This invention relates to anti-p-selectin antibodies and, in particular, to anti-p-selectin antibodies and variants thereof that contain an Fc part derived from human origin and do not bind complement factor C1q. These antibodies have new and inventive properties causing a benefit for a patient suffering from critical limb ischemia or peripheral arterial occlusive disease (CLI/PAOD).
|
0. 137. An antibody that binds to p-selectin comprising a light chain variable region, a light chain constant region, a heavy chain variable region, and a heavy chain constant region, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:3, the light chain constant region has the amino acid sequence of SEQ ID NO:23, the heavy chain variable region has the amino acid sequence of SEQ ID NO:4, and the heavy chain constant region has the amino acid sequence of SEQ ID NO:28.
0. 117. An antibody that binds to p-selectin comprising a light chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; and SEQ ID NO: 21; or a heavy chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; and SEQ ID NO: 22.
0. 83. A human antibody that binds to p-selectin comprising a light chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; and SEQ ID NO: 21; or a heavy chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; and SEQ ID NO: 22.
0. 49. A humanized antibody that binds to p-selectin comprising a light chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; and SEQ ID NO: 21; or a heavy chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; and SEQ ID NO: 22.
15. A humanized or human antibody that binds to p-selectin comprising a light chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 1; SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; and SEQ ID NO: 21; or a heavy chain variable region having an amino acid sequence selected from the group consisting of: SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; and SEQ ID NO: 22.
0. 120. An antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20; and
(b) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 86. A human antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20; and
(b) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 52. A humanized antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20; and
(b) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
18. A humanized or human antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20; and
(b) an antibody wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 103. An antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3;
(b) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3;
(c) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(d) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(e) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(f) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(g) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3;
(h) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3; and
(i) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 69. A human antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3;
(b) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3;
(c) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(d) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(e) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(f) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(g) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3;
(h) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3; and
(i) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 35. A humanized antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3;
(b) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3;
(c) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(d) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(e) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(f) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(g) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3;
(h) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3; and
(i) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
1. A humanized or human antibody that binds to p-selectin comprising 3 complementarity determining regions in the light chain variable region and 3 complementarity determining regions in the heavy chain variable region selected from the group consisting of:
(a) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3;
(b) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3;
(c) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(d) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(e) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3;
(f) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3;
(g) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3;
(h) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3; and
(i) an antibody wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
2. An antibody of
3. An antibody of
4. An antibody of
5. An antibody of
6. An antibody of
7. An antibody of
(a) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 1 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 2;
(b) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4;
(c) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 5 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 6;
(d) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 8;
(e) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 9 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 10;
(f) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 11 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 12;
(g) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 13 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 14;
(h) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 16; and
(i) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 17 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 18.
8. An antibody of
9. An antibody of
10. An antibody of
11. An antibody of
12. An antibody of
13. An antibody of
14. An antibody of
16. An antibody of
17. An antibody of
19. An antibody of
20. An antibody of
21. An antibody of
22. An antibody of
23. An antibody of
24. An antibody of
25. An antibody of
26. An antibody of
27. An antibody of
28. An antibody of
30. The pharmaceutical composition of
32. The pharmaceutical composition of
0. 36. An antibody of claim 35 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 37. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3.
0. 38. An antibody of claim 37 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 39. An antibody of claim 37 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 40. An antibody of claim 37 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 41. An antibody of claim 35 selected from the group consisting of:
(a) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 1 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 2;
(b) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4;
(c) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 5 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 6;
(d) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 8;
(e) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 9 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 10;
(f) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 11 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 12;
(g) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 13 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 14;
(h) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 16; and
(i) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 17 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 18.
0. 42. An antibody of claim 41 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 43. An antibody of claim 41 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 44. An antibody of claim 41 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28; and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 45. An antibody of claim 41 wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4.
0. 46. An antibody of claim 45 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 47. An antibody of claim 45 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 48. An antibody of claim 45 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 50. An antibody of claim 49 comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 3.
0. 51. An antibody of claim 49 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4.
0. 53. An antibody of claim 52 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20.
0. 54. An antibody of claim 52 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 55. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3.
0. 56. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 57. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 58. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 59. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 60. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 61. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 62. An antibody of claim 35 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 63. A pharmaceutical composition comprising an antibody of claim 35.
0. 64. The pharmaceutical composition of claim 63, further comprising at least one pharmaceutically acceptable excipient.
0. 65. A pharmaceutical composition comprising an antibody of claim 37.
0. 66. The pharmaceutical composition of claim 65, further comprising at least one pharmaceutically acceptable excipient.
0. 67. A kit to detect the presence of p-selectin protein comprising an antibody of claim 35.
0. 68. A kit to detect the presence of p-selectin protein comprising an antibody of claim 37.
0. 70. An antibody of claim 69 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 71. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3.
0. 72. An antibody of claim 71 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 73. An antibody of claim 71 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 74. An antibody of claim 71 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 75. An antibody of claim 69 selected from the group consisting of:
(a) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 1 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 2;
(b) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4;
(c) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 5 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 6;
(d) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 8;
(e) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 9 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 10;
(f) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 11 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 12;
(g) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 13 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 14;
(h) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 16; and
(i) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 17 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 18.
0. 76. An antibody of claim 75 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 77. An antibody of claim 75 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 78. An antibody of claim 75 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28; and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 79. An antibody of claim 75 wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4.
0. 80. An antibody of claim 79 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 81. An antibody of claim 79 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 82. An antibody of claim 79 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 84. An antibody of claim 83 comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 3.
0. 85. An antibody of claim 83 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4.
0. 87. An antibody of claim 86 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20.
0. 88. An antibody of claim 86 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 89. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3.
0. 90. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 91. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 92. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 93. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 94. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 95. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 96. An antibody of claim 69 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 97. A pharmaceutical composition comprising an antibody of claim 69.
0. 98. The pharmaceutical composition of claim 97, further comprising at least one pharmaceutically acceptable excipient.
0. 99. A pharmaceutical composition comprising an antibody of claim 71.
0. 100. The pharmaceutical composition of claim 99, further comprising at least one pharmaceutically acceptable excipient.
0. 101. A kit to detect the presence of p-selectin protein comprising an antibody of claim 69.
0. 102. A kit to detect the presence of p-selectin protein comprising an antibody of claim 71.
0. 104. An antibody of claim 103 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 105. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 48 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 30 for CDR1, SEQ ID NO: 34 for CDR2, and SEQ ID NO: 39 for CDR3.
0. 106. An antibody of claim 105 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 107. An antibody of claim 105 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 108. An antibody of claim 105 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 109. An antibody of claim 103 selected from the group consisting of:
(a) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 1 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 2;
(b) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4;
(c) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 5 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 6;
(d) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 7 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 8;
(e) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 9 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 10;
(f) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 11 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 12;
(g) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 13 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 14;
(h) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 15 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 16; and
(i) an antibody wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 17 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 18.
0. 110. An antibody of claim 109 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28.
0. 111. An antibody of claim 109 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 112. An antibody of claim 109 further comprising a heavy chain constant region having an amino acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 28; and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 113. An antibody of claim 109 wherein the light chain variable region has the amino acid sequence of SEQ ID NO: 3 and the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4.
0. 114. An antibody of claim 113 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28.
0. 115. An antibody of claim 113 further comprising a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 116. An antibody of claim 113 further comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 28 and a light chain constant region having the amino acid sequence of SEQ ID NO: 23.
0. 118. An antibody of claim 117 comprising a light chain variable region having the amino acid sequence of SEQ ID NO: 3.
0. 119. An antibody of claim 117 comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 4.
0. 121. An antibody of claim 120 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 19; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 20.
0. 122. An antibody of claim 120 wherein the light chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 21; and the heavy chain variable region comprises the 3 complementarity determining regions in SEQ ID NO: 22.
0. 123. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 47 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 33 for CDR2, and SEQ ID NO: 38 for CDR3.
0. 124. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 125. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 49 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 126. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 32 for CDR1, SEQ ID NO: 36 for CDR2, and SEQ ID NO: 41 for CDR3.
0. 127. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 50 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 31 for CDR1, SEQ ID NO: 35 for CDR2, and SEQ ID NO: 40 for CDR3.
0. 128. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 129. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 44 for CDR1, SEQ ID NO: 46 for CDR2, and SEQ ID NO: 51 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 130. An antibody of claim 103 wherein the light chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 43 for CDR1, SEQ ID NO: 45 for CDR2, and SEQ ID NO: 52 for CDR3; and the heavy chain variable region comprises 3 complementarity determining regions having the amino acid sequences of: SEQ ID NO: 29 for CDR1, SEQ ID NO: 37 for CDR2, and SEQ ID NO: 42 for CDR3.
0. 131. A pharmaceutical composition comprising an antibody of claim 103.
0. 132. The pharmaceutical composition of claim 131, further comprising at least one pharmaceutically acceptable excipient.
0. 133. A pharmaceutical composition comprising an antibody of claim 105.
0. 134. The pharmaceutical composition of claim 133, further comprising at least one pharmaceutically acceptable excipient.
0. 135. A kit to detect the presence of p-selectin protein comprising an antibody of claim 103.
0. 136. A kit to detect the presence of p-selectin protein comprising an antibody of claim 105.
0. 138. A pharmaceutical composition comprising an antibody of claim 137.
0. 139. The pharmaceutical composition of claim 138, further comprising at least one pharmaceutically acceptable excipient.
0. 140. A kit to detect the presence of p-selectin protein comprising an antibody of claim 137.
0. 141. A kit to detect the presence of p-selectin protein comprising a pharmaceutical composition according to claim 139.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||
Amino acids are abbreviated either in the three (Leu) or one letter code (L) Antibody HuMab 00X is also named antibody 00X
Culture of Hybridomas
HuMab hybridomas were cultured in IMDM (Cambrex), Fetal clone 1 Bovine serum (Perbio Science), origin Hybridoma cloning factor (Igen), sodium pyruvate, penicillin/streptomycin, 2-mercaptoethanol, HAT (Sigma-Aldrich) and Kanamycin (Invitrogen) in 37° C. and 5% CO2.
Generation of a Hybridoma Cell Line Producing Anti-P-Selectin Antibodies
Immunization Procedure of Transgenic Mice
Protocol A:
10 HCo7 transgenic mice (5 males and 5 females), strain GG2201 (Medarex, San Jose, Calif., USA) were immunized with a recombinant truncated form of P-selectin which lacks the transmembrane and cytoplasmic domain of P-selectin and which was purchased from R&D Systems. For the first immunization 50 μg recombinant P-selectin, dissolved in 100 μl PBS, was mixed with 100 μl complete Freunds' adjuvant. For the remaining immunizations recombinant P-selectin coupled to KLH was used. For the second immunization 50 μg KLH-coupled recombinant P-selectin was dissolved in 100 μl PBS and mixed with 100 μl incomplete Freunds' adjuvant. For the remaining immunizations 20 μg KLH-coupled recombinant P-selectin was dissolved in 100 μl PBS and mixed with 100 μl incomplete Freunds' adjuvant. Immunizations were administered alternating interperitoneal and subcutaneous starting with an interperitoneal immunization.
Protocol B:
3 HCo7 (all female) and 3 KM (all male) transgenic mice, strain GG2489 (Medarex, San Jose, Calif., USA) were immunized with full-length P-selectin purified from human outdated platelets by immunoaffinity chromatography (s. below). For the first immunization, 50 μg of the purified P-selectin, dissolved in 100 μl PBS, was mixed with 100 μl complete Freunds' adjuvant (CFA; Difco Laboratories, Detroit, USA). For the second immunization, 50 μg of the purified P-selectin, dissolved in 100 μl PBS, was mixed with 100 μl incomplete Freunds' adjuvant (ICFA; Difco).
For all other immunizations, 20 μg of the purified P-selectin was used and mixed with 100 μl incomplete Freunds' adjuvant.
Antigen Specific ELISA
Anti-P-selectin titers in sera of immunized mice were determined by antigen specific ELISA. Plate (96 flat bottom ELISA plate, Greiner) was coated with 0.1 μg/ml purified P-selectin dissolved in PBS and coated overnight at room temperature. Thereafter, wells were blocked with PBSTC (PBS containing 0.05% Tween 20 (Sigma-Aldrich Chemie BV) and 2% chickenserum (Gibco)) for 1 hour at room temperature.
Tested serum taps were diluted 1:100 in PBSTC and added to the wells. Serum obtained from mice prior to immunization was dissolved 1:100 in PBSTC and used as negative control. A mouse antibody directed against human P-selectin ( 1/7, produced in house by Roche Basel) was dissolved 1:100 in PBSTC and used as a positive control. Plates were incubated for 1 hour at room temperature. Subsequently, plates were washed twice using PBST (PBS containing 0.05% Tween 20. Gt-α-huIgG-HRP (Jackson) was diluted 1:5000 in PBSTC and added to the wells containing the tested taps and the negative control. Rb-α-mIgG (Jackson) was diluted 1:3000 in PBSTC and added to the wells containing the positive control. Plates were incubated for 1 hour at room temperature. Finally, plates were washed twice using PBST and developed with freshly prepared ABTS® solution (1 mg/ml) (ABTS: 2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) for 30 minutes at room temperature (RT) in the dark. Absorbance was measured at 405 nm.
Boosting of Mice
When serum titers of anti-P-selectin were sufficient, mice were additionally boosted twice with 20 μg recombinant human P-selectin in 100 μl PBS, intraveneously 4 and 3 days before fusion.
Hybridoma Generation
Mice were sacrificed and the spleen and lymph nodes flanking the abdominal aorta and vena cava were collected. Fusion of splenocytes and lymph node cells with the fusion partner SP 2.0 cells was performed according to standard operating procedures.
Human monoclonal antibodies with variable heavy and light sequences of SEQ ID NOs 1-22 were obtained by the immunization procedure.
κ-ELISA
To determine whether hybridomas that resulted from the fusion generate human antibodies, a κ-ELISA was performed. ELISA plates were coated with rat anti-human IgG κ-light chain antibody (DAKO) diluted 1/10000 in PBS by overnight incubation at 4° C. After discarding the wells, plates were blocked by incubation with PBSTC for 1 hour at room temperature. Thereafter, wells were incubated with hybridoma culture supernatant, 1/2 diluted in PBSTC. Culture medium 1/2 diluted in PBSTC was used as negative control, κ-light positive mouse serum 1/100 diluted in PBSTC served as positive control. Subsequently, wells were washed thrice and were incubated with HRP-conjugated rat anti-human IgG F(ab′)2 (DAKO), diluted 1/2000 in PBSTC for 1 h at 37° C. Wells were washed thrice and assays were developed with freshly prepared ABTS® solution (1 mg/ml) for 30 minutes at room temperature (RT) in the dark. Absorbance was measured at 405 nm in an ELISA plate reader.
The nucleotide sequences coding for the light chain variable region VL and the heavy chain variable region VH of the P-selectin HuMabs were isolated by a standard cDNA synthesis/PCR procedure.
Total RNA was prepared from 1×106−1×107 hybridoma cells using the RNeasy® Mini Kit (Qiagen). Hybridoma derived RNA was used as a template for the 1st strand cDNA synthesis which was performed according to a conventional method making use of an oligo dT primer. 2nd-strand cDNA synthesis and further PCR amplification of VL and VH encoding cDNA fragments were performed with reverse light and heavy chain primers complementary to nucleotide sequences of the κ-light and γ1-heavy chain constant region and 5′-specific light and heavy chain primers, respectively. The PCR products were cloned using the TOPO® TA cloning kit from Invitrogen™ life technologies and pCR4-TOPO® as a cloning vector. Cloned PCR products were identified by restriction mapping of the appropriate plasmids using EcoRi for digestion and expected/calculated DNA fragment sizes of about 740 and 790 bp for VL and VH, respectively.
The DNA sequence of cloned PCR fragments was determined by double strand sequencing.
The GCG® (Genetics Computer Group, Madison, Wis.) software package version 10.2 was used for general data processing. DNA and protein sequences were aligned using the GCG® modul CLUSTALW. Sequence alignments were tabulated, edited and color-coded using the program GENEDOC® (version 2.1).
The anti-P-selectin HuMab light and heavy chain encoding genes were separately assembled in mammalian cell expression vectors.
Thereby the gene segments encoding the anti-P-selectin HuMab light chain variable region (VL) and the human κ-light chain constant region (CL) were joined as were gene segments for the anti-P-selectin HuMab heavy chain variable region (VH) and the human γ1-heavy chain constant region (CH1-Hinge-CH2-CH3).
General information regarding the nucleotide sequences of human light and heavy chains from which the codon usage can be deduced is given in: Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242.
The transcription unit of the anti-P-selectin HuMab κ-light chain is composed of the following elements:
The transcription unit of the anti-P-selectin HuMab γ1-heavy chain is composed of the following elements:
Functional elements of the anti-P-selectin HuMab κ-light chain and γ1-heavy chain expression plasmids: Beside the anti-P-selectin HuMab κ-light chain or γ1-heavy chain expression cassette these plasmids contain
An anti-P-selectin γ4-heavy chain prototype expression plasmid was derived from the anti-P-selectin γ1-heavy chain expression plasmid by replacing the human genomic γ1-constant region and γ1-immunoglobulin polyadenylation (“poly A”) signal sequence by the human genomic γ4-constant region and γ4-immunoglobulin polyadenylation-signal sequence.
For the expression of anti-P-selectin HuMab κ-light chains the same expression plasmids were used as described for IgG1 (see above).
Expression plasmids encoding mutant anti-P-selectin γ1- and γ4-heavy chains were created by site-directed mutagenesis of the wild type expression plasmids using the QuickChange™ Site-Directed mutagenesis Kit (Stratagene).
The following mutants were generated for LC1004-002. Amino acids are numbered according to EU numbering [Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb, P. D., Rutishauser, U., and Waxdal, M. J. (1969) Proc Natl Acad Sci U S A 63, 78-85; Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S., and Foeller, C. (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242].
TABLE 1
Isotype
Abbreviation
Mutations
Description
IgG1
IgG1v1
PVA-236;
The amino acid sequence
GLPSS331
Glu233Leu234Leu235G1y236 (SEQ ID
as specified
NO: 53) of the human γ1-heavy
by
chain is replaced by the amino acid
E233P;
sequence Pro233Val234Ala235 of the
L234V;
human γ2-heavy chain.
