lead candidate antibody is that it cross-reacts with equal or near-equal potency to the homologous protein in a relevant toxicology species. According to the general regulatory guidelines, binding to one rodent and one non-rodent would be the preferred scenario, but for antibodies this is rarely the case, and instead many labs struggle to identify any relevant toxicology species except for primates.

[0351] For the present study, cross reactivity to non-human primates like Macaca mulatta (rhesus) or Macaca fascicularis (cynomolgus) was expected since the IL1RAP protein in these species share 99% homology to the human IL1RAP protein.

[0352] A number of potential lead antibodies were selected and tested for binding to recombinant M. fascicularis IL1RAP (aa21-367) in an ELISA assay.

[0353] One exemplary antibody of the invention (CAN04) was tested along with eight comparator anti-IL1RAP monoclonal antibodies (CAN01, CAN02, CAN03, CAN07, CAN08, CAN09, Mab676 from R&D, and a polyclonal anti-IL1RAP antibody (KMT-1).

Results & Conclusions

[0354] Surprisingly, several of the comparator anti-L1 RAP antibodies tested were found not to cross-react with cynomolgus IL1RAP, amongst them the commercial reference antibody mAb676 from R&D, Table 5.

TABLE-US-00009 TABLE 5 Binding to cynomolgus IL1RAP (Values in bold denotes clones identified to cross-react with IL1RAP from M. fascicularis) Binding to rec. M. fascicularis Clone IL1RAP (OD.sub.405) CAN01 0.324 CAN02 0.014 CAN09 0.022 CAN03 0.870 CAN04 0.416 CAN07 0.111 CAN08 0.375 mAb676 (R&D) 0.037 KMT-1 0.481

E. Inhibition of IL-1α, IL-1β and IL-33 Signalling by Exemplary Antibodies of the Invention

(i) Effect of CAN04 on IL-1 Signalling in HEK-Blue IL-33/IL-1β Cell Line

Materials & Methods

[0355] As IL1RAP is a functional part of the IL-1 receptor complex, antibodies binding to IL1RAP may also inhibit IL-1 signalling. Since a number of tumour cell types have been shown to use IL-1 as a growth factor, this may be an important additional mechanism for mediating anti-tumour effects.

[0356] In order to test for the capability of potential lead candidate antibodies to block IL-1 signalling, an IL-1 dependent reporter gene assay was set up. HEK-Blue IL-33/IL-1β cells (InvivoGen) respond to IL-1 signalling by the release of alkaline phosphatase that can be quantified by a colorimetric assay. To test the inhibitory capacity of the lead candidates HEK-Blue cells were plated at 50 000 cells/well and incubated with the test antibodies 45 minutes prior to stimulation with IL-1α, IL-1β or IL-33 in a final concentration in assay of 0.3 ng/ml for each ligand. Final assay concentrations of antibodies were 100 nM-0.01 nM. In the control wells, the antibodies were replaced by PBS. The cells were incubated at 37° C. o/n before measuring the amount of alkaline phosphatase released. Antibodies were also tested for potential agonistic effects by incubating the cells in the presence of a high concentration of antibody (10 mg/ml) in the absence of additional stimuli. Any IL-1R agonistic effects would thus be recorded as a release of alkaline phosphatase.

[0357] One exemplary antibody of the invention (CAN04) was tested along with two comparator anti-IL1RAP monoclonal antibodies (CAN01 and CAN 03) and an isotype negative control antibody.

Results & Conclusions

[0358] As depicted in FIG. 4(A), the exemplary antibody CAN04 induced a pronounced inhibition of IL-1β signalling. Comparator antibody CAN03 also produced a detectable inhibition but significantly less than CAN04. Neither the comparator CAN01 nor the isotype control produced any measurable inhibition of IL1 signalling.

[0359] In addition to blocking IL-1β, CAN04 is also a potent inhibitor of IL-1α and IL-33 signalling; see FIGS. 4(B) and (C), respectively.

[0360] None of the tested candidates showed any agonistic effect.

(ii) Effect in Primary CML Cells

Materials & Methods

[0361] Bone marrow aspirates or peripheral blood was drawn from three patients with chronic myeloid leukemia (CML) in chronic phase. Mononuclear cells were isolated by centrifugation over Lymphoprep, and samples were enriched for CD34+ cells using an anti-CD34 antibody and magnetic beads (Miltenyi biotech.) The CD34+ cells were kept in liquid nitrogen until use, when they were thawed and stained with antibodies against CD34 and CD38. Propidium iodide was used as viability marker. Using a FACS Aria cell sorter, viable CD34+CD38− cells were sorted at a density of 2500 cells per well into 96 wells tissue culture treated plates containing 100 μl serum free Stemspan culturing medium without supplements. After sorting, IL1b and the test antibody were added to the wells in a total of 100 ul Stemspan to produce a final volume of 200 μl per well with 0-0.4 ng/mL IL1b and 0-10 μg/mL antibody. The plates were incubated at 37° C., 5% CO.sub.2, for 7 days after which the number of viable (7AAD-) cells were counted using Countbright counting beads and a FACS Canto.

