Antibodies that recognize sortilin

Abstract

The present invention provides an antibody that specifically binds to Sortilin. The antibody inhibits or delays pathology associated with altered levels of Progranulin and associated disease progression. In some embodiments, the humanized antibody comprises the three light chain CDRs and the three heavy chain CDRs of monoclonal antibody 5E20. In some embodiments, the CDRs are of a definition selected from the group of Kabat, Chothia, Kabat / Chothia combined, AbM and Contact. In some embodiments, the humanized mature heavy chain variable region comprises the three Kabat / Chothia combined heavy chain CDRs of 5E20 (SEQ ID NOs: 5-7) and the humanized mature light chain variable region comprises the three Kabat / Chothia combined light chain CDRs of 5E20 (SEQ ID NOs: 11-13).

Claims

1. A humanized antibody that specifically binds to human sortilin, (a) a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO:4 and a light chain variable region having an amino acid sequence comprising SEQ ID NO:10; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 28 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 34; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 52, and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 58, except that Kabat position L54 can be L, G, or I; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 78 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 84; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 90 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 96; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 102 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 108; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 114 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 120; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 126 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 132; a murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 138 and a light chain variable region having an amino acid sequence comprising SEQ ID NO: 144; or A murine antibody characterized by a heavy chain variable region having an amino acid sequence comprising SEQ ID NO: 150 and a light chain variable region having an amino acid sequence comprising SEQ ID NO:

156. three light chain CDRs and three heavy chain CDRs of (b)(i) any one of SEQ ID NOs: 257-262, with or without the C-terminal lysine; or (ii) location according to EU numbering, an alanine at position 234, an alanine at position 235, and an alanine at position 322; an alanine at position 234, an alanine at position 235, an alanine at position 322, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256; an alanine at position 234, an alanine at position 235, a leucine at position 428, and a serine at position 434; an alanine at position 234, an alanine at position 235, an alanine at position 322, a leucine at position 428, and a serine at position 434; an alanine at position 234 and an alanine at position 235; or an alanine at position 234, an alanine at position 235, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256 A humanized antibody comprising a heavy chain constant region comprising:

2. The humanized antibody described in claim 1, wherein the humanized antibody comprises a humanized mature heavy chain variable region comprising three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 5 to 7, and a humanized mature light chain variable region comprising three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 11 to 13.

3. 2. The humanized antibody of claim 1, comprising a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 163-169, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 173-176.

4. At least one of the following positions is occupied by a specified amino acid; Kabat position H5 is occupied by L or V, Kabat position H40 is occupied by A or T, Kabat position H42 is occupied by G or D, Kabat position H44 is occupied by G or R, Kabat position H49 is occupied by A, Kabat position H77 is occupied by T or S, Kabat position H83 is occupied by R or K, Kabat position H93 is occupied by S, and Kabat position H94 is occupied by R; and / or At least one of the following positions is occupied by the specified amino acid: Kabat position L11 is L or V, Kabat position L36 is L, Kabat position L44 is F, Kabat position L46 is G, Kabat position L69 is A, Kabat position L85 is T or D, Kabat position L87 is F, Kabat position L100 is G or Q, and Kabat position L106 is I or K. The humanized antibody of claim 3.

5. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 173; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 174; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

174. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

175. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

175. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 175; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 175; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

175. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

175. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

175. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 176; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

176. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

176. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 176; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 176; the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168 and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 176; or the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 176; The humanized antibody of claim 3.

6. 2. The humanized antibody of claim 1, wherein the humanized antibody comprises a humanized mature heavy chain variable region comprising three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 29-31, and a humanized mature light chain variable region comprising three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 35-37.

7. The humanized antibody of claim 6, comprising a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 180 or SEQ ID NO: 181, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to SEQ ID NO: 185 or SEQ ID NO:

186.

8. At least one of the following positions is occupied by a specified amino acid: Kabat position H2 is occupied by A, Kabat position H12 is occupied by K or V, Kabat position H48 is occupied by I, Kabat position H67 is occupied by A, Kabat position H71 is occupied by V, Kabat position H91 is occupied by F, and Kabat position H108 is occupied by T; and / or At least one of the following positions is occupied by the specified amino acid: Kabat position L2 is V, Kabat position L9 is L or S, and Kabat position L74 is K or T. The humanized antibody of claim 7.

9. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

185. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

186. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181 and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 185; or the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

186. The humanized antibody of claim 7.

10. The humanized antibody of claim 1, wherein the humanized antibody comprises a humanized mature heavy chain variable region comprising three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 53 to 55, and a humanized mature light chain variable region comprising three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 59 to 61, except that Kabat position L54 can be L, G, or I.

11. The humanized antibody of claim 10, comprising a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 190-192, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 196-199.

12. At least one of the following positions is occupied by a specified amino acid: Kabat position H49 is occupied by A, Kabat position H80 is occupied by L or G, and Kabat position H82c is occupied by L or G; and / or At least one of the following positions is occupied by the specified amino acid: Kabat position L43 is A or S, Kabat position L48 is V, Kabat position L54 is L, G or I, Kabat position L71 is Y, and Kabat position L76 is N or S. The humanized antibody of claim 11.

13. The humanized antibody of claim 11, wherein the humanized mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 190 to 192, and the humanized mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 196 to 199.

14. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

196. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

197. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

198. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

199. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

196. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

197. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

198. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

199. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

196. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

197. the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192 and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO: 198; or the humanized mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the humanized mature light chain variable region has the amino acid sequence of SEQ ID NO:

199. The humanized antibody of claim 13.

15. The humanized antibody, a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs:79-81, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs:85-87; a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 91-93, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 97-99; a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 103-105, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 109-111; a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 115-117, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 121-123; a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 127-129, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 133-135; a humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 139-141, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 145-147; or A humanized mature heavy chain variable region comprising the three Kabat / Chothia composite heavy chain CDRs of SEQ ID NOs: 151-153, and a humanized mature light chain variable region comprising the three Kabat / Chothia composite light chain CDRs of SEQ ID NOs: 157-159. The humanized antibody of claim 1, comprising:

16. The humanized antibody of claim 1, wherein the humanized antibody has an isotype of human IgG1, human IgG2, or human IgG4.

17. The humanized antibody of claim 1, wherein the heavy chain constant region is a mutant form of the natural human heavy chain constant region that has reduced binding to Fcγ receptors compared to the natural human heavy chain constant region.

18. The humanized antibody of claim 1, wherein the heavy chain constant region is a mutant form of the native human heavy chain constant region that has enhanced binding to neonatal Fcγ receptors compared to the native human heavy chain constant region.

19. 2. The humanized antibody of claim 1, wherein the humanized antibody has a heavy chain constant region comprising, in EU numbering, (a) an alanine at position 234, an alanine at position 235, and an alanine at position 322; (b) an alanine at position 234, an alanine at position 235, an alanine at position 322, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256; (c) an alanine at position 234, an alanine at position 235, a leucine at position 428, and a serine at position 434; or (d) an alanine at position 234, an alanine at position 235, an alanine at position 322, a leucine at position 428, and a serine at position 434.

20. 2. The humanized antibody of claim 1, wherein the heavy chain constant region has a sequence comprising any one of SEQ ID NOs: 257-262, with or without the C-terminal lysine.

21. a heavy chain of SEQ ID NO: 244 with or without the C-terminal lysine and a light chain of SEQ ID NO: 245; a heavy chain of SEQ ID NO: 246 with or without the C-terminal lysine and a light chain of SEQ ID NO: 247; a heavy chain of SEQ ID NO: 248 with or without the C-terminal lysine and a light chain of SEQ ID NO: 249; a heavy chain of SEQ ID NO: 250 and a light chain of SEQ ID NO: 245, with or without the C-terminal lysine; a heavy chain of SEQ ID NO: 251 and a light chain of SEQ ID NO: 247, with or without the C-terminal lysine; a heavy chain of SEQ ID NO: 252 and a light chain of SEQ ID NO: 249, with or without the C-terminal lysine; a heavy chain of SEQ ID NO: 253 and a light chain of SEQ ID NO: 245, with or without the C-terminal lysine; a heavy chain of SEQ ID NO: 254 and a light chain of SEQ ID NO: 245, with or without the C-terminal lysine; a heavy chain of SEQ ID NO: 255 and a light chain of SEQ ID NO: 245, with or without the C-terminal lysine; or A heavy chain of SEQ ID NO: 256 with or without the C-terminal lysine and a light chain of SEQ ID NO: 245 The humanized antibody of claim 1, comprising:

22. A pharmaceutical composition comprising the humanized antibody of any one of claims 1 to 21 and a pharmaceutically acceptable carrier.

23. A nucleic acid encoding the heavy and light chains of the humanized antibody of any one of claims 1 to 21.

24. 1. A method for humanizing a murine antibody, said method comprising: (a) selecting one or more acceptor antibody sequences; (b) identifying the amino acid residues of the mouse antibody to be retained; (c) synthesizing a nucleic acid encoding a humanized heavy chain comprising the CDRs of the murine antibody heavy chain and a nucleic acid encoding a humanized light chain comprising the CDRs of the murine antibody light chain; and (d) expressing the nucleic acid in a host cell to produce the humanized antibody. Including, (i) the mouse antibody is a mature heavy chain variable region of SEQ ID NO: 52 and a mature light chain variable region of SEQ ID NO: 58; a mature heavy chain variable region of SEQ ID NO: 4 and a mature light chain variable region of SEQ ID NO: 10; a mature heavy chain variable region of SEQ ID NO: 28 and a mature light chain variable region of SEQ ID NO: 34; a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 84; a mature heavy chain variable region of SEQ ID NO: 90 and a mature light chain variable region of SEQ ID NO: 96; a mature heavy chain variable region of SEQ ID NO: 102 and a mature light chain variable region of SEQ ID NO: 108; a mature heavy chain variable region of SEQ ID NO: 114 and a mature light chain variable region of SEQ ID NO: 120; a mature heavy chain variable region of SEQ ID NO: 126 and a mature light chain variable region of SEQ ID NO: 132; A mature heavy chain variable region of SEQ ID NO: 138 and a mature light chain variable region of SEQ ID NO: 144; or The mature heavy chain variable region of SEQ ID NO: 150 and the mature light chain variable region of SEQ ID NO: 156 characterized by (ii) the humanized antibody (A) any one of SEQ ID NOs: 257-262, with or without the C-terminal lysine; or (B) Position according to EU numbering, an alanine at position 234, an alanine at position 235, and an alanine at position 322; an alanine at position 234, an alanine at position 235, an alanine at position 322, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256; an alanine at position 234, an alanine at position 235, a leucine at position 428, and a serine at position 434; an alanine at position 234, an alanine at position 235, an alanine at position 322, a leucine at position 428, and a serine at position 434; an alanine at position 234 and an alanine at position 235; or an alanine at position 234, an alanine at position 235, a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256 The method of claim 1, wherein the heavy chain constant region comprises:

25. 1. A method for producing a humanized antibody, the method comprising: (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of the humanized antibody in a cell culture medium so that the cells secrete the humanized antibody; and (b) purifying the humanized antibody from the cell culture medium. Including, The method, wherein the humanized antibody is a humanized antibody according to any one of claims 1 to 21.

26. 1. A method for producing a cell line that produces a humanized antibody, said method comprising: (a) introducing into a cell a vector encoding the heavy and light chains of the humanized antibody and a selectable marker; (b) growing the cells under conditions to select for cells with increased copy numbers of the vector; (c) isolating a single cell from the selected cells; and (d) banking cells cloned from single cells selected based on the yield of humanized antibodies; Including, The method, wherein the humanized antibody is a humanized antibody according to any one of claims 1 to 21.

27. ​​A composition for use in a method for increasing progranulin levels in a subject having or at risk of developing a disease or disorder associated with altered progranulin levels, comprising a therapeutically effective amount of a humanized antibody described in any one of claims 1 to 21.

28. A composition for use in a method for treating or preventing a disease or disorder associated with altered progranulin levels in a subject, comprising a therapeutically effective amount of a humanized antibody described in any one of claims 1 to 21.

29. 29. The composition of claim 28, wherein the disease or disorder associated with altered progranulin levels is frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, a neurodegenerative disorder, or an age-related neurodegenerative disorder.

30. A composition for use in a method for detecting sortilin in a subject having or at risk of having a disease associated with a disease or disorder associated with altered progranulin levels, comprising a therapeutically effective amount of a humanized antibody described in any one of claims 1 to 21.

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