L235A;
The amino acid sequence
delta G236;
Ala327Leu328Pro329A1a330Pro331
A327G;
(SEQ ID NO: 54) of the human
A330S;
γ1-heavy chain is replaced by the
P331S
amino acid sequence
SEQ ID
Gly327Leu328Pro329Ser330Ser331
NO: 25
(SEQ ID NO: 55) of the human
γ4-heavy chain
IgG1
IgG1v2
L234A;
The amino acid sequence
L235A
Leu234Leu235 of the human γ1-heavy
SEQ ID
chain is replaced by the amino acid
NO: 26
sequence Ala234A1a235
IgG4
IgG4v1
S228P;
Ser228 of the human γ4-heavy chain
L235E
is replaced by Pro228 and Leu235 of
SEQ ID
the human γ4-heavy chain is
NO: 28
replaced by Glu235
Recombinant HuMabs were generated by transient transfection of adherent HEK293-EBNA cells (ATTC # CRL-10852) cultivated in DMEM (Gibco) supplemented with 10% ultra-low IgG FCS (Gibco), 2 mM Glutamine (Gibco), 1% v/v nonessential aminoacids (Gibco) and 250 μg/ml G418 (Roche). For transfection Fugene™ 6 (Roche) Transfection Reagent was used in a ratio of reagent (μl) to DNA (μg) ranging from 3:1 to 6:1. Immunoglobulin light and heavy chains were expressed from two different plasmids using a molar ratio of light chain to heavy chain encoding plasmid from 1:2 to 2:1. HuMab containing cell culture supernatants were harvested at day 4 to 11 after transfection. Supernatants were stored at −20° C. until purification.
General information regarding the recombinant expression of human antibody in e.g. HEK293 is given in: Meissner, P., Pick, H., Kulangara, A., Chatellard, P., Friedrich, K., and Wurm, F. M. (2001) Biotechnol Bioeng 75, 197-203.
Equipment:
For affinity measurements rabbit anti human Fcγ antibodies (Dianova) have been coupled by amine coupling to the chip surface for presentation of the antibody against P-selectin. Approximately 400 RU of anti P-selectin antibodies were bound. Recombinant P-selectin (R&D Systems) was added in various concentrations between 0-50 nM. Association was measured by P-selectin-injection for 120 seconds; dissociation was measured by washing the chip surface with buffer for 180 seconds. The affinity data for different P-selectin antibodies are shown in Table 2. Using Biaevaluation Software the kinetic data were fitted to a 1:1 Langmuir binding model of P-selectin to the presented monoclonal antibody.
TABLE 2
Affinity data measured by SPR (BIACORE ® 2000)
Antibody
HuMab
ka (1/Ms)
kd (1/s)
KA (1/M)
KD (M)
001
6.08 × 105
4.19 × 10−4
1.45 × 109
6.89 × 10−10
002
8.10 × 105
2.13 × 10−3
3.81 × 109
2.63 × 10−9
003
6.60 × 105
2.91 × 10−4
2.27 × 109
4.41 × 10−10
005
8.42 × 105
2.89 × 10−4
2.91 × 109
3.43 × 10−10
011
1.77 × 106
2.38 × 10−3
7.44 × 108
1.34 × 10−9
012
1.08 × 106
1.25 × 10−4
8.65 × 109
1.16 × 10−10
013
1.46 × 106
2.02 × 10−4
7.22 × 109
1.39 × 10−10
017
7.79 × 105
1.39 × 10−5
5.59 × 109
1.79 × 10−11
Materials and Methods:
Cell adhesion assay: In the adhesion assay the effect of the HuMabs on the adhesion of leukocyte-like HL60 cells (ATCC CCL 240) to P-selectin coated onto microtiter plates was evaluated. The HL60 cells were labeled with BCECF-AM (2′, 7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester; Cat.no 216254, Calbiochem). Full-length purified P-selectin (purification procedure s. above) at a concentration of 1 μg/ml in buffer containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris (pH 7.4) plus 0.0005% T×100 was coated overnight at 4° C. to 96 wells plates (Nunc Immunoplate Maxisorp™ F96). Thereafter, the wells were blocked with the above-mentioned buffer containing 3.5% bovine serum albumin (BSA, Fluka) for 2 h at room temperature (RT). The wells were preincubated with 50 μl of different dilutions of the P-selectin HuMabs or reference mouse P-selectin antibodies (WAPS 12.2, respective hybridoma cell line provided by ATCC) in the above-mentioned buffer containing 1% BSA for 20 minutes at RT. The labeled HL60 cells (50 μl, 70,000 cells/well) were added and allowed to bind for 45 min at RT. In some experiments the HL60 cells were preincubated with 20 μg/ml of human IgG1 for 30 minutes prior to their addition to the wells in order to block Fc receptors. After removal of the unbound HL60 cells by gentle washing (4 times with the above-mentioned buffer), the adherent cells were lysed with 120 μl NP-40 (Fluka; 1% in H2O). 100 μl of the supernates were transferred to plates to measure the respective fluorescence at an excitation wavelength of 485 nm and an emission of 538 nm using a luminescence spectrometer LS 50B (Perkin Elmer).
Rosetting assay: To evaluate the effect of the antibodies on the interaction of activated platelets with HL60 cells a rosetting assay (Jungi et al, Blood 67:629 (1986)) in combination with double color cytofluorimetric analysis (Evangelista et al., Blood 88:4183 (1996) was applied. Washed human platelets were prepared as described (Fox et al, Methods Enzymol 215:45 (1992)). They were activated with thrombin (final conc 1 U/ml) for 5 min and labeled with a FITC-conjugated anti-human GPIIb antibody pl-36 (Kouns et al., J Biol Chem 267:18844 (1992)). Then 1.4−2×106 platelets within 70 μl of tyrode solution were incubated with different dilutions of HuMabs (100 μl) in the dark for 30 min at RT. 50 μl of HL60 cell suspension (in tyrode solution) adjusted to 20×106/ml was added. The HL60 cells were labeled by incubation with 20 μl of a PE (phycoerythrin)-conjugated anti-human CD45 Ab (Code No. 555483, Pharmingen). After having incubated the labeled HL60 cells with the platelets and the HuMabs for 30 min at room temperature in FACS® tubes (Becton Dickinson), the formation of mixed aggregates or rosettes was analyzed by measuring both platelet and HL60 cell marker fluorescence using a FACScan™ (Becton Dickinson). Forward and side scatter, as well as green (FITC) and red (PE) signals were acquired by logarithmic amplification with excitation wavelength of 488 nm and emission wavelength of 530 nm (FITC) and 570 nm (PE), respectively. Electronic compensation was used to remove spectral overlap. HL60 cells were identified on the basis of forward and side scatter. Gating on events identified as HL60 cells was performed to exclude single platelets. Five thousand HL60 cell-gated events were measured for each sample. A sample in which non-activated or thrombin-activated platelets were mixed with HL60 cells in the presence of EDTA (10 mmol/l) was used to set a threshold on the green fluorescence scale. The percentage of HL60 cells above the threshold represents the percentage of HL60 cell binding platelets. The shift of the platelet marker fluorescence towards lower fluorescence values reflects the reduction of the number of mixed aggregates with a higher number of adhering platelets in favor of an increase of the number of mixed aggregates with a low number of adhering platelets.
Results:
In the HL60 cell adhesion assay the P-selectin antibodies inhibited the adhesion of the HL60 cells to purified P-selectin with IC50 values in the range of 0.08-0.5 μg/ml, Although the mutations were introduced in the Fc portion of the antibody, both the IgG4 and IgG1 variants of HuMabs were more potent than the parent antibody with IC50 values of 0.08-0.11 μg/ml as illustrated in
In the rosetting assay evaluating the adhesion of human activated platelets expressing P-selectin to HL60 cells the IC50 values of the HuMabs were even below those of the adhesion assay due to the lower number of P-selectin receptors in this assay (IC50: 0.05-0.3 μg/ml, preferably between 0.05 and 0.2 μg/ml). The efficacy of the Fc variants of the respective HuMabs tends to be increased as compared to the non-mutated parent antibody (
Materials and Methods: The cross-reactivity of the P-selectin HuMabs was evaluated by measuring (i) the binding of the HuMabs to activated platelets from rat and cynomologus monkey using FACS® analysis and (ii) their inhibitory activity in the rosetting assay evaluating the adhesion of rat and cynomologus platelets to HL60 cells.
To measure the binding of the HuMabs to activated rat and cynomologus platelets, washed rat and cynomologus platelets were prepared similar to preparing washed human platelets (s. above). They were activated with thrombin (final conc 1 U/ml) for 5 min. Activated platelets were incubated with different dilutions of the HuMabs (20 μl) for 30 min at RT. After binding of the HuMabs the platelets were fixed with PFA 2% at RT for 15 min. Samples were washed with Tyrode buffer and resuspended in 300 ml Tyrode. The binding of the HuMabs was detected with a FITC-conjugated F(ab′)2 fragment of rabbit anti-human IgG (Code No. F0056, Dako). As a control antibody inhibiting rat P-selectin a rabbit anti-human polyclonal anti-P-selectin antibody (Code No. 09361A, Pharmingen) was used.
To measure the inhibitory effect of the P-selectin HuMabs in the resetting assay, washed rat and cynomologus platelets were prepared as described above for human platelets. The rosetting assay was performed essentially as described for human platelets. For the labelling of the cynomologus platelets the FITC-conjugated anti-human GPIIb antibody pl-36 was used, whereas the rat platelets were labeled with the FITC-conjugated mouse anti-rat CD61 antibody (Code No. 554952, Pharmingen).
Results: None of the P-selectin specific antibodies of the invention which inhibit human P-selectin-mediated functions was shown to bind to rat P-selectin or to inhibit the formation of mixed aggregates consisting of rat platelets and HL60 cells, as shown for some examples in
Materials and Methods: The selectivity of the P-selectin HuMabs vs E- and L-selectin was determined in a cell-free ELISA measuring the binding of the antibodies to recombinant E- and L-selectin (ADP1 and ADP2, R&D Systems) and a cell-based ELISA measuring the binding of the antibodies to E-selectin-CHO transfectants and L-selectin-300.19 transfectants (transfectants were generated as described in Goetz et al., J Cell Biol 137:509 (1997); Ley et al., Blood 82:1632 (1993)).
In the cell-free ELISA recombinant P-, E-, or L-selectin at a concentration of 1 μg/ml in buffer containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 20 mM Tris (pH 7.4) plus 0.0005% Tx100 was coated overnight at 4° C. to 96 well plates (Nunc Immunoplate Maxisorp F96). Thereafter, the wells were blocked with the above-mentioned buffer containing 3.5% bovine serum albumin (BSA, Fluka) for 2 h at RT. The wells were preincubated with 50 μl of different dilutions of the P-selectin HuMabs or reference mouse P-, E-selectin antibody (BBA26; R&D Systems) and goat L-selectin antibody (AF728; R&D Systems) in the above-mentioned buffer containing 1% BSA overnight at RT. The binding of the HuMabs was detected by using a biotinylated anti-human IgG (Amersham, RPN1003, Final concentration 1:1000) or for the control antibodies the corresponding biotinylated anti-mouse or anti-goat IgG. After 1 h incubation, the wells were washed (3 times) with the above-mentioned buffer, and 0.1 ml of streptavidin-biotinylated peroxidase complex (Amersham, RPN1051), diluted 1:750 in the mentioned buffer containing 0.1% BSA was added for 30 min. The wells were then washed and 0.2 ml of peroxidase substrate solution containing ABTS (2.2′-azino-di-(3-ethylbenzthiazoline sulfonate, Boehringer, Mannheim) was added (ABTS stock solution: 1 ml 40 mM ABTS, 5 μl 30% H2O2 and 20 ml 0.1M Na-Acetat, 0.05 NaH2PO4). The reaction was stopped after around 10 min using 50 μl of 0.1 M citrate and 0.01% NaN3. The color reaction was read at 405 nm.
In the cell-based ELISA P- and E-selectin-CHO-transfectants, after detaching the cells with cell-dissociation solution (Sigma 05914), were seeded into each well of 96 well plates (TC Microwell F96 Nunc 167008) adjusted to 100,000 cells/well and cultivated in respective media overnight at 37° (medium for P-CHO-transfectants: DMEM+10% FCS+2 mM Glutamine+Penicillin 100 U/ml/Streptomycin 100 μg/ml; medium for E-selctin transfectants: HAM F-12+10% FCS+2 mM Glutamine+Penicillin 100 U/ml /Streptomycin 100 μg/ml+0.1% Fungizone+100 μg/ml Neomycin). After removal of the media and blocking the wells with A-T buffer (150mM NaCl, 1mM GaCl2, 1mM MgCl2, 20 mM Tris (pH 7.4)) containing 3% TopBlock™ (Code No. TB23201 0; Juro) for 1 h, 50 μl of different dilutions of the P-selectin HuMabs or reference mouse P- and E-selectin antibody (s. above) in the above-mentioned buffer containing 1% TopBlock™ and 0.1% azide were added and incubated for 60 min at RT. After washing the wells (4 times), the bound antibodies were detected using the same steps as mentioned above for the cell-free ELISA.
Since the L-selectin 300.19 cells are suspension cells, the cell-based ELISA format had to be modified by plating the L-selectin-300.19 transfectants into wells of 96 well polystyrene filter plates (Corning 3510). Using the filter plates blocking and incubation solutions were removed by filtering them through the bottom of the plates, but otherwise the protocol was similar to that using P- and E-selectin-CHO cells. As controls non-transfected CHO and 300.19 were used.
Results:
The antibodies of the invention were highly selective vs E- and L-selectin. They bound to P-selectin-CHO cells with EC50 values in the range of 0.01 to 0.08 μg/ml, preferably in the range of 0.01 to 0.04 μg/ml, whereas the EC50 values on E-selectin-CHO cells and L-selectin-300.19 were dearly above 50 μg/ml, preferably above 100 μg/ml. HuMab 002 had highest selectivity with a selectivity factor vs E- and L-selectin of more than 4,000 fold in the cell-based ELISA. Furthermore HuMab 002 does not bind to E- and L-selectin transfectants above baseline levels up to a concentration of 100 μg/ml. The selectivity of the Fc variants IgG4v1 and IgG1v1 of HuMab 002 is similar to that of the parent HuMab 002 (
Materials and Methods:
To address the effect of the P-selectin antibodies on the recruitment of leukocytes to sites of vessel wall injury and platelet thrombi, a human blood flow system which allows the measurement of the interaction of human leukocytes with human platelets at different shear rates was used essentially as described (Kirchhofer et al., Blood 89:1270 (1997)). In a parallel plate perfusion device human whole blood drawn from the antecubital vein of a healthy donor was perfused over a collagen surface simulating an injured denuded vessel wall. Collagen-coated coverslips were prepared as described (Kirchhofer et al., Blood 89:1270 (1997)). They were positioned in three parallel plate perfusion chambers. To allow the measurement of different shear rates (65/s and 280/s) different dimensions of perfusion chambers were used and the blood was perfused over the collagen-coated coverslips at a constant blood flow of 1 ml/min which was controlled by individual roller pumps positioned distal to the perfusion device. Immediately after drawing the blood from the vein and separating the blood into three tubings, a GPIIb/IIIa inhibitor (0.5 μmol/lamifiban) is added to prevent platelet aggregation and to generate platelet monolayers. At the same time, the P-selectin antibodies (the HuMabs, mutants, respective reference antibodies or human IgG1 and IgG4 as controls) were administered at different concentrations and the blood-inhibitor mixture then entered the perfusion chamber containing the collagen-coated coverslips. After a 5.5 minute perfusion period, PBS is perfused through the perfusion chamber without interrupting the flow for 3 min. After a brief interruption of flow the chambers were fixed with 3% paraformaldehyde in PBS at 1 ml/min for 2 min. Then the coverslips were removed from the chambers, fixed again for 1 h in 3% paraformaldehyde in PBS at 4° C. and stored in PBS-0.03% sodium azide. To evaluate the number of leukocytes adhering to the platelet monolayer, after air-drying the coverslips were stained with Duff-Quick™ solution (Dade Behring AG) and embedded in Merckoglas™ (Merck, Germany). An image analysis system (MCID, Imaging Research Inc.) was used to determine the number of leukocytes adhering to a standard area oriented perpendicular to the blood flow 1 mm apart from the beginning of the coverslip. At a shear rate of 65/s and 280/s the area on which the number of leukocytes was counted comprised 3.1 mm2 and 2.1 mm2, respectively.
Results:
The P-selectin HuMabs inhibited the adhesion of leukocytes to the platelet monolayer in a concentration-dependent manner. At a shear rate of 65/s and a concentration of 10 μg/ml the HuMabs inhibited the adhesion of leukocytes by 60-99%, preferably 70-99 %. The inhibitory effect of the HuMabs was more pronounced at the higher shear rate of 280/s (closer to the arterial situation) as compared to the venous shear rate of 65/s. Overall, at a shear rate of 280/s the number of adhering leukocytes was lower than at 65/s. When comparing the Fc variants with the respective parent antibodies, they had similar inhibitory activity in the ex vivo perfusion chamber, as demonstrated for HuMab 002 and its variants IgG4v1 and IgG1v1 (
Materials and Methods:
To address the anti-inflammatory potential of the P-selectin HuMabs under shear conditions, the above-mentioned human blood flow system was used in a set up in which endothelial cells were coated onto the coverslips. Human umbilical vein endothelial cells (HUVEC) from umbilical cords were isolated by digestion with collagenase Type II (Roche Switzerland) according to the method of Jaffe et al, Culture of human endothelial cells derived from umbilical veins. J. Olin. Invest. 52, 2745-2756 (1973). They were cultivated in 1% gelatine-coated tissue culture flasks in medium 199 (M199, Sigma, Germany) supplemented with 20% fetal calf serum (Gibco, Auckland), 100 IU/ml penicillin (Gibco, Auckland), 0.1 mg/ml streptomycin (Gibco, Auckland), 2mmol/l L-glutamine (Gibco, Auckland), 10 U/ml heparin (Sigma) and 50 μg/ml EC growth supplement (Sigma, Germany). HUVECs were grown to confluency (approx. 4 days), passaged with trypsin/ethylendiaminetetraacetic acid (Gibco, Auckland) and seeded onto Thermanox® plastic coverslips (approx 200,000 ECs/coverslip) previously coated with 1% gelatine (Fluka, Germany). The HUVECs were allowed to settle and became confluent over 1-2 days. They were stimulated with 20 ng/ml IL-4 (R&D Systems) 24 h before starting the perfusion and with 10−4 M histamine (Fluka, Germany) 5-10 min prior to the perfusion. Each experiment was performed with HUVECs at passage 1. The coverslips with confluent monolayers of stimulated HUVECs were positioned into the parallel plate perfusion chambers as described above. Similar to the perfusion experiments described above, whole blood was drawn from healthy donors. However in these experiments, the blood was anticoagulated with a thrombin inhibitor Ro-46-6240 (10 μM) and preincubated with different concentrations of the P-selectin antibodies (HuMabs, mutants, respective reference antibodies) or human IgG1 and IgG4 as controls for 5 min just prior to the perfusion over the activated endothelial cells. The blood flow was adjusted to 1 ml/min, the shear rate 65/s and the perfusion time 5.5 min. After a washing period of 3 min with PBS, the HUVECs with the adhering leukocytes were fixed with 3% paraformaldehyde for 2 min under the same flow conditions as described. Then the coverslips were removed from the chambers, immersed in fresh fixative for 1 h, and stored in PBS-0.02% sodium azide. For morphometric analysis, the leukocytes were stained with a mouse antibody against the leukocyte common antigen CD45, which was labeled beforehand using a modified biotinylated anti-mouse immunoglobulin (Animal Research Kit, Dako, USA). The nuclei were counterstained with hematoxylin (J.T Baker, Holland).
Results:
The stimulation of the HUVECs with the combination of IL-4 and histamine resulted in the expression of P-selectin and the adhesion of different types of leukocytes with granulocytes (including PMNs and eosinophils) constituting the prevailing portion of adhering leukocytes. The HuMabs of the invention inhibited the adhesion of the total leukocyte population by 60-90% at 3 μg/ml. Overall the inhibitory activity of the Fc variants was not significantly different from that of the non-mutated HuMabs.
The P-selectin HuMabs demonstrate a differential effect on the different leukocyte subtypes. The effect on granulocytes is more pronounced as compared to mononuclear leukocytes. The antibodies according to the invention inhibited the adhesion of granulocytes (including PMNs and eosinophils) by 90-99%, monocytes by 50-88%, and lymphocytes by 5-40%. The respective decrease in the absolute numbers of the different leukocyte subtypes is representatively given for IgG4v1 in
C1q and C3c Binding ELISA:
To determine the ability of the antibodies of the invention to induce C1q binding and C3 activation, an ELISA approach was used. C1q is part of the adaptive immune system and, upon binding to immune complexes, triggers the sequential activation of several zymogens. The enzymes in turn, cause the cleavage of C3 molecules, which can result in the onset of inflammatory reactions, opsonization of foreign or aberrant particles and lysis of cell membranes.
In principle, the ELISA plate is coated with concentration ranges of the antibody, to which human C1q or human pooled serum, as a source of C3, is added. C1q or C3ε binding is detected by an antibody directed against human C1q or C3ε followed by a peroxidase-labeled conjugate.
HuMab 002 (the hybridoma- and the transient transfectoma-derived material, its mutant variants, and control antibodies were tested in concentrations of 0.16-20 μg/ml. As a negative control a human IgG4 (CLB, the Netherlands, 0.5 μg/ml stock), that binds C1q very weakly, was used. Human IgG1 (Sigma, 2 ug/ml stock) was incorporated as positive control. For the detection of C1q, a rabbit antibody directed against C1q (Dako) and a swine anti-rabbit IgG antibody, conjugated with horseradish peroxidase (Sigma) were used. For the detection of C3ε a mouse anti-human C3 antibody and a rabbit anti-mouse IgG antibody, conjugated with horseradish peroxidase (Sigma) were applied.
Calculations concerning EC50 values or maximum binding at 10 μg/ml (Bmax) of the HuMab tested were determined using nonlinear regression curve fitting (one site binding) using Graphpad® Prism software.
Results:
HuMab 002 according to the invention was able to bind C1q efficiently as indicated by EC50 values of 0.946 μg/ml and 1.159 μg/ml, and Bmax (OD405) values of 0.987 and 0.711 for the hybridoma- and transfectoma-derived material, respectively. As expected, the negative control human IgG4 did not bind C1q, as indicated by a Bmax value of 0.222 at OD405. However, all three Fc-variants tested (IgG4v1, IgG1v1, IgG1v2) had lost the capacity to bind C1q, as shown by OD405 Bmax values of 0.132, 0.119, and 0.132, respectively (Table 3). In line with the C1q binding capacities, C3 deposition to HuMab 002 (hybridoma- and transfectoma-derived) occurred in an antibody-concentration dependent manner, and EC50 values ranged between 2.7 μg/ml and 8.3 μg/ml. However, all three Fc-variants were unable to initiate C3 deposition, as indicated by OD405 Bmax values of 0.104, 0.156 and 0.133, respectively (Table 3).
As HuMab 002 interacts with complement components, this antibody has the intrinsic potential to induce CDC in vivo. Therefore, the Fc part of this antibody is modified according to the invention.
TABLE 3
Clq ELISA
C3 ELISA
Bmax
Bmax
(OD405 at
Background
(OD405 at
Background
10 μg/ml)
(OD405)
10 μg/ml)
(OD405)
HuMab 002
0.987
0.079
4.47
0.098
(hybridoma)
IgG4v1
0.132
0.104
IgG1v1
0.0119
0.156
IgG1v2
0.132
0.133
HuMab 002
0.711
4.071
(transient)
IgG4
0.222
0.182
IgG antibody dependent cytotoxicity effects are mediated by Fcγ receptors on effector cells. Binding of hybridoma- and transfectoma-derived HuMab 002 as well as the mutant variants and control antibodies to FcγR expressing effector cells from human blood was studied by FACS® analysis.
Materials and Methods:
FcγRI IIA1.6 transfectants or freshly isolated effector cells were incubated with antibodies, and binding of antibody was detected with FITC-labeled rabbit-anti-human IgG F(ab)2 (DAKO), or FITC-labeled rabbit-anti-human IgG F(ab)2 (BD/Pharmingen). HuMab 002 (transient transfectoma- and/or hybridoma-derived material, and mutant variants) wase tested at a concentration of 1 μg/ml (IIA1.6 transfectants) or 10 μg/ml (effector cells). Absence of primary antibody or human IgG4 (10 μg/ml) was used as negative control. To detect FcγRI expression on IIA1.6 cells, FITC-labeled mouse anti-human CD64 (BD/Pharmingen) was used. In experiments using NK cell-enriched peripheral blood mononuclear cells, NK cells were identified by double staining using PE-labeled mouse-anti-human CD56 (BD/Pharmingen). Granulocytes and monocytes were identified based on FSC/SSC profile.
IIA1.6 cells, IIA1.6-FcγRI transfectant and freshly isolated effector cells were incubated with antibodies. Binding of antibody was detected with FITC-labeled Rb-α-huIgG F(ab)2 (DAKO), or FITC-labeled Rb-α-huIgG F(ab)2 (BD/Pharmingen).
HuMab 002 (transient transfectoma-, hybridoma derived- and mutant variant material) was tested at a concentration of 1 μg/ml in the IIA1.6-FcγRI transfectant binding assay. The IIA1.6 wild type cells were used as a negative control. As a control for FcγRI expression m-α-huCD64-FITC (BD/Pharmingen) was used.
HuMab 002 (transient transfectoma-, hybridoma derived- and mutant variant material) was tested at a concentration of 10 μg/ml in the effector cell binding assays. Transient transfectoma material was not tested in the granulocyte binding assay. IgG4 (10 μg/ml) was used as a negative control in all effector cell binding assays with the exception of the granulocyte binding assay.
Whole blood was enriched for NK cells using an NK isolation kit (Dynal Biotech ASA, Oslo, Norway). NK cells were identified by m-α-huCD56-FITC staining.
PBMCs (peripheral blood mononuclear cells) were obtained from whole blood using Ficoll procedure as described in the protocol enclosed with the NK isolation kit (Dynal Biotech ASA, Oslo, Norway). Monocytes were identified based on FSC/SSC profile. Granulocytes were isolated from whole blood using FACS lysis buffer and identified based on FSC/SSC profile.
Freshly isolated effector cells were incubated with antibodies, and binding of antibody was detected with FITC-labeled rabbit-anti-human IgG F(ab)2 (DAKO), or FITC-labeled rabbit-anti-human IgG F(ab)2 (BD/Pharmingen). HuMab 002 (transient transfectoma- and/or hybridoma-derived material, and mutant variants) were tested at a concentration of 10 μg/ml. Absence of primary antibody or human IgG4 (10 μg/ml) was used as negative control. NK cells were isolated from MNC samples by a NK isolation kit (Miltenyi Biotec, USA). In experiments using NK cell-enriched peripheral blood mononuclear cells, NK cells were identified by double staining using PE-labeled mouse-anti-human CD56 (BD/Pharmingen). Granulocytes and monocytes were isolated according to the state of the art from PBMC (e.g. Monocyte isolation kit (Miltenyi, see above). Granulocytes and monocytes were identified based on FSC/SSC profile.
Results:
HuMab 002 according to the invention was able to bind to FcR as indicated by binding to granulocytes, monocytes and NK cells. All three Fc-variants tested (IgG4v1, IgG1v1 and IgG1v2) had completely lost the capacity to bind to NK cells (Table 4). In addition, HuMab 002 bound efficiently to granulocytes and monocytes, whereas the mutant variants showed binding levels comparable to absence of primary antibody or human IgG4, as indicated by percentages of cells binding antibody in Tables 5 and 6. This indicates that the mutant variants lost the capacity to interact with FcR on effector cells.
As HuMab 002 can efficiently interact with FcR, this antibody has the intrinsic potential to induce antibody dependent cell-mediated cytotoxicity in vivo. Inactivation of the interaction with FcR as performed for the Fc-variants according to the invention prevents ADCC in an effective manner.
TABLE 4
NK cell binding
Antibody
(% NK cells binding antibody)
No antibody
0.03
HuMab 002 (hybridoma)
90.92
HuMab 002 (transient)
37.40
Human IgG4
0.06
IgG4v1
0.06
IgG1v1
0.12
IgG1v2
0.00
TABLE 5
Monocyte binding
Antibody
(% monocytes binding antibody)
No antibody
8.5
HuMab 002 (hybridoma)
38.4
HuMab 002 (transient)
31.3
Human IgG4
9.4
IgG4v1
14.5
IgG1v1
12.3
IgG1v2
14.0
TABLE 6
Granulocyte binding
Antibody
(% granulocytes binding antibody)
No antibody
1.2
HuMab 002 (hybridoma)
63.6
IgG4v1
1.6
IgG1v1
2.1
IgG1v2
2.0
Himber, Jacques, Parren, Paul, Steiner, Beat, Stern, Anne, Kopetzki, Erhard, Stubenrauch, Kay-Gunnar, Van De Winkel, Jan, Graus, Yvo, Jansen-Molenaar, Miranda, Kling, Dorothee, Rebers, Frank, Strein, Pamela, van Vugt, Martine
| Patent | Priority | Assignee | Title |
| RE44359, | Apr 13 2004 | Hoffmann-La Roche Inc | Nucleic acid molecules encoding anti-P-selectin antibodies |
| RE44389, | Apr 13 2004 | Hoffman-La Roche Inc. | Methods of inhibiting the binding of P-selectin to PSGL-1 with anti-P-selectin antibodies |
| Patent | Priority | Assignee | Title |
| 4783399, | May 04 1984 | SCRIPPS RESEARCH INSTITUTE, THE, A CORP OF CA | Diagnostic system for the detection of cytomegalovirus |
| 5800815, | May 05 1903 | AERES BIOMEDICAL LIMITED | Antibodies to P-selectin and their uses |
| 7317091, | Mar 01 2002 | XENCOR, INC | Optimized Fc variants |
| 7563441, | Apr 13 2004 | Hoffmann-La Roche Inc | Anti-P-selectin antibodies |
| 20060024298, | |||
| EP182634, | |||
| WO3064606, | |||
| WO3074679, | |||
| WO3075840, | |||
| WO9306863, | |||
| WO9321956, | |||
| WO9425067, | |||
| WO9429351, | |||
| WO9720932, |
| Executed on | Assignor | Assignee | Conveyance | Frame | Reel | Doc |
| Jul 21 2011 | Hoffmann-La Roche Inc. | (assignment on the face of the patent) | / |
| Date | Maintenance Fee Events |
| Sep 27 2012 | M1551: Payment of Maintenance Fee, 4th Year, Large Entity. |
| Dec 28 2016 | M1552: Payment of Maintenance Fee, 8th Year, Large Entity. |
| Sep 28 2020 | M1553: Payment of Maintenance Fee, 12th Year, Large Entity. |
| Date | Maintenance Schedule |
| Aug 07 2015 | 4 years fee payment window open |
| Feb 07 2016 | 6 months grace period start (w surcharge) |
| Aug 07 2016 | patent expiry (for year 4) |
| Aug 07 2018 | 2 years to revive unintentionally abandoned end. (for year 4) |
| Aug 07 2019 | 8 years fee payment window open |
| Feb 07 2020 | 6 months grace period start (w surcharge) |
| Aug 07 2020 | patent expiry (for year 8) |
| Aug 07 2022 | 2 years to revive unintentionally abandoned end. (for year 8) |
| Aug 07 2023 | 12 years fee payment window open |
| Feb 07 2024 | 6 months grace period start (w surcharge) |
| Aug 07 2024 | patent expiry (for year 12) |
| Aug 07 2026 | 2 years to revive unintentionally abandoned end. (for year 12) |