[0362] One exemplary antibody of the invention (CAN04) was tested along with one comparator anti-IL1RAP monoclonal antibodies (CAN01) and an isotype negative control antibody.

Results & Conclusions

[0363] As shown in FIG. 5, culturing of CD34+CD38− primary chronic phase CML cells in the presence of IL1b results in increased cell expansion. The IL1-induced increase in cell expansion is significantly reduced upon addition of the exemplary CAN04 antibody to the culture (p<0.0001). Also in comparison with another IL1RAP-targeting antibody, CAN01, CAN04 is significantly more effective in reducing the total cell expansion (p=0.0022). We conclude that binding of CAN04 to IL1RAP interferes with the stimulation in cellular expansion induced by IL1 in primary primitive CML cells.

F. ADCC Effect of Exemplary Antibodies of the Invention

(i) Chronic Myeloid Leukemia (CML) Cell Lines

Materials & Methods

[0364] Chronic myeloid leukemia (CML) cell lines KU812, LAMA84 and BV173, or primary cells from three patients with CML in blast crisis were used as target cells in the in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Briefly, target cells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions, and seeded into a 96-well plate at a density of 5,000-10,000 cells per well. Subsequently, the exemplar antibody of the invention, CAN04, or isotype control antibody was added to wells in different concentrations and incubated for 30 min before 100,000 NK effector cells were added to each well. NK-cells were extracted from healthy volunteers after informed consent by using an NK-cell negative cell isolation kit according to manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). A non-specific human IgG1 antibody was used as an isotype negative control in the experiments (Eureka Therapeutics, Emeryville, Calif.). The degree of cell death was assessed by detection of 7-AAD positive cells using a FACS CANTO flow cytometer (BD). Each experiment was performed at least twice with NK cells from different donors.

Results & Conclusions

[0365] The in vitro ADCC assay shows that the exemplary antibody of the invention, CAN04, directs NK-cells to kill CML cell lines KU812, LAMA84 and BV173 to a higher degree than the isotype control (FIG. 6A). A dose titration of CAN04 using BV173 target cells shows that the effect on cell killing is dose dependent with a higher degree of cell killing with increasing CAN04 concentration (FIG. 6B). Chronic myeloid leukemia that has progressed into blast crisis display only transient effect to treatment with tyrosine kinase inhibitors and thus imposes a major treatment problem. The ADCC assay with primary cells from two individual CML blast crisis patients shows that these cells were sensitive to the cellular cytotoxicity induced by CAN04 and NK-cells (FIG. 6C). In addition, primary cells from a third CML blast crisis patient harbouring the T315I mutation that cause resistance to several tyrosine kinase inhibitors display similar sensitivity (FIG. 6D). Altogether, the experiments show that CAN04 has the ability to direct NK-cells to specific cell killing of CML cell lines as well as primary blast crisis CML cells, and that the cytotoxic effect induced by CAN04 is dose dependent.

(ii) Melanoma Cell Lines

Materials & Methods

[0366] The malign melanoma cell line SKMEL-5 was used as a target for in vitro antibody dependent cellular cytotoxicity (ADCC) assay. Briefly, target cells were labelled with PKH26 (Sigma-Aldrich, St Louis, Mo.) according to manufacturer's instructions, and seeded into a 96-well plate at a density of 5,000-10,000 cells per well. Subsequently, CAN04 or isotype control antibody were added to wells in different concentrations and incubated for 30 min before 100,000 NK effector cells were added to each well. NK-cells were extracted from healthy volunteers after informed consent by using an NK-cell negative cell isolation kit according to manufacturer's instructions (Miltenyi Biotech, Bergisch Gladbach, Germany). A non-specific human IgG1 antibody was used as control in the experiments (Eureka Therapeutics, Emeryville, Calif.). The degree of cell death was assessed by detection of 7-AAD positive cells using a FACS CANTO flow cytometer (BD). Each experiment was performed at least twice with NK cells from different donors.

Results & Conclusions

[0367] The in vitro ADCC assay showed that CAN04 directs NK-cells to killing of the SKMEL-5 cell line to a much higher degree than a matching isotype control (FIG. 7). The dose titration of CAN04 showed that CAN04 is efficient in inducing ADCC, at low concentrations (FIG. 7). In summary, these data demonstrate that CAN04 has the ability to direct NK-cells to specific cell killing of SKMEL-5 in a dose dependent manner and that CAN04.

G. Internalisation of Exemplary Antibodies of the Invention

Materials & Methods

[0368] Cells and Culture Conditions: LAMA-84 cells, a cell-line established from a patient with chronic myeloid leukemia in blast crisis, were obtained from DSMZ (Braunschweig, Germany) and cultured according to the recommendation by the supplier. Briefly, cells were cultured in RPMI 1640 with 10% FBS, 1% Glutamine and 1% Penicillin/Streptomycin in 5% CO.sub.2, 37° C. Cell cultures were split to a density of 0.5×10.sup.6 cells/ml every 2-3 days. Cells were used for up to 12 passages after they were received from DSMZ.

[0369] Cells from the suspension cell-line LAMA-84 were washed once in phosphate buffered saline (PBS) supplemented with 1% Bovine serum albumin (BSA) and resuspended in PBS-BSA supplemented with 5% human AB+ serum from Sigma and incubated for 5 minutes at room temperature (RT). The AlexaFluor488 (AF488) labelled IL-1RAP selective antibody CAN04, or isotype matched control antibody, was added to a final concentration of 10 μg/ml. Cells were placed (incubated) on ice or at 37° C. for 2 or 16 hours.

[0370] For the image analysis with confocal microscopy (LSM 510 Meta Zeiss confocal microscope), cells were washed twice in PBS-1% BSA, were briefly spun down followed by resuspension in 3% paraformaldehyde (in PBS) fixation for 20 minutes (at 4 C.°). Cells were then spun down, resuspended in PBS containing 0.001% Triton X-100 (PBS-TX) and a nuclear marker (DAPI), and were let to incubate for 5 minutes at RT. After a brief centrifugation cells were resuspended in PBS-TX and placed in glass-bottomed microscope wells. Cells were then let to adhere for one hour. Image data were collected via confocal scanning of cells providing high-resolution images of AlexaFluor488 fluorescence in thin optical sections through the centre of cells (depicted by nuclear marker). Further analyses of antibody binding to cell membrane and/or internalized antibodies were performed via software image analyses (Zeiss Zen2010).

Results & Conclusions

[0371] The structural relation of CAN04 binding to the cell membrane and its capacity to enter the cells (“internalize”) was demonstrated with high resolution imaging data recorded by means of confocal laser scanning microscopy, and by image analyses of this data.

[0372] Image data representations are shown in FIG. 8.

H. Therapeutic Efficacy In Vivo of an Exemplary Antibody of the Invention

Materials & Methods

[0373] Unconditioned NOD/SCID mice were engrafted with lethal doses of MA9Ras cells, previously generated by transformation of human umbilical cord blood CD34.sup.+ cells by retroviral integration of cDNAs directing the expression of an MLL/AF9 fusion and an activated NRAS gene. Leukemic mice were treated with the exemplary CAN04 antibody targeting IL1RAP, or a corresponding isotype control antibody. The antibodies were administered by intraperitoneal injections twice weekly throughout the experiment with first treatment given day three after transplantation. Each dose of antibody was 500 μg, except for the first that was given as a bolus of 1000 μg. Mice were sacrificed upon signs of severe disease as judged by hunchback, untidy fur, and decreased mobility, or due to solid tumours.

Results & Conclusions

[0374] Immunodeficient mice were engrafted with human leukemic cells and treated with CAN04, a monoclonal antibody targeting IL1RAP. The frequency of leukemic cells in peripheral blood was significantly reduced at day 36 after transplantation, and the platelet counts remained normal in mice given CAN04 compared to isotype control antibody indicating a more functional haematopoiesis (FIG. 9A-B). CAN04 treatment resulted in a significant reduction of leukemic cells in bone marrow and spleen (FIG. 9C-D). We conclude that anti-IL1RAP immunotherapy reduces human leukemia in peripheral blood, bone marrow, and spleen, in the MA9Ras xenograft model. The results support anti-IL1RAP immunotherapy as a new promising therapeutic strategy for AML.

I. Effect of (a) Low Fucose Antibodies and (b) Fc Mutations on ADCC Activity

Materials & Methods

[0375] The humanised heavy chain variable domain variant of CAN04 (comprising SEQ ID No: 9) was sub-cloned into vector containing the of IgG1za human constant domain (SEQ ID NO:19).

[0376] The humanised light chain variable domain variant of CAN04 (comprising SEQ ID No: 16) was sub-cloned into vector containing the Kappa human constant domain (SEQ ID NO:18).

[0377] To examine the effect of low fucose, hCAN04 antibody “Fc-1” (see below) were transiently expressed by co-transfection of the resulting vectors into HEK293 cells cultured in medium containing Kinfunensine. Antibody Fc-1, produced by expression in the absence of Kinfunensine, was used as the ‘Normal Fucose’ control

[0378] To address how various Fc-engineered mutants of hCAN04 affect the ADCC activity, genetically engineered hCAN04 variants were generated as follows: [0379] Fc-1=(Humanised CAN04 with wildtype Fc; i.e. hCAN04 variant 6 in Table 3) [0380] Fc-2=(As Fc-1 but with mutations S239D/S298A/I332E) [0381] Fc-3=(As Fc-1 but with mutations S239D/A330L/I332E) [0382] Fc-4=(As Fc-1 but with mutations S239D/I332E) [0383] Fc-5=(As Fc-1 but with mutations S298A/E333A/K334A) [0384] Fc-6=(As Fc-1 but with mutation N297Q) [0385] Fc-7=(As Fc-1 but with mutation N297S) [0386] Fc-8=(As Fc-1 but with mutations P247I/A339Q)

[0387] wherein the position of the amino acid mutations is defined using the Eu Numbering Scheme, which differs from the numbering in SEQ ID NOS: 18 and 19 above; see

[0388] Edelman et al., 1969, Proc. Natl. Acad. Sci. USA, 63:78-85).

[0389] Antibodies were purified using Protein A affinity chromatography.

[0390] ADCC assays were performed using the SKMEL5 cell line.

Results & Conclusions

[0391] A low fucose variant of hCAN04, produced in the presence of kinfunensine, was superior to a normal fucose CAN04-variant (FIG. 10).

[0392] The effect of Fc mutation on ADCC activity was dependent upon the mutation(s) made. Thus: [0393] (a) ADCC-activity of the Fc-6 and Fc-7 variants of humanised CAN04 was completely abrogated; [0394] (b) ADCC-activity of the Fc-3, Fc-4, and Fc-5 variants of humanised CAN04 was enhanced, even relative to the low fucose variant of CAN04; [0395] (c) ADCC-activity of the Fc-8 variant of humanised CAN04 was similar to that of the low fucose form of CAN04 [0396] (d) ADCC-activity of the Fc-2 variant of humanised CAN04 was similar to that of the normal fucose form of CAN04.

Example J—Analysis of Competitive Binding by ELISA

Protocol

[0397] All samples should be analysed in duplicate. [0398] Coat a Nunc-MaxiSorp 96 Micro Well™ Plate with 100 ul/well of recombinant hIL1RAP 21-367 (1 ug/ml) diluted in 0.01M PBS, pH 7.4. [0399] Incubate the plate overnight at 4° C. [0400] Wash the plate with ELISA washing buffer [0401] (0.01M PBS, 0.05% Tween 20, pH 7.4). [0402] Add 150 μl/well of ELISA blocking solution [0403] (PBS, 0.5% BSA, 0.05% Tween 20, pH 7.4). [0404] Incubate the plate for 1 h at room temperature (RT) under agitation. [0405] Wash the plate with ELISA washing buffer. [0406] Add samples of test items (e.g. mAb 1, mAb 2) to wells (100 ul/well, 10 ug/ml) [0407] Incubate the plate for 1 h at RT. [0408] Wash the plate with ELISA washing solution. [0409] Add a solution of reference antibody CAN04 (100 ul/well, 1 ug/ml) to all wells. [0410] Incubate the plate for 1 h at RT. [0411] Wash the plate with ELISA washing buffer. [0412] Add 100 μl/well of a suitable secondary antibody conjugated to Alkaline rabbit anti-mouse IgG conjugated to Alkaline Phosphatase (If the test items are human antibodies, a suitable secondary antibody would be Goat Anti-Mouse IgG (Fc specific)-Alkaline Phosphatase antibody, SIGMA, A1418) [0413] Incubate the plate for 1 h at RT under agitation. [0414] Wash the plate with washing buffer. [0415] Add 100 μl of pNPP substrate per well. [0416] (4-Nitrophenyl phosphatise disodium salt hexahydrate, SIGMA, 1 mg/ml). [0417] Incubate the plate at RT under agitation and measure absorbance at 405 nm consecutively for 30 min. Absorbance at 0 min should be taken as background signal.