Antibodies that recognize sortilin

Abstract

The present invention provides antibodies that specifically bind to Sortilin. The antibodies inhibit or delay pathologies associated with altered Progranulin levels and the resulting deterioration of disease. In one aspect, the present invention provides isolated monoclonal antibodies that compete with antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 for binding to human Sortilin. Some antibodies bind to the same epitope on human Sortilin as antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 043,481, filed June 24, 2020, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing Reference The sequence listing set forth in file 561308SEQLIST.txt is 353,813 bytes, was created on June 22, 2021, and is incorporated herein by reference. [Background technology]

[0003] Background of the Invention Frontotemporal dementia (FTD) is an early-onset dementia affecting 50,000-60,000 individuals in the United States. Decreased brain levels of progranulin (PGRN) are associated with genetic forms of FTD. Heterozygous loss-of-function mutations in the human progranulin gene cause FTD through a haploinsufficient mechanism (Baker, M. et al., Nature 2006;442:916-9; Cruts, M. et al., Nature 2006,442:920-924). The FTD genotype, lacking a functional copy of the progranulin gene (FTD-GRN) and resulting in approximately 50% reduced progranulin levels, accounts for approximately 10% of the total FTD population. Mutations that moderately reduce progranulin levels increase the risk of Alzheimer's disease and Parkinson's disease (Sheng, J. et al., Gene; 2014, 542(2):141-5; Mendsaikhan, A. et al., Cells: 2019, 8(3), 230). Progranulin is a neurotrophic / anti-inflammatory factor, and deficiency can disrupt homeostasis between microglia and neurons and promote neurodegeneration. Progranulin may also play a role in regulating lysosome formation and function. Sortilin (SORT1) has been identified as a regulator of progranulin levels by mediating its endocytosis (Carrasquillo, M.M. et al., Am J Hum Genet 2010;87(6):890-7; Hu, F. et al., Neuron 2010;68(4):654-67). Targeted disruption of the sortilin-progranulin interaction increases extracellular progranulin levels and could potentially reverse disease phenotypes in FTD-GRN patients. Modulation of progranulin levels has been reported with antibodies targeting the progranulin receptor sortilin (SORT1) in healthy humans. For example, Alector's AL001 has completed Phase 1 and is currently in Phase 2 (see https: / / investors.alector.com / static-files / 7418b689-c5b7-43ac-a16a-3c64e1a14e80). Increases in plasma and CSF progranulin compared to baseline have been reported in healthy volunteers administered anti-sortilin antibodies. Sortilin is also a receptor / transporter for other factors (e.g., neurotensin). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Baker, M. et al., Nature 2006;442:916-9 [Non-patent document 2] Cruts, M. et al., Nature 2006,442,:920-924 [Non-patent document 3] Sheng, J. et al., Gene;2014,542(2):141-5 [Non-patent document 4] Mendsaikhan, A. et al., Cells:2019,8(3),230 [Non-patent document 5] Carrasquillo, MM et al. Am J Hum Genet 2010;87(6):890-7 [Non-patent document 6] Hu, F. et al., Neuron 2010;68(4):654-67 Summary of the Invention [Means for solving the problem]

[0005] Brief summary of the claimed invention In one aspect, the invention provides isolated monoclonal antibodies that compete with antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 for binding to human sortilin. Some antibodies bind to the same epitope on human sortilin as antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2.

[0006] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 5E20, where 5E20 is 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. For example, the antibody can be a humanized antibody.

[0007] In some antibodies, the CDRs are defined as selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat / Chothia combination heavy chain CDRs of 5E20 (SEQ ID NOs: 5-7), and the humanized mature light chain variable region comprises the three Kabat / Chothia combination light chain CDRs of 5E20 (SEQ ID NOs: 11-13). In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 5E20 (SEQ ID NOs: 14, 6, and 7), and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 5E20 (SEQ ID NOs: 11-13). In some antibodies, the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 5E20 (SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 7), and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 5E20 (SEQ ID NO: 11-13). In some antibodies, the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 5E20 (SEQ ID NO: 5, SEQ ID NO: 17, and SEQ ID NO: 7), and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 5E20 (SEQ ID NO: 11-13). In some antibodies, the humanized mature heavy chain variable region comprises the three Contact heavy chain CDRs of 5E20 (SEQ ID NO: 18-20), and the humanized mature light chain variable region comprises the three Contact light chain CDRs of 5E20 (SEQ ID NO: 21-23).

[0008] Some antibodies comprise 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.

[0009] In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H5 is occupied by L or V, H40 is occupied by A or T, H42 is occupied by G or D, H44 is occupied by G or R, H49 is occupied by A, H77 is occupied by T or S, H83 is occupied by R or K, H93 is occupied by S, and H94 is occupied by R.

[0010] In some antibodies, provided positions H49, H93, and H94 in the VH region are occupied by A, S, and R, respectively. In some antibodies, positions H5, H49, H77, H93, and H94 in the VH region are occupied by V, A, S, S, and R, respectively. In some antibodies, positions H5, H44, H49, H77, H93, and H94 in the VH region are occupied by V, R, A, S, S, and R, respectively. In some antibodies, positions H5, H42, H44, H49, H77, H93, and H94 in the VH region are occupied by V, D, R, A, S, S, and R, respectively. In some antibodies, positions H5, H42, H44, H49, H77, H83, H93, and H94 in the VH region are occupied by V, D, R, A, S, K, S, and R, respectively. In some antibodies, positions H5, H40, H44, H49, H77, H93, and H94 in the VH region are occupied by V, T, R, A, S, S, and R, respectively. In some antibodies, H5, H40, H42, H44, H49, H77, H93, and H94 in the VH region are occupied by V, T, D, R, A, S, S, and R, respectively.

[0011] In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L11 is L or V, L36 is L, L44 is F, L46 is G, L69 is A, L85 is T or D, L87 is F, L100 is G or Q, and L106 is I or K. In some antibodies, positions L36, L44, L46, L69, and L87 in the VL region are occupied by L, F, G, A, and F, respectively. In some antibodies, positions L11, L36, L44, L46, L69, and L87 in the VL region are occupied by V, L, F, G, A, and F, respectively. In some antibodies, positions L11, L36, L44, L46, L69, L87, L100, and L106 in the VL region are occupied by V, L, F, G, A, F, Q, and K, respectively. In some antibodies, positions L11, L36, L44, L46, L69, L85, L87, L100, and L106 in the VL region are occupied by V, L, F, G, A, D, F, Q, and K, respectively.

[0012] Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 163 to 169, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 173 to 176. Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 163 to 169, and a mature light chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 173 to 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 163 to 169, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 173 to 176.

[0013] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173.

[0014] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174.

[0015] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175.

[0016] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO:169, and the mature light chain variable region has the amino acid sequence of SEQ ID NO:176.

[0017] In another aspect, the invention provides an antibody that specifically binds to human sortilin comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 8H24, where 8H24 is 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. For example, the antibody can be a humanized antibody.

[0018] In some antibodies, the CDRs are defined as selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat / Chothia combination heavy chain CDRs of 8H24 (SEQ ID NOs:29-31), and the humanized mature light chain variable region comprises the three Kabat / Chothia combination light chain CDRs of 8H24 (SEQ ID NOs:35-37). In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 8H24 (SEQ ID NOs:38, 30, and 31), and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 8H24 (SEQ ID NOs:35-37). In some antibodies, the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 8H24 (SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:31), and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 8H24 (SEQ ID NO:35-37). In some antibodies, the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 8H24 (SEQ ID NO:29, SEQ ID NO:41, and SEQ ID NO:31), and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 8H24 (SEQ ID NO:35-37). In some antibodies, the humanized mature heavy chain variable region comprises the three Contact heavy chain CDRs of 8H24 (SEQ ID NO:42-44), and the humanized mature light chain variable region comprises the three Contact light chain CDRs of 8H24 (SEQ ID NO:45-47).

[0019] Some antibodies comprise a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 180-181, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 185-186.

[0020] In some antibodies, at least one of the following positions in the VH region is occupied by an amino acid as specified: H2 is occupied by A, H12 is occupied by K or V, H48 is occupied by I, H67 is occupied by A, H71 is occupied by V, H91 is occupied by F, and H108 is occupied by T.

[0021] In some antibodies, positions H2, H48, H67, H71, H91 and H108 in the VH region are occupied by A, I, A, V, F and T, respectively. In some antibodies, positions H2, H12, H48, H67, H71, H91 and H108 in the VH region are occupied by A, V, I, A, V, F and T, respectively. In some antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is V, L9 is L or S, and L74 is K or T.

[0022] In some antibodies, position L2 in the VL region is occupied by V. In some antibodies, positions L2, L9, and L74 of the VL region are occupied by V, S, and T, respectively.

[0023] Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 180-181, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 185-186. Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 180-181, and a mature light chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 185-186. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 180-181, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 185-186.

[0024] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 185. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 186. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 185. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 186.

[0025] In another aspect, the invention provides a murine antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 11M14, wherein 11M14 is 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, with the proviso that position L54 can be L, G, or I. For example, the antibody can be a humanized antibody.

[0026] In some antibodies, the CDRs have a definition selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat / Chothia combination heavy chain CDRs of 11M14 (SEQ ID NOs: 53-55), and the humanized mature light chain variable region comprises the three Kabat / Chothia combination light chain CDRs of 11M14 (SEQ ID NOs: 59-61), except that position L54 can be L, G, or I. In some antibodies, the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 11M14 (SEQ ID NOs: 62, 54, and 55), and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 11M14 (SEQ ID NOs: 59-61), except that position L54 can be L, G, or I. In some antibodies, the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 11M14 (SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:55), and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 11M14 (SEQ ID NOs:59-61), except that position L54 can be L, G, or I. In some antibodies, the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 11M14 (SEQ ID NOs:53, SEQ ID NO:65, and SEQ ID NO:55), and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 11M14 (SEQ ID NOs:59-61), except that position L54 can be L, G, or I. In some antibodies, the humanized mature heavy chain variable region comprises the three Contact heavy chain CDRs of 11M14 (SEQ ID NOs: 66-68), and the humanized mature light chain variable region comprises the three Contact light chain CDRs of 11M14 (SEQ ID NOs: 69-71), except that position L54 can be L, G, or I.

[0027] Some antibodies comprise 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.

[0028] In some antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H49 is occupied by A, H80 is occupied by L or G, and H82c is occupied by L or G. In some antibodies, position H49 in the VH region is occupied by A. In some antibodies, positions H49 and H82c in the VH region are occupied by A and G, respectively. In some antibodies, positions H49 and H80 in the VH region are occupied by A and G, respectively.

[0029] In some antibodies, at least one of the following positions in the VL region is occupied by a specified amino acid: L43 is A or S, L48 is V, L54 is L, G, or I, L71 is Y, and L76 is N or S. In some antibodies, positions L48 and L71 in the VL region are occupied by V and Y, respectively. In some antibodies, positions L43, L48, L71, and L76 in the VL region are occupied by S, V, Y, and S, respectively. In some antibodies, positions L43, L48, L54, L71, and L76 in the VL region are occupied by S, V, G, Y, and S, respectively. In some antibodies, positions L43, L48, L54, L71, and L76 in the VL region are occupied by S, V, I, Y, and S, respectively.

[0030] Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 190 to 192, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 196 to 199. Some antibodies comprise a mature heavy chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 190 to 192, and a mature light chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 196 to 199. In some antibodies, the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 190 to 192, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 196 to 199.

[0031] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197.

[0032] In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. In some antibodies, the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192, and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199.

[0033] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 5M13, where 5M13 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 79-81) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 85-87).

[0034] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2F18, where 2F18 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 91-93) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 97-99).

[0035] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2P22, where 2P22 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 103-105) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 109-111).

[0036] In another aspect, the invention provides an antibody that specifically binds to human sortilin comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 6B15, where 6B15 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 115-117) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 121-123).

[0037] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2C14, where 2C14 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 127-129) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 133-135).

[0038] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 9N18, where 9N18 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 139-141), and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 145-147).

[0039] In another aspect, the invention provides an antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 4N2, where 4N2 is 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. In some antibodies, the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 151-153) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 157-159).

[0040] For example, the antibody can be a humanized antibody.

[0041] The antibodies may be intact antibodies or binding fragments. Some antibodies have the human IgG1 isotype, while others may have the human IgG2 or IgG4 isotype. Some antibodies have a mature light chain variable region fused to a light chain constant region and a mature heavy chain variable region fused to a heavy chain constant region. The heavy chain constant region of some antibodies is a mutant form of a natural human heavy chain constant region that has reduced binding to Fcγ receptors compared to the natural human heavy chain constant region.

[0042] The heavy chain constant regions of some antibodies are variants of naturally occurring human heavy chain constant regions that have enhanced binding to neonatal Fcγ receptors compared to naturally occurring human heavy chain constant regions.

[0043] Some antibodies comprise a heavy chain of SEQ ID NO: 244 and a light chain of SEQ ID NO: 245. Some antibodies comprise a heavy chain of SEQ ID NO: 246 and a light chain of SEQ ID NO: 247. Some antibodies comprise a heavy chain of SEQ ID NO: 248 and a light chain of SEQ ID NO: 249. Some antibodies comprise a heavy chain of SEQ ID NO: 250 and a light chain of SEQ ID NO: 245. Some antibodies comprise a heavy chain of SEQ ID NO: 251 and a light chain of SEQ ID NO: 247. Some antibodies comprise a heavy chain of SEQ ID NO: 252 and a light chain of SEQ ID NO: 249.

[0044] In another aspect, the invention provides a pharmaceutical composition comprising any of the antibodies disclosed herein and a pharmaceutically acceptable carrier. In another aspect, the invention provides nucleic acids encoding the heavy and / or light chains of any of the antibodies disclosed herein.

[0045] In yet another aspect, the invention provides a method for the production of any non-human antibody described herein, such as murine antibody 5E20 (wherein 5E20 is characterized by a mature heavy chain variable region of SEQ ID NO: 4 and a mature light chain variable region of SEQ ID NO: 10), murine antibody 8H24 (wherein 8H24 is characterized by a mature heavy chain variable region of SEQ ID NO: 28 and a mature light chain variable region of SEQ ID NO: 34), murine antibody 11M14 (wherein 11M14 is characterized by a mature heavy chain variable region of SEQ ID NO: 52 and a mature light chain variable region of SEQ ID NO: 58), murine antibody 5M13 (wherein 5M13 is characterized by a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 84), murine antibody 2F18 (wherein 2F18 is characterized by a mature heavy chain variable region of SEQ ID NO: 90 and a mature light chain variable region of SEQ ID NO: 96), or the like. The present invention provides methods for humanizing murine antibody 6B15 (featuring a mature heavy chain variable region of SEQ ID NO: 114 and a mature light chain variable region of SEQ ID NO: 120), murine antibody 2C14 (featuring a mature heavy chain variable region of SEQ ID NO: 126 and a mature light chain variable region of SEQ ID NO: 132), murine antibody 9N18 (featuring a mature heavy chain variable region of SEQ ID NO: 138 and a mature light chain variable region of SEQ ID NO: 144), and murine antibody 4N2 (featuring a mature heavy chain variable region of SEQ ID NO: 150 and a mature light chain variable region of SEQ ID NO: 156). Such methods may include the steps of selecting one or more acceptor antibody sequences, identifying the amino acid residues of the murine antibody to be retained, synthesizing nucleic acids encoding a humanized heavy chain comprising the CDRs of the murine antibody heavy chain and a humanized light chain comprising the CDRs of the murine antibody heavy chain, and expressing the nucleic acids in a host cell to produce the humanized antibody.

[0046] Also provided are methods for producing humanized, chimeric, or veneered forms of antibodies, such as 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. In such methods, cells transformed with nucleic acids encoding the antibody heavy and light chains are cultured so that the cells secrete the antibody. The antibody can then be purified from the cell culture medium.

[0047] Cell lines producing humanized, chimeric or veneered forms of any of the antibodies disclosed herein, e.g., 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2, can be produced by introducing vectors encoding the antibody heavy and light chains and a selectable marker into cells, growing the cells under conditions to select for cells with increased vector copy number, isolating single cells from the selected cells, and banking the cloned cells from the selected single cells based on antibody yield.

[0048] Some cells were grown under selective conditions and exposed to at least 100 mg / L / 10 6 Cells / 24 hours can be screened for cell lines that naturally express and secrete the protein.

[0049] The present invention also provides a method for increasing progranulin levels in a subject having or at risk of developing a disease or disorder associated with altered progranulin levels, the method comprising administering to the subject an effective amount of an antibody disclosed herein, thereby increasing progranulin levels in the subject.

[0050] The present invention also provides methods for treating or preventing a disease or disorder associated with altered progranulin levels in a subject, comprising administering an effective amount of an antibody disclosed herein, thereby treating or preventing the disease or disorder. Some methods further comprise detecting progranulin levels in the subject. Some methods further comprise monitoring progranulin levels in the subject.

[0051] In some methods, the disease or disorder associated with changes in progranulin levels is frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders.In some methods, the disease or disorder associated with changes in progranulin levels is frontotemporal dementia.

[0052] The present invention also provides a method of detecting Sortilin in a subject having or at risk for a disease associated with a disease or disorder associated with altered levels of Progranulin, the method comprising administering to the subject an antibody disclosed herein and detecting antibody bound to Sortilin in the subject.

[0053] In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues FTESFLT (SEQ ID NO: 202). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues ESFL (SEQ ID NO: 203). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to human sortilin at an epitope within a motif of the formula E(S / Q / D)FL (SEQ ID NO: 206). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues DGCILGYKEQFL (SEQ ID NO: 204). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues PSICLCSLEDFL (SEQ ID NO: 205). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues RTEFGMAIGP (SEQ ID NO: 213). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to a peptide consisting of residues WGFTESFLTS (SEQ ID NO: 214). In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to an epitope defined by amino acid residues D74, R76, F97, K110, Y535, L560, and E557 of SEQ ID NO: 215. In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to an epitope defined by amino acid residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559, and L560 of SEQ ID NO: 215. In another aspect, the present invention provides an isolated monoclonal antibody that specifically binds to an epitope defined by amino acid residues E557, S558, F559, L560, P510, and Y535 of SEQ ID NO: 215.

[0054] The present invention also provides a method of treating or preventing a disease or disorder associated with altered progranulin levels in a subject, the method comprising administering an immunogen comprising a sortilin peptide of up to 20 contiguous amino acids of SEQ ID NO: 1 to which antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2 specifically binds, wherein the peptide induces the formation of antibodies in the subject that specifically bind to sortilin. The present invention provides, for example, the following items. (Item 1) An isolated monoclonal antibody that competes with antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 for binding to human sortilin. (Item 2) 2. The antibody of item 1, which binds to the same epitope on human sortilin as antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2. (Item 3) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 5E20, wherein 5E20 is 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. (Item 4) 4. The antibody of item 3, wherein the antibody is a humanized antibody. (Item 5) 5. The humanized antibody of item 4, wherein the CDRs are defined as selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. (Item 6) 6. The humanized antibody of item 5, wherein the humanized mature heavy chain variable region comprises three 5E20 Kabat / Chothia combined heavy chain CDRs (SEQ ID NOs: 5 to 7), and the humanized mature light chain variable region comprises three 5E20 Kabat / Chothia combined light chain CDRs (SEQ ID NOs: 11 to 13). (Item 7) 6. The humanized antibody according to Item 5, wherein the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 5E20 (SEQ ID NO: 14, SEQ ID NO: 6, and SEQ ID NO: 7), and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 5E20 (SEQ ID NOs: 11 to 13). (Item 8) 6. The humanized antibody according to Item 5, wherein the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 5E20 (SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 7), and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 5E20 (SEQ ID NOs: 11 to 13). (Item 9) 6. The humanized antibody according to Item 5, wherein the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 5E20 (SEQ ID NO: 5, SEQ ID NO: 17, and SEQ ID NO: 7), and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 5E20 (SEQ ID NOs: 11 to 13). (Item 10) 6. The humanized antibody according to Item 5, wherein the humanized mature heavy chain variable region comprises three Contact heavy chain CDRs of 5E20 (SEQ ID NOs: 18 to 20), and the humanized mature light chain variable region comprises three Contact light chain CDRs of 5E20 (SEQ ID NOs: 21 to 23). (Item 11) 11. The humanized antibody according to any one of items 4 to 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: 163 to 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 to 176. (Item 12) 12. The humanized antibody of item 11, wherein at least one of the following positions in the VH region is occupied by a specified amino acid: H5 is occupied by L or V, H40 is occupied by A or T, H42 is occupied by G or D, H44 is occupied by G or R, H49 is occupied by A, H77 is occupied by T or S, H83 is occupied by R or K, H93 is occupied by S, and H94 is occupied by R. (Item 13) 13. The humanized antibody of item 12, wherein the provided positions H49, H93, and H94 of the VH region are occupied by A, S, and R, respectively. (Item 14) 13. The humanized antibody of item 12, wherein the provided positions H5, H49, H77, H93 and H94 of the VH region are occupied by V, A, S, S and R, respectively. (Item 15) 13. The humanized antibody of item 12, wherein the provided positions H5, H44, H49, H77, H93 and H94 of the VH region are occupied by V, R, A, S, S and R, respectively. (Item 16) 13. The humanized antibody of item 12, wherein the provided positions H5, H42, H44, H49, H77, H93 and H94 of the VH region are occupied by V, D, R, A, S, S and R, respectively. (Item 17) 13. The humanized antibody of item 12, wherein the provided positions H5, H42, H44, H49, H77, H83, H93 and H94 of the VH region are occupied by V, D, R, A, S, K, S and R, respectively. (Item 18) 13. The humanized antibody of item 12, wherein the provided positions H5, H40, H44, H49, H77, H93 and H94 of the VH region are occupied by V, T, R, A, S, S and R, respectively. (Item 19) 13. The humanized antibody of item 12, wherein the provided positions H5, H40, H42, H44, H49, H77, H93 and H94 of the VH region are occupied by V, T, D, R, A, S, S and R, respectively. (Item 20) 12. The humanized antibody of item 11, wherein at least one of the following positions in the VL region is occupied by a specified amino acid: L11 is L or V, L36 is L, L44 is F, L46 is G, L69 is A, L85 is T or D, L87 is F, L100 is G or Q, and L106 is I or K. (Item 21) 27. The humanized antibody of item 26, wherein the provided positions L36, L44, L46, L69 and L87 of the VL region are occupied by L, F, G, A and F, respectively. (Item 22) 27. The humanized antibody of item 26, wherein the provided positions L11, L36, L44, L46, L69 and L87 of the VL region are occupied by V, L, F, G, A and F, respectively. (Item 23) 27. The humanized antibody of item 26, wherein the provided positions L11, L36, L44, L46, L69, L87, L100 and L106 of the VL region are occupied by V, L, F, G, A, F, Q and K, respectively. (Item 24) 27. The humanized antibody of item 26, wherein the provided positions L11, L36, L44, L46, L69, L85, L87, L100 and L106 of the VL region are occupied by V, L, F, G, A, D, F, Q and K, respectively. (Item 25) 12. The humanized antibody according to Item 11, comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 163 to 169, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 173 to 176. (Item 26) 26. The humanized antibody according to Item 25, comprising a mature heavy chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NOs: 163 to 169 and a mature light chain variable region having an amino acid sequence at least 98% identical to any of SEQ ID NOs: 173 to 176. (Item 27) 27. The humanized antibody according to Item 26, wherein the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 163 to 169, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 173 to 176. (Item 28) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 29) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 30) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 31) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 32) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 33) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 34) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 173. (Item 35) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 36) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 37) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 38) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 39) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 40) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 41) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 174. (Item 42) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 43) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 44) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 45) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 46) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 47) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 48) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 175. (Item 49) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 163 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 50) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 164 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 51) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 165 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 52) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 166 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 53) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 167 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 54) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 168 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 55) 28. The humanized antibody of claim 27, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 169 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 176. (Item 56) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 8H24, wherein 8H24 is 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. (Item 57) 57. The antibody of item 56, which is a humanized antibody. (Item 58) 58. The humanized antibody of item 57, wherein the CDRs are defined as selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. (Item 59) 59. The humanized antibody of Item 58, wherein the humanized mature heavy chain variable region comprises the three Kabat / Chothia combined heavy chain CDRs of 8H24 (SEQ ID NOs: 29 to 31) and the humanized mature light chain variable region comprises the three Kabat / Chothia combined light chain CDRs of 8H24 (SEQ ID NOs: 35 to 37). (Item 60) 59. The humanized antibody of Item 58, wherein the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 8H24 (SEQ ID NO: 38, SEQ ID NO: 30, and SEQ ID NO: 31), and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 8H24 (SEQ ID NOs: 35 to 37). (Item 61) 59. The humanized antibody of Item 58, wherein the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 8H24 (SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 31), and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 8H24 (SEQ ID NOs: 35 to 37). (Item 62) 59. The humanized antibody of Item 58, wherein the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 8H24 (SEQ ID NO: 29, SEQ ID NO: 41, and SEQ ID NO: 31), and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 8H24 (SEQ ID NOs: 35 to 37). (Item 63) 59. The humanized antibody according to Item 58, wherein the humanized mature heavy chain variable region comprises the three Contact heavy chain CDRs of 8H24 (SEQ ID NOs: 42 to 44), and the humanized mature light chain variable region comprises the three Contact light chain CDRs of 8H24 (SEQ ID NOs: 45 to 47). (Item 64) 64. The humanized antibody of any one of Items 57 to 63, comprising a humanized mature heavy chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 180 to 181, and a humanized mature light chain variable region having an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 185 to 186. (Item 65) 65. The humanized antibody of item 64, wherein at least one of the following positions in the VH region is occupied by a specified amino acid: H2 is occupied by A, H12 is occupied by K or V, H48 is occupied by I, H67 is occupied by A, H71 is occupied by V, H91 is occupied by F, and H108 is occupied by T. (Item 66) 66. The humanized antibody of item 65, wherein the provided positions H2, H48, H67, H71, H91 and H108 of the VH region are occupied by A, I, A, V, F and T, respectively. (Item 67) 66. The humanized antibody of item 65, wherein H2, H12, H48, H67, H71, H91, and H108 of the VH region are occupied by A, V, I, A, V, F, and T, respectively. (Item 68) 65. The humanized antibody of item 64, wherein at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is V, L9 is L or S, and L74 is K or T. (Item 69) 69. The humanized antibody of item 68, wherein the provided position L2 of the VL region is occupied by V. (Item 70) 69. The humanized antibody of item 68, wherein the provided positions L2, L9 and L74 of the VL region are occupied by V, S and T, respectively. (Item 71) 65. The humanized antibody according to Item 64, comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 180 to 181, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 185 to 186. (Item 72) 72. The humanized antibody according to Item 71, comprising a mature heavy chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 180 to 181, and a mature light chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 185 to 186. (Item 73) 73. The humanized antibody according to Item 72, wherein the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 180 to 181, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 185 to 186. (Item 74) 74. The humanized antibody of claim 73, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 185. (Item 75) 74. The humanized antibody of claim 73, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 180 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 186. (Item 76) 74. The humanized antibody of claim 73, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 185. (Item 77) 74. The humanized antibody of claim 73, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 181 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 186. (Item 78) An antibody that specifically binds to sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 11M14, wherein 11M14 is 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, with the proviso that position L54 can be L, G, or I. (Item 79) 79. The antibody of item 78, which is a humanized antibody. (Item 80) 80. The humanized antibody of item 79, wherein the CDRs are defined as selected from the group consisting of Kabat, Chothia, Kabat / Chothia combination, AbM, and Contact. (Item 81) 81. The humanized antibody of item 80, wherein the humanized mature heavy chain variable region comprises the three Kabat / Chothia combined heavy chain CDRs of 11M14 (SEQ ID NOs: 53-55) and the humanized mature light chain variable region comprises the three Kabat / Chothia combined light chain CDRs of 11M14 (SEQ ID NOs: 59-61), with the proviso that position L54 can be L, G, or I. (Item 82) 81. The humanized antibody of Item 80, wherein the humanized mature heavy chain variable region comprises the three Kabat heavy chain CDRs of 11M14 (SEQ ID NO: 62, SEQ ID NO: 54, and SEQ ID NO: 55) and the humanized mature light chain variable region comprises the three Kabat light chain CDRs of 11M14 (SEQ ID NOs: 59-61), with the proviso that position L54 can be L, G, or I. (Item 83) 81. The humanized antibody of Item 80, wherein the humanized mature heavy chain variable region comprises the three Chothia heavy chain CDRs of 11M14 (SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 55) and the humanized mature light chain variable region comprises the three Chothia light chain CDRs of 11M14 (SEQ ID NOs: 59-61), with the proviso that position L54 can be L, G, or I. (Item 84) 81. The humanized antibody of Item 80, wherein the humanized mature heavy chain variable region comprises the three AbM heavy chain CDRs of 11M14 (SEQ ID NO: 53, SEQ ID NO: 65, and SEQ ID NO: 55) and the humanized mature light chain variable region comprises the three AbM light chain CDRs of 11M14 (SEQ ID NOs: 59-61), with the proviso that position L54 can be L, G, or I. (Item 85) 81. The humanized antibody of Item 80, wherein the humanized mature heavy chain variable region comprises the three Contact heavy chain CDRs of 11M14 (SEQ ID NOs: 66 to 68) and the humanized mature light chain variable region comprises the three Contact light chain CDRs of 11M14 (SEQ ID NOs: 69 to 71), with the proviso that position L54 can be L, G, or I. (Item 86) 86. The humanized antibody of any one of Items 79 to 85, 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 to 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 to 199. (Item 87) 87. The humanized antibody of paragraph 86, wherein at least one of the following positions in the VH region is occupied by a specified amino acid: H49 is occupied by A, H80 is occupied by L or G, and H82c is occupied by L or G. (Item 88) 88. The humanized antibody of item 87, wherein the provided position H49 in the VH region is occupied by A. (Item 89) 88. The humanized antibody of item 87, wherein the provided positions H49 and H82c of the VH region are occupied by A and G, respectively. (Item 90) 88. The humanized antibody of item 87, wherein positions H49 and H80 of the VH region are occupied by A and G, respectively. (Item 91) 87. The humanized antibody of item 86, wherein at least one of the following positions in the VL region is occupied by a specified amino acid: L43 is A or S; L48 is V; L54 is L, G or I; L71 is Y; and L76 is N or S. (Item 92) 92. The humanized antibody of item 91, wherein the provided positions L48 and L71 of the VL region are occupied by V and Y, respectively. (Item 93) 92. The humanized antibody of item 91, wherein the provided positions L43, L48, L71 and L76 of the VL region are occupied by S, V, Y and S, respectively. (Item 94) 92. The humanized antibody of item 91, wherein the provided positions L43, L48, L54, L71 and L76 of the VL region are occupied by S, V, G, Y and S, respectively. (Item 95) 92. The humanized antibody of item 91, wherein the provided positions L43, L48, L54, L71 and L76 of the VL region are occupied by S, V, I, Y and S, respectively. (Item 96) 87. The humanized antibody of Item 86, comprising a mature heavy chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 190 to 192, and a mature light chain variable region having an amino acid sequence at least 95% identical to at least one of SEQ ID NOs: 196 to 199. (Item 97) 97. The humanized antibody according to Item 96, comprising a mature heavy chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 190 to 192, and a mature light chain variable region having an amino acid sequence at least 98% identical to SEQ ID NOs: 196 to 199. (Item 98) 98. The humanized antibody according to Item 97, wherein the mature heavy chain variable region has the amino acid sequence of any one of SEQ ID NOs: 190 to 192, and the mature light chain variable region has the amino acid sequence of any one of SEQ ID NOs: 196 to 199. (Item 99) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. (Item 100) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197. (Item 101) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. (Item 102) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 190 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199. (Item 103) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. (Item 104) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197. (Item 105) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. (Item 106) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 191 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199. (Item 107) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 196. (Item 108) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 197. (Item 109) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 198. (Item 110) 99. The humanized antibody of claim 98, wherein the mature heavy chain variable region has the amino acid sequence of SEQ ID NO: 192 and the mature light chain variable region has the amino acid sequence of SEQ ID NO: 199. (Item 111) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 5M13, wherein 5M13 is 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. (Item 112) 112. The antibody of item 111, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 79 to 81) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 85 to 87). (Item 113) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2F18, wherein 2F18 is 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. (Item 114) 114. The antibody of item 113, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 91 to 93) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 97 to 99). (Item 115) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2P22, wherein 2P22 is 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. (Item 116) 116. The antibody of item 115, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 103 to 105) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 109 to 111). (Item 117) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 6B15, wherein 6B15 is 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. (Item 118) 18. The antibody of item 117, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 115 to 117) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 121 to 123). (Item 119) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 2C14, wherein 2C14 is 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. (Item 120) 119. The antibody of claim 119, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 127 to 129) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 133 to 135). (Item 121) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 9N18, wherein 9N18 is 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. (Item 122) 122. The antibody of item 121, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 139 to 141) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 145 to 147). (Item 123) An antibody that specifically binds to human sortilin, comprising the three light chain CDRs and three heavy chain CDRs of monoclonal antibody 4N2, wherein 4N2 is 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. (Item 124) 124. The antibody of item 123, wherein the three heavy chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 151 to 153) and the three light chain CDRs are as defined by the Kabat-Chothia combination (SEQ ID NOs: 157 to 159). (Item 125) 125. The antibody of any one of items 111 to 124, which is a humanized antibody. (Item 126) 126. The antibody of any one of items 1 to 125, which is an intact antibody. (Item 127) 126. The antibody of any one of items 1 to 125, which is a binding fragment. (Item 128) Item 10. The antibody of any one of the preceding items, wherein the isotype is human IgG1. (Item 129) 126. The antibody of any one of paragraphs 1 to 125, wherein the mature light chain variable region is fused to a light chain constant region and the mature heavy chain variable region is fused to a heavy chain constant region. (Item 130) 130. The antibody of claim 129, wherein the heavy chain constant region is a variant of a native human heavy chain constant region that has reduced binding to Fcγ receptors compared to a native human heavy chain constant region. (Item 131) 130. The antibody of claim 129, wherein the heavy chain constant region is a variant of a native human heavy chain constant region that has enhanced binding to neonatal Fcγ receptors compared to the native human heavy chain constant region. (Item 132) 129. The antibody of item 129, comprising a heavy chain of SEQ ID NO: 244 and a light chain of SEQ ID NO: 245. (Item 133) 129. The antibody of item 129, comprising a heavy chain of SEQ ID NO: 246 and a light chain of SEQ ID NO: 247. (Item 134) 129. The antibody of claim 129, comprising a heavy chain of SEQ ID NO: 248 and a light chain of SEQ ID NO: 249. (Item 135) 130. The antibody of item 129, comprising a heavy chain of SEQ ID NO: 250 and a light chain of SEQ ID NO: 245. (Item 136) 129. The antibody of item 129, comprising a heavy chain of SEQ ID NO: 251 and a light chain of SEQ ID NO: 247. (Item 137) 130. The antibody of item 129, comprising a heavy chain of SEQ ID NO: 252 and a light chain of SEQ ID NO: 249. (Item 138) 138. The antibody of any one of items 1 to 127 and 129 to 137, wherein the isotype is a human IgG2 or IgG4 isotype. (Item 139) 139. A pharmaceutical composition comprising an antibody as defined in any of items 1 to 138 and a pharmaceutically acceptable carrier. (Item 140) 139. A nucleic acid encoding the heavy and / or light chain of the antibody of any one of items 1 to 139. (Item 141) 1. A method for humanizing a murine antibody, said method comprising: (a) selecting one or more acceptor antibody sequences; (b) identifying amino acid residues of the mouse antibody to be retained; (c) synthesizing a nucleic acid encoding a humanized heavy chain comprising the CDRs of a murine antibody heavy chain and a nucleic acid encoding a humanized light chain comprising the CDRs of a murine antibody light chain; (d) expressing the nucleic acid in a host cell to produce the humanized antibody; Including, the murine antibody is 5E20, which is characterized by a mature heavy chain variable region of SEQ ID NO: 4 and a mature light chain variable region of SEQ ID NO: 10; the murine antibody is 8H24, which is characterized by a mature heavy chain variable region of SEQ ID NO: 28 and a mature light chain variable region of SEQ ID NO: 34; the murine antibody is 11M14, which is characterized by a mature heavy chain variable region of SEQ ID NO: 52 and a mature light chain variable region of SEQ ID NO: 58; the murine antibody is 5M13, which is characterized by a mature heavy chain variable region of SEQ ID NO: 78 and a mature light chain variable region of SEQ ID NO: 84; the murine antibody is 2F18, which is characterized by a mature heavy chain variable region of SEQ ID NO: 90 and a mature light chain variable region of SEQ ID NO: 96; the murine antibody is 2P22, characterized by a mature heavy chain variable region of SEQ ID NO: 102 and a mature light chain variable region of SEQ ID NO: 108; the murine antibody is 6B15, which is characterized by a mature heavy chain variable region of SEQ ID NO: 114 and a mature light chain variable region of SEQ ID NO: 120; the murine antibody is 2C14, characterized by a mature heavy chain variable region of SEQ ID NO: 126 and a mature light chain variable region of SEQ ID NO: 132; the murine antibody is 9N18, which is characterized by a mature heavy chain variable region of SEQ ID NO: 138 and a mature light chain variable region of SEQ ID NO: 144; The method, wherein the murine antibody is 4N2, which is characterized by a mature heavy chain variable region of SEQ ID NO: 150 and a mature light chain variable region of SEQ ID NO: 156. (Item 142) 1. A method for producing a humanized, chimeric or veneered antibody, said method comprising: (a) culturing cells transformed with nucleic acids encoding the heavy and light chains of an antibody so that the cells secrete the antibody; (b) purifying the antibody from the cell culture medium; Including, The method wherein the antibody is a humanized, chimeric, or veneered form of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. (Item 143) 1. A method for producing a cell line that produces a humanized, chimeric or veneered antibody, said method comprising: (a) introducing into cells a vector encoding the antibody heavy and light chains and a selectable marker; (b) growing the cells under conditions that select for cells with increased copy number of the vector; (c) isolating a single cell from the selected cells; (d) banking cells cloned from the single cells selected based on antibody yield; Including, The method wherein the antibody is a humanized, chimeric, or veneered form of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. (Item 144) The cells are grown under selective conditions and treated with at least 100 mg / L / 10 6 144. The method of claim 143, further comprising screening for cell lines that naturally express and secrete the cells / 24h. (Item 145) 140. 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 administering to the subject an effective amount of an antibody described in any one of items 1 to 140, thereby increasing progranulin levels in the subject. (Item 146) 140. A method for treating or preventing a disease or disorder associated with altered progranulin levels in a subject, the method comprising administering an effective amount of an antibody as defined by any one of items 1 to 140, thereby treating or preventing said disease or disorder. (Item 147) Item 147. The method of item 146, further comprising detecting progranulin levels in the subject. (Item 148) Item 148. The method of item 147, further comprising monitoring progranulin levels in the subject. (Item 149) Item 147. The method of item 146, 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. (Item 150) Item 149. The method of item 149, wherein the disease or disorder associated with altered progranulin levels is frontotemporal dementia. (Item 151) 141. 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, the method comprising administering to the subject an antibody as defined by any one of items 1 to 140, and detecting said antibody bound to sortilin in said subject. (Item 152) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues FTESFLT (SEQ ID NO: 202). (Item 153) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues ESFL (SEQ ID NO: 203). (Item 154) An isolated monoclonal antibody that specifically binds to human sortilin at an epitope within the motif of the formula E(S / Q / D)FL (SEQ ID NO: 206). (Item 155) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues DGCILGYKEQFL (SEQ ID NO: 204). (Item 156) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues PSICLCSLEDFL (SEQ ID NO: 205). (Item 157) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues RTEFGMAIGP (SEQ ID NO: 213). (Item 158) An isolated monoclonal antibody that specifically binds to a peptide consisting of residues WGFTESFLTS (SEQ ID NO: 214). (Item 159) An isolated monoclonal antibody that specifically binds to the epitope defined by amino acid residues D74, R76, F97, K110, Y535, L560 and E557 of SEQ ID NO:215. (Item 160) An isolated monoclonal antibody that specifically binds to an epitope defined by amino acid residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of SEQ ID NO:215. (Item 161) An isolated monoclonal antibody that specifically binds to the epitope defined by amino acid residues E557, S558, F559, L560, P510 and Y535 of SEQ ID NO:215. (Item 162) 1. A method of treating or preventing a disease or disorder associated with altered progranulin levels in a subject, comprising administering an immunogen comprising a sortilin peptide of up to 20 consecutive amino acids of SEQ ID NO: 1 to which antibody 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2 specifically binds, wherein the peptide induces the formation of antibodies in the subject that specifically bind to sortilin. [Brief explanation of the drawings]

[0055] [Figure 1]FIG. 1 shows the alignment of the heavy chain variable regions of the murine 5E20 antibody (5E20VH; SEQ ID NO: 4) and humanized versions of the 5E20 antibody (hu5E20VHv1, hu5E20VHv2, hu5E20VHv3, hu5E20VHv4, hu5E20VHv5, hu5E20VHv6 and hu5E20VHv7) with the human germline heavy chain variable region sequence IGHV3-21*01 (SEQ ID NO: 162) and the human acceptor heavy chain variable region sequence AEX29086-VH_huFrwk (AEX29086VH; SEQ ID NO: 161). hu5E20VHv1 (5E20VHv1) is SEQ ID NO: 163, hu5E20VHv2 (5E20VHv2) is SEQ ID NO: 164, hu5E20VHv3 (5E20VHv3) is SEQ ID NO: 165, hu5E20VHv4 (5E20VHv4) is SEQ ID NO: 166, hu5E20VHv5 (5E20VHv5) is SEQ ID NO: 167, hu5E20VHv6 (5E20VHv6) is SEQ ID NO: 168, and hu5E20VHv7 (5E20VHv7) is SEQ ID NO: 169. The CDRs of murine 5E20VH as defined by the Kabat / Chothia combination are in bold.

[0056] [Figure 2] Figure 2 shows the alignment of the light chain variable region of the murine 5E20 antibody (5E20VL, SEQ ID NO: 10) and the humanized versions of the 5E20 antibody (hu5E20VLv1, hu5E20VLv2, hu5E20VLv3 and hu5E20VLv4) with the human germline light chain variable region sequence #IGKV1-12*01 (SEQ ID NO: 172) and the human acceptor BAH04687-VL_huFrwk (BAH04687VL; SEQ ID NO: 171). hu5E20VLv1 (5D20VLv1) is SEQ ID NO: 173, hu5E20VLv2 (5D20VLv2) is SEQ ID NO: 174, hu5E20VLv3 (5E20VLv3) is SEQ ID NO: 175, and hu5E20VLv4 (5E20VLv4) is SEQ ID NO: 176. The CDRs of murine 5E20VL as defined by the Kabat / Chothia combination are in bold.

[0057] [Figure 3] 3 shows the alignment of the heavy chain variable regions of the murine 8H24 antibody (8H24_VH, SEQ ID NO: 28) and the humanized versions of the 8H24 antibody (hu8H24VHv1 and hu8H24VHv2) with the human germline heavy chain variable region sequence IGHV1-69*08_IGHJ1*01 (SEQ ID NO: 179) and the human acceptor heavy chain variable region sequence AAC51714-VH_huFrwk (AAC51714VH Frwk, SEQ ID NO: 178). hu8H24VHv1 (h8H24VLv1) is SEQ ID NO: 180, and hu8H24VHv2 (h8H24VHv2) is SEQ ID NO: 181. The CDRs of murine 8H24VH, as defined by the Kabat / Chothia combination, are in bold.

[0058] [Figure 4] Figure 4 shows the alignment of the light chain variable region of murine 8H24 (8H24VL) SEQ ID NO: 34) and the humanized versions of the 8H24 antibody (hu8H24VLv1 and hu8H24VLv2) with the human germline light chain variable region sequence IGKV2-40*01 (IGKV2-40*01_IGKJ4*01, SEQ ID NO: 184) and the human acceptor ABC66914-VL_huFrwk (ABC66914VL Frwk SEQ ID NO: 183). hu8H24VLv1 (hH824VLv1) is SEQ ID NO: 185, and hu8H24VLv2 (hH824VLv2) is SEQ ID NO: 186. The CDRs of murine 8H24VL, as defined by the Kabat / Chothia combination, are in bold.

[0059] [Figure 5]Figure 5 shows the alignment of the heavy chain variable regions of the murine 11M14 antibody (11M14_VH; SEQ ID NO: 52) and the humanized versions of the 11M14 antibody (hu11M14VHv1b, hu11M14VHv2b, and hu11M14VHv3b) with the human germline heavy chain variable region sequence IGHV3-48*03 (SEQ ID NO: 189) and the human acceptor heavy chain variable region sequence ACS96198-VH_huFrwk (ASC96198VH hFrwk, SEQ ID NO: 188), where hu11M14VHv1b (11M14VHv1b) is SEQ ID NO: 190, hu11M14VHv2b (11M14VHv2b) is SEQ ID NO: 191, and hu11M14VHv3b (11M14VHv3b) is SEQ ID NO: 192. The CDRs of mouse 11M14VH as defined by the Kabat / Chothia combination are in bold.

[0060] [Figure 6] Figure 6 shows the alignment of the light chain variable region of the murine 11M14 antibody (11M14VL, SEQ ID NO: 58) and humanized versions of the 11M14 antibody (hu11M14VLv1b, hu11M14VLv2b, hu11M14VLv3b and hu11M14VLv4b) with the human germline light chain variable region sequence IGKV1-39*01 (SEQ ID NO: 195) and the human acceptor CBZ39892-VL_huFrwk (CBZ39892VL hFrwk, SEQ ID NO: 194). hu11M14VLv1b (11M14VLv1b) is SEQ ID NO: 196, hu11M14VLv2b (11M14VLv2b) is SEQ ID NO: 197, hu11M14VLv3b (11M14VLv3b) is SEQ ID NO: 198, and hu11M14VLv4b (11M14VLv4b) is SEQ ID NO: 199. The CDRs of murine 11M14VL as defined by the Kabat / Chothia combination are in bold.

[0061] [Figure 7] FIG. 7 shows the inhibition of human progranulin binding to human sortilin by ELISA by nonclonal mouse antibody supernatants (percentage PGRN binding).

[0062] [Figure 8A] Figure 8 shows the binding of selected purified monoclonal antibodies to Sortilin (left graph) and inhibition of Progranulin binding to Sortilin (percentage PGRN blocking, right graph) by ELISA. The table on the right shows the EC50 (nM) of Ab binding to human Sortilin-ECD for selected purified antibodies and percent blocking at 7.4 nM mAb. [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 8D] Same as above.

[0063] [Figure 9A] Figure 9 shows the results of a Biacore competition assay to determine whether selected monoclonal antibodies block the binding of neurotensin to sortilin. The table summarizes the assay results for selected monoclonal antibodies. Example data are shown in three graphs: no competition with 8H24, weak competition with 1M16, and competition with 4J22. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above.

[0064] [Figure 10A] Figure 10 shows the results of binding of selected purified monoclonal antibodies to human sortilin overexpressed by HEK293 cells versus the parental HEK cell line and U251MG cells (a human glioblastoma cell line) that endogenously express sortilin. The plots on the left show binding of monoclonal antibodies 6B15, 7B18, 10B6, and 10O16 in the HEK293 cell assay (left column of plots) and the U251MG cell assay (right column of plots). The table on the right summarizes the assay results for selected monoclonal antibodies. [Figure 10B] Same as above. [Figure 10C] Same as above.

[0065] [Figure 11] FIG. 11 shows the inhibition of huPGRN binding to human sortilin-expressing HEK293 cells by selected purified monoclonal antibodies (PGRN blocking rate).

[0066] [Figure 12A] Figure 12 shows the dose response of inhibition of huPGRN binding to cell surface human sortilin-expressing HEK293 cells. The plot on the left shows the binding curves of antibodies with different potencies. The table on the right summarizes the results of the assay for selected monoclonal antibodies. (Maximum binding (%) at 11 nM mAb) [Figure 12B] Same as above.

[0067] [Figure 13A] Figure 13 shows extracellular PGRN and cell surface sortilin levels after treatment of U251MG cells with selected purified monoclonal antibodies. The plot on the left shows the assay results for selected monoclonal antibodies as a correlation graph. The table on the right summarizes the assay results for selected monoclonal antibodies. [Figure 13B] Same as above.

[0068] [Figure 14] Figure 14 shows plasma levels of anti-Sortilin antibody hu8H24 H1L2 IgG1 LALA, hu5E20 H7L4 IgG1 LALA, or hu11M14 H1bL3b IgG1 LALA after 60 mg / kg and 30 mg / kg administration in cynomolgus monkeys (pharmacokinetic study). Plasma anti-Sortilin antibody levels after administration of 30 mg / kg or 60 mg / kg of antibody in cynomolgus monkeys. N=3 animals per Ab group. Animals were administered a 30 mg / kg dose and 48 days later administered a 60 mg / kg dose of anti-Sortilin antibody.

[0069] [Figure 15]Figure 15 shows plasma PGRN levels after 60 mg / kg and 30 mg / kg administration of hu8H24 H1L2 IgG1 LALA, hu5E20 H7L4 IgG1 LALA, or hu11M14 H1bL3b IgG1 LALA in non-human primates (pharmacodynamic study). Fold-up of plasma PGRN levels after a single dose of 30 mg / kg or 60 mg / kg antibody in cynomolgus monkeys. N=3 animals per Ab group. Triangles indicate animals that showed detectable levels of anti-drug antibodies.

[0070] [Figure 16] Figure 16 shows CSF PGRN levels after 60 mg / kg and 30 mg / kg administration of hu8H24 H1L2 IgG1 LALA, hu5E20 H7L4 IgG1 LALA, or hu11M14 H1bL3b IgG1 LALA in cynomolgus monkeys (pharmacodynamics study). Fold-up of PGRN levels in CSF after a single dose of 30 mg / kg or 60 mg / kg of antibody in cynomolgus monkeys. N=3 animals per Ab group. Triangles indicate animals that showed the presence of anti-drug antibodies. No CSF ​​samples were collected on day 7 in animals treated with 30 mg / kg anti-Sortilin antibody.

[0071] [Figure 17] Figure 17 shows plasma anti-Sortilin antibody levels after four weekly doses of 60 mg / kg hu11M14 H1bL3b_IgG1_LALA or hu11M14 H1bL3b_IgG1_LALA_YTE in cynomolgus monkeys (pharmacokinetic study). Mean ± SD anti-Sortilin antibody plasma levels following four weekly doses of 60 mg / kg Ab. N=4.

[0072] [Figure 18]Figure 18 shows anti-Sortilin antibody levels in CSF after four weekly doses of 60 mg / kg hu11M14 H1bL3b_IgG1_LALA or hu11M14 H1bL3b_IgG1_LALA_YTE in cynomolgus monkeys (pharmacokinetic study). Mean ± SD Anti-Sortilin Ab CSF levels with four weekly doses of 60 mg / kg Ab. N=4 for hu11M14 H1bL3b_IgG1_LALA and N=3 for hu11M14 H1bL3b_IgG1_LALA_YTE.

[0073] [Figure 19] Figure 19 shows plasma progranulin levels in cynomolgus monkeys after four weekly repeated doses of 60 mg / kg hu11M14 H1bL3b_IgG1_LALA or hu11M14 H1bL3b_IgG1_LALA_YTE (pharmacodynamic study). Mean ± SD plasma fold PGRN levels in cynomolgus monkeys. N=4 animals per Ab group.

[0074] [Figure 20] Figure 20 shows CSF progranulin levels in cynomolgus monkeys after four weekly repeated doses of 60 mg / kg hu11M14 H1bL3b_IgG1_LALA or hu11M14 H1bL3b_IgG1_LALA_YTE (pharmacodynamic study). Mean ± SD. Fold PGRN levels in CSF in cynomolgus monkeys. N=4 animals in the hu11M14 H1bL3b_IgG1_LALA group, N=3 animals in the hu11M14 H1bL3b_IgG1_LALA_YTE group.

[0075] [Figure 21]Figure 21 shows sortilin levels in cynomolgus monkey PBMCs after four weekly repeated administrations of a 60 mg / kg dose of hu11M14 H1bL3b_IgG1_LALA or hu11M14 H1bL3b_IgG1_LALA_YTE. Mean ± SD Sortilin levels in cynomolgus monkey PBMCs as percent of baseline following four weekly administrations of 60 mg / kg anti-sortilin antibody. N=4 animals per group. Sortilin levels normalized to total protein levels. DETAILED DESCRIPTION OF THE INVENTION

[0076] A brief description of arrays SEQ ID NO: 1 shows the amino acid sequence of the human sortilin extracellular domain.

[0077] SEQ ID NO: 2 shows the nucleotide sequence of mouse 5E20VH(mIgG1).

[0078] SEQ ID NO: 3 shows the amino acid sequence of the signal peptide for mouse 5E20VH.

[0079] SEQ ID NO: 4 shows the amino acid sequence of mouse 5E20VH.

[0080] SEQ ID NO: 5 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-H1.

[0081] SEQ ID NO: 6 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-H2.

[0082] SEQ ID NO: 7 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-H3.

[0083] SEQ ID NO: 8 shows the nucleotide sequence of mouse 5E20VL(kappa).

[0084] SEQ ID NO: 9 shows the amino acid sequence of the signal peptide for mouse 5E20VL.

[0085] SEQ ID NO: 10 shows the amino acid sequence of mouse 5E20VLVk.

[0086] SEQ ID NO: 11 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-L1.

[0087] SEQ ID NO: 12 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-L2.

[0088] SEQ ID NO: 13 shows the amino acid sequence of mouse 5E20_Kabat Chothia combined CDR-L3.

[0089] SEQ ID NO: 14 shows the amino acid sequence of Kabat CDR-H1 of the mouse 5E20 antibody.

[0090] SEQ ID NO: 15 shows the amino acid sequence of Chothia CDR-H1 of the mouse 5E20 antibody.

[0091] SEQ ID NO: 16 shows the amino acid sequence of Chothia CDR-H2 of the mouse 5E20 antibody.

[0092] SEQ ID NO: 17 shows the amino acid sequence of AbM CDR-H2 of the mouse 5E20 antibody.

[0093] SEQ ID NO: 18 shows the amino acid sequence of Contact CDR-H1 of the mouse 5E20 antibody.

[0094] SEQ ID NO: 19 shows the amino acid sequence of Contact CDR-H2 of the mouse 5E20 antibody.

[0095] SEQ ID NO: 20 shows the amino acid sequence of Contact CDR-H3 of the mouse 5E20 antibody.

[0096] SEQ ID NO: 21 shows the amino acid sequence of Contact CDR-L1 of the mouse 5E20 antibody.

[0097] SEQ ID NO: 22 shows the amino acid sequence of Contact CDR-L2 of the mouse 5E20 antibody.

[0098] Sequence number 23: Contact CDR-L3 of mouse 5E20 antibody.

[0099] SEQ ID NO: 24 shows the amino acid sequence of the chimeric 5E20 heavy chain.

[0100] SEQ ID NO: 25 shows the amino acid sequence of the chimeric 5E20 light chain.

[0101] SEQ ID NO: 26 shows the nucleotide sequence of mouse 8H24VH (IgG2c).

[0102] SEQ ID NO: 27 shows the amino acid sequence of mouse 8H24VH signal peptide.

[0103] SEQ ID NO: 28 shows the amino acid sequence of mouse 8H24Vh.

[0104] SEQ ID NO: 29 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-H1.

[0105] SEQ ID NO: 30 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-H2.

[0106] SEQ ID NO: 31 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-H3.

[0107] SEQ ID NO: 32 shows the nucleotide sequence of mouse 8H24VL (kappa).

[0108] SEQ ID NO: 33 shows the amino acid sequence of mouse 8H24VL signal peptide.

[0109] SEQ ID NO: 34 shows the amino acid sequence of mouse 8H24Vk.

[0110] SEQ ID NO: 35 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-L1.

[0111] SEQ ID NO: 36 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-L2.

[0112] SEQ ID NO: 37 shows the amino acid sequence of mouse 8H24_Kabat Chothia combined CDR-L3.

[0113] SEQ ID NO: 38 shows the amino acid sequence of the Kabat CDR-H1 of the murine 8H24 antibody.

[0114] SEQ ID NO: 39 shows the amino acid sequence of Chothia CDR-H1 of the murine 8H24 antibody.

[0115] SEQ ID NO: 40 shows the amino acid sequence of Chothia CDR-H2 of the murine 8H24 antibody.

[0116] SEQ ID NO: 41 shows the amino acid sequence of AbM CDR-H2 of the mouse 8H24 antibody.

[0117] SEQ ID NO: 42 shows the amino acid sequence of Contact CDR-H1 of mouse 8H24 antibody.

[0118] SEQ ID NO: 43 shows the amino acid sequence of Contact CDR-H2 of mouse 8H24 antibody.

[0119] SEQ ID NO: 44 shows the amino acid sequence of Contact CDR-H3 of mouse 8H24 antibody.

[0120] SEQ ID NO: 45 shows the amino acid sequence of Contact CDR-L1 of mouse 8H24 antibody.

[0121] SEQ ID NO: 46 shows the amino acid sequence of Contact CDR-L2 of mouse 8H24 antibody.

[0122] SEQ ID NO: 47 shows the amino acid sequence of Contact CDR-L3 of mouse 8H24 antibody.

[0123] SEQ ID NO: 48 shows the amino acid sequence of the chimeric 8H24 heavy chain.

[0124] SEQ ID NO: 49 shows the amino acid sequence of the chimeric 8H24 light chain.

[0125] SEQ ID NO: 50 shows the nucleotide sequence of mouse 11M14VH(IgG1).

[0126] SEQ ID NO: 51 shows the amino acid sequence of mouse 11M14VH signal peptide.

[0127] SEQ ID NO: 52 shows the amino acid sequence of mouse 11M14Vh.

[0128] SEQ ID NO: 53 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-H1.

[0129] SEQ ID NO: 54 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-H2.

[0130] SEQ ID NO: 55 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-H3.

[0131] SEQ ID NO: 56 shows the nucleotide sequence of mouse 11M14Vk (kappa).

[0132] SEQ ID NO: 57 shows the amino acid sequence of mouse 11M14Vk signal peptide.

[0133] SEQ ID NO: 58 shows the amino acid sequence of mouse 11M14Vk.

[0134] SEQ ID NO: 59 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-L1.

[0135] SEQ ID NO: 60 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-L2.

[0136] SEQ ID NO: 61 shows the amino acid sequence of mouse 11M14_Kabat Chothia combined CDR-L3.

[0137] SEQ ID NO: 62 shows the amino acid sequence of the Kabat CDR-H1 of the murine 11M14 antibody.

[0138] SEQ ID NO: 63 shows the amino acid sequence of Chothia CDR-H1 of the murine 11M14 antibody.

[0139] SEQ ID NO: 64 shows the amino acid sequence of Chothia CDR-H2 of the mouse 11M14 antibody.

[0140] SEQ ID NO: 65 shows the amino acid sequence of AbM CDR-H2 of the mouse 11M14 antibody.

[0141] SEQ ID NO: 66 shows the amino acid sequence of Contact CDR-H1 of mouse 11M14 antibody.

[0142] SEQ ID NO: 67 shows the amino acid sequence of Contact CDR-H2 of mouse 11M14 antibody.

[0143] SEQ ID NO: 68 shows the amino acid sequence of Contact CDR-H3 of mouse 11M14 antibody.

[0144] SEQ ID NO: 69 shows the amino acid sequence of Contact CDR-L1 of mouse 11M14 antibody.

[0145] SEQ ID NO: 70 shows the amino acid sequence of Contact CDR-L2 of mouse 11M14 antibody.

[0146] SEQ ID NO: 71 shows the amino acid sequence of Contact CDR-L3 of mouse 11M14 antibody.

[0147] SEQ ID NO: 72 shows the amino acid sequence of Alternate Kabat-Chothia CDR-L2 (Hu11M14VLv3b, present in SEQ ID NO: 198).

[0148] SEQ ID NO: 73 shows the amino acid sequence of Alternate Kabat-Chothia CDR-L2 (Hu11M14VLv4b, present in SEQ ID NO: 199).

[0149] SEQ ID NO: 74 shows the amino acid sequence of Alternate Contact CDR-L2 (Hu11M14VLv3b, present in SEQ ID NO: 198).

[0150] SEQ ID NO: 75 shows the amino acid sequence of Alternate Contact CDR-L2 (Hu11M14VLv4b, present in SEQ ID NO: 199).

[0151] SEQ ID NO: 76 shows the nucleotide sequence of mouse 5M13VH(IgG1).

[0152] SEQ ID NO: 77 shows the amino acid sequence of mouse 5M13VH signal peptide.

[0153] SEQ ID NO: 78> Mouse 5M13VH amino acid sequence.

[0154] SEQ ID NO: 79 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-H1.

[0155] SEQ ID NO: 80 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-H2.

[0156] SEQ ID NO: 81 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-H3.

[0157] SEQ ID NO: 82 shows the nucleotide sequence of mouse 5M13VL(kappa).

[0158] SEQ ID NO: 83 shows the amino acid sequence of mouse 5M13VL signal peptide.

[0159] SEQ ID NO: 84 shows the amino acid sequence of mouse 5M13VL Vk.

[0160] SEQ ID NO: 85 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-L1.

[0161] SEQ ID NO: 86 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-L2.

[0162] SEQ ID NO: 87 shows the amino acid sequence of mouse 5M13_Kabat Chothia combined CDR-L3.

[0163] SEQ ID NO: 88 shows the nucleotide sequence of mouse 2F18VH (mIgG1).

[0164] SEQ ID NO: 89 shows the amino acid sequence of mouse 2F18VH signal peptide.

[0165] SEQ ID NO: 90 shows the amino acid sequence of mouse 2F18VH.

[0166] SEQ ID NO: 91 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-H1.

[0167] SEQ ID NO: 92 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-H2.

[0168] SEQ ID NO: 93 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-H3.

[0169] SEQ ID NO: 94 shows the nucleotide sequence of mouse 2F18VL (kappa).

[0170] SEQ ID NO: 95 shows the amino acid sequence of mouse 2F18VL signal peptide.

[0171] SEQ ID NO: 96 shows the amino acid sequence of mouse Vk_2F18VL.

[0172] SEQ ID NO: 97 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-L1.

[0173] SEQ ID NO: 98 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-L2.

[0174] SEQ ID NO: 99 shows the amino acid sequence of mouse 2F18_Kabat Chothia combined CDR-L3.

[0175] SEQ ID NO: 100 shows the nucleotide sequence of mouse 2P22VH (IgG2b).

[0176] SEQ ID NO: 101 shows the amino acid sequence of mouse 2P22 VH signal peptide.

[0177] SEQ ID NO: 102 shows the amino acid sequence of mouse 2P22VH.

[0178] SEQ ID NO: 103 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-H1.

[0179] SEQ ID NO: 104 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-H2.

[0180] SEQ ID NO: 105 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-H3.

[0181] SEQ ID NO: 106 shows the nucleotide sequence of mouse 2P22VL(kappa).

[0182] SEQ ID NO: 107 shows the amino acid sequence of mouse 2P22VL signal peptide.

[0183] SEQ ID NO: 108 shows the amino acid sequence of mouse Vk_2P22VL.

[0184] SEQ ID NO: 109 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-L1.

[0185] SEQ ID NO: 110 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-L2.

[0186] SEQ ID NO: 111 shows the amino acid sequence of mouse 2P22_Kabat Chothia combined CDR-L3.

[0187] SEQ ID NO: 112 shows the nucleotide sequence of murine 6B15VH(IgG1).

[0188] SEQ ID NO: 113 shows the amino acid sequence of the 6B15VH signal peptide.

[0189] SEQ ID NO: 114 shows the amino acid sequence of mouse 6B15VH.

[0190] SEQ ID NO: 115 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-H1.

[0191] SEQ ID NO: 116 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-H2.

[0192] SEQ ID NO: 117 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-H3.

[0193] SEQ ID NO: 118 shows the nucleotide sequence of mouse 6B15VL(kappa).

[0194] SEQ ID NO: 119 shows the amino acid sequence of the 6B15VL signal peptide.

[0195] SEQ ID NO: 120 shows the amino acid sequence of mouse 6B15VLVk_6B15.

[0196] SEQ ID NO: 121 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-L1.

[0197] SEQ ID NO: 122 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-L2.

[0198] SEQ ID NO: 123 shows the amino acid sequence of mouse 6B15_Kabat Chothia combined CDR-L3.

[0199] SEQ ID NO: 124 shows the nucleotide sequence of mouse 2C14VH(IgG1).

[0200] SEQ ID NO: 125 shows the amino acid sequence of the mouse 2C14VH signal peptide.

[0201] SEQ ID NO: 126 shows the amino acid sequence of mouse 2C14VH.

[0202] SEQ ID NO: 127 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-H1.

[0203] SEQ ID NO: 128 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-H2.

[0204] SEQ ID NO: 129 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-H3.

[0205] SEQ ID NO: 130 shows the nucleotide sequence of mouse 2C14VL(kappa).

[0206] SEQ ID NO: 131 shows the amino acid sequence of the mouse 2C14VL signal peptide.

[0207] SEQ ID NO: 132 shows the amino acid sequence of mouse 2C14VLVk_2C14.

[0208] SEQ ID NO: 133 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-L1.

[0209] SEQ ID NO: 134 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-L2.

[0210] SEQ ID NO: 135 shows the amino acid sequence of mouse 2C14_Kabat Chothia combined CDR-L3.

[0211] SEQ ID NO: 136 shows the nucleotide sequence of murine 9N18VH (IgG2b).

[0212] SEQ ID NO: 137 shows the amino acid sequence of mouse 9N18VH signal peptide.

[0213] SEQ ID NO: 138 shows the amino acid sequence of mouse 9N18VH.

[0214] SEQ ID NO: 139 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-H1.

[0215] SEQ ID NO: 140 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-H2.

[0216] SEQ ID NO: 141 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-H3.

[0217] SEQ ID NO: 142 shows the nucleotide sequence of mouse 9N18VL (kappa).

[0218] SEQ ID NO: 143 shows the amino acid sequence of mouse 9N18VL signal peptide.

[0219] SEQ ID NO: 144 shows the amino acid sequence of mouse 9N18VLVk_9N18.

[0220] SEQ ID NO: 145 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-L1.

[0221] SEQ ID NO: 146 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-L2.

[0222] SEQ ID NO: 147 shows the amino acid sequence of mouse 9N18_Kabat Chothia combined CDR-L3.

[0223] SEQ ID NO: 148 shows the nucleotide sequence of mouse 4N2VH(IgG3).

[0224] SEQ ID NO: 149 shows the amino acid sequence of the mouse 4N2VH signal peptide.

[0225] SEQ ID NO: 150 shows the amino acid sequence of mouse 4N2VHVh_4N2.

[0226] SEQ ID NO: 151 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-H1.

[0227] SEQ ID NO: 152 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-H2.

[0228] SEQ ID NO: 153 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-H3.

[0229] SEQ ID NO: 154 shows the nucleotide sequence of mouse 4N2VL (kappa).

[0230] SEQ ID NO: 155 shows the amino acid sequence of the mouse 4N2VL signal peptide.

[0231] SEQ ID NO: 156 shows the amino acid sequence of mouse 4N2VLVk_4N2.

[0232] SEQ ID NO: 157 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-L1.

[0233] SEQ ID NO: 158 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-L2.

[0234] SEQ ID NO: 159 shows the amino acid sequence of mouse 4N2_Kabat Chothia combined CDR-L3.

[0235] SEQ ID NO: 160 shows the amino acid sequence of 3V6F-VH_mSt.

[0236] SEQ ID NO: 161 shows the amino acid sequence of AEX29086-VH_huFrwk.

[0237] SEQ ID NO: 162 is IGHV3-21 * The amino acid sequence of 01 is shown below.

[0238] SEQ ID NO: 163 shows the amino acid sequence of h5E20VHv1.

[0239] SEQ ID NO: 164 shows the amino acid sequence of h5E20VHv2.

[0240] SEQ ID NO: 165 shows the amino acid sequence of h5E20VHv3.

[0241] SEQ ID NO: 166 shows the amino acid sequence of h5E20VHv4.

[0242] SEQ ID NO: 167 shows the amino acid sequence of h5E20VHv5.

[0243] SEQ ID NO: 168 shows the amino acid sequence of h5E20VHv6.

[0244] SEQ ID NO: 169 shows the amino acid sequence of h5E20VHv7.

[0245] SEQ ID NO: 170 shows the amino acid sequence of 3V6F-VL_mSt.

[0246] SEQ ID NO: 171 shows the amino acid sequence of BAH04687-VL_huFrwk.

[0247] SEQ ID NO: 172 is IGKV1-12 * The amino acid sequence of 01 is shown below.

[0248] SEQ ID NO: 173 shows the amino acid sequence of h5E20VLv1.

[0249] SEQ ID NO: 174 shows the amino acid sequence of h5E20VLv2.

[0250] SEQ ID NO: 175 shows the amino acid sequence of h5E20VLv3.

[0251] SEQ ID NO: 176 shows the amino acid sequence of h5E20VLv4.

[0252] SEQ ID NO: 177 shows the amino acid sequence of 1MRC-VH_mSt.

[0253] SEQ ID NO: 178 shows the amino acid sequence of AAC51714-VH_huFrwk.

[0254] SEQ ID NO: 179 is IGHV1-69 * 08_IGHJ1 * The amino acid sequence of 01 is shown below.

[0255] SEQ ID NO: 180 shows the amino acid sequence of h8H24VHv1.

[0256] SEQ ID NO: 181 shows the amino acid sequence of h8H24VHv2.

[0257] SEQ ID NO: 182 shows the amino acid sequence of 1MRC-VL_mSt.

[0258] SEQ ID NO: 183 shows the amino acid sequence of ABC66914-VL_huFrwk.

[0259] SEQ ID NO: 184 is IGKV2-40 * The amino acid sequence of 01 is shown below.

[0260] SEQ ID NO: 185 shows the amino acid sequence of h8H24VLv1.

[0261] SEQ ID NO: 186 shows the amino acid sequence of h8H24VLv2.

[0262] SEQ ID NO: 187 shows the amino acid sequence of 1 MQK-VH_mSt.

[0263] SEQ ID NO: 188 shows the amino acid sequence of ACS96198-VH_huFrwk.

[0264] SEQ ID NO: 189 is IGHV3-48 * The amino acid sequence of 03 is shown.

[0265] SEQ ID NO: 190 shows the amino acid sequence of h11M14VHv1b.

[0266] SEQ ID NO: 191 shows the amino acid sequence of h11M14VHv2b.

[0267] SEQ ID NO: 192 shows the amino acid sequence of h11M14VHv3b.

[0268] SEQ ID NO: 193 shows the amino acid sequence of 1 MQK-VL_mSt.

[0269] SEQ ID NO: 194 shows the amino acid sequence of CBZ39892-VL_huFrwk.

[0270] SEQ ID NO: 195 is IGKV1-39 * The amino acid sequence of 01 is shown below.

[0271] SEQ ID NO: 196 shows the amino acid sequence of h11M14VLv1b.

[0272] SEQ ID NO: 197 shows the amino acid sequence of h11M14VLv2b.

[0273] SEQ ID NO: 198 shows the amino acid sequence of h11M14VLv3b.

[0274] SEQ ID NO: 199 shows the amino acid sequence of h11M14VLv4b.

[0275] SEQ ID NO: 200 shows the amino acid sequence of the HA peptide.

[0276] SEQ ID NO: 201 shows the amino acid sequence of the c-Myc peptide.

[0277] SEQ ID NO: 202 shows the amino acid sequence of the consensus motif of the peptide bound by antibody 5E20.

[0278] SEQ ID NO: 203 shows the amino acid sequence of the consensus motif of the peptide bound by antibody 5E20.

[0279] SEQ ID NO: 204 shows the amino acid sequence of the peptide bound by antibody 5E20.

[0280] SEQ ID NO: 205 shows the amino acid sequence of the peptide bound by antibody 5E20.

[0281] SEQ ID NO: 206 shows the amino acid sequence of the sequence motif bound by antibody 5E20.

[0282] SEQ ID NO: 207 shows the amino acid sequence of the linker.

[0283] SEQ ID NO: 208 shows the amino acid sequence of the chimeric 11M14 heavy chain.

[0284] SEQ ID NO: 209 shows the amino acid sequence of the chimeric 11M14 light chain.

[0285] SEQ ID NO: 210 shows the amino acid sequence of residues 523 to 610 of sortilin.

[0286] SEQ ID NO: 211 shows the amino acid sequence of a sortilin peptide.

[0287] SEQ ID NO: 212 shows the amino acid sequence of a sortilin peptide.

[0288] SEQ ID NO: 213 shows the amino acid sequence of the peptide bound by antibody 8H24.

[0289] SEQ ID NO: 214 shows the amino acid sequence of the peptide bound by antibody 11M14.

[0290] SEQ ID NO: 215 shows the amino acid sequence of the human sortilin extracellular domain minus the signal peptide.

[0291] SEQ ID NO: 216 shows the amino acid sequence of hSORT1_ECD_Emut1.

[0292] SEQ ID NO: 217 shows the amino acid sequence of hSORT1_ECD_Emut2a.

[0293] SEQ ID NO: 218 shows the amino acid sequence of hSORT1_ECD_Emut2b.

[0294] SEQ ID NO: 219 shows the amino acid sequence of hSORT1_ECD_Emut3.

[0295] SEQ ID NO: 220 shows the amino acid sequence of hSORT1_ECD_Emut4.

[0296] SEQ ID NO: 221 shows the amino acid sequence of hSORT1_ECD_Emut5.

[0297] SEQ ID NO: 222 shows the amino acid sequence of hSORT1_ECD_Emut6N.

[0298] SEQ ID NO: 223 shows the amino acid sequence of hSORT1_ECD_Emut8N.

[0299] SEQ ID NO: 224 shows the amino acid sequence of hSORT1_ECD_Emut11N.

[0300] SEQ ID NO: 225 shows the amino acid sequence of hSORT1_ECD_Emut14N.

[0301] SEQ ID NO: 226 shows the amino acid sequence of hSORT1_ECD_Emut16.

[0302] SEQ ID NO: 227 shows the amino acid sequence of hSORT1_ECD_Emut17.

[0303] SEQ ID NO: 228 shows the amino acid sequence of hSORT1_ECD_Emut18.

[0304] SEQ ID NO: 229 shows the amino acid sequence of hSORT1_ECD_Emut19.

[0305] SEQ ID NO: 230 shows the amino acid sequence of hSORT1_ECD_Emut20.

[0306] SEQ ID NO: 231 shows the amino acid sequence of hSORT1_ECD_Emut21.

[0307] SEQ ID NO: 232 shows the amino acid sequence of hSORT1_ECD_Emut22.

[0308] SEQ ID NO: 233 shows the amino acid sequence of hSORT1_ECD_Emut23.

[0309] SEQ ID NO: 234 shows the amino acid sequence of hSORT1_ECD_Emut24.

[0310] SEQ ID NO: 235 shows the amino acid sequence of hSORT1_ECD_Emut25.

[0311] SEQ ID NO: 236 shows the amino acid sequence of hSORT1_ECD_Emut26.

[0312] SEQ ID NO: 237 shows the amino acid sequence of hSORT1_ECD_Emut27.

[0313] SEQ ID NO: 238 shows the amino acid sequence of hSORT1_ECD_Emut28.

[0314] SEQ ID NO: 239 shows the amino acid sequence of hSORT1_ECD_Emut29.

[0315] SEQ ID NO: 240 shows the amino acid sequence of hSORT1_ECD_Emut30.

[0316] SEQ ID NO: 241 shows the amino acid sequence of hSORT1_ECD_Emut31.

[0317] SEQ ID NO: 242 shows the amino acid sequence of hSORT1_ECD_Emut32.

[0318] SEQ ID NO: 243 shows the amino acid sequence of hSORT1_ECD_Emut33.

[0319] SEQ ID NO: 244 shows the amino acid sequence of hu11M14_H1b_IgG1 LALA_YTE-heavy chain.

[0320] SEQ ID NO: 245 shows the amino acid sequence of the hu11M14_L3 b-light chain.

[0321] SEQ ID NO: 246 shows the amino acid sequence of the hu8H24_H1_IgG1 LALA_YTE-heavy chain.

[0322] SEQ ID NO: 247 shows the amino acid sequence of the hu8H24_L2_light chain.

[0323] SEQ ID NO: 248 shows the amino acid sequence of the hu5E20_H7_IgG1 LALA_YTE-heavy chain.

[0324] SEQ ID NO: 249 shows the amino acid sequence of the hu5E20_L4_light chain.

[0325] SEQ ID NO: 250 shows the amino acid sequence of the hu11M14_H1b_IgG1 LALA_-heavy chain.

[0326] SEQ ID NO: 251 shows the amino acid sequence of the hu8H24_H1_IgG1 LALA-heavy chain.

[0327] SEQ ID NO: 252 shows the amino acid sequence of the hu5E20_H7_IgG1 LALA_heavy chain.

[0328] definition Monoclonal antibodies or other biological entities are typically provided in isolated form. This means that the antibody or other biological entity is typically at least 50% w / w pure from interfering proteins and other impurities resulting from its production or purification, but does not exclude the possibility that the monoclonal antibody may be combined with an excess of pharmaceutically acceptable carriers or other excipients intended to facilitate its use. Monoclonal antibodies may be at least 60%, 70%, 80%, 90%, 95%, or 99% w / w pure from interfering proteins and impurities resulting from its production or purification. In many cases, an isolated monoclonal antibody or other biological entity is the predominant macromolecular species remaining after its purification.

[0329] Specific binding of an antibody to its target antigen is at least 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12M -1 Specific binding refers to the affinity and / or avidity of an antibody to at least one target. Specific binding is detectably greater than or distinguishable from nonspecific binding that occurs to at least one unrelated target. Specific binding can be the result of bond formation between specific functional groups or a specific spatial fit (e.g., lock and key type), whereas nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody binds to only one target.

[0330] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain contains a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. This variable region is first expressed linked to a cleavable signal peptide. A variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region refers to a light chain variable region without the light chain signal peptide. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0331] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with heavy chains also including a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology, Paul, W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7 (incorporated by reference in its entirety for all purposes).

[0332] An immunoglobulin light or heavy chain variable region (also referred to herein as a "light chain variable domain" ("VL domain") or a "heavy chain variable domain" ("VH domain"), respectively) consists of a "framework" region interrupted by three "complementarity-determining regions" or "CDRs." The framework regions serve to align the CDRs for specific binding to an epitope of an antigen. The CDRs contain the amino acid residues of an antibody primarily responsible for antigen binding. From the amino to carboxyl terminus, both VL and VH domains comprise the following framework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDR1, 2, and 3 of the VL domain are also referred to herein as CDR-L1, CDR-L2, and CDR-L3, respectively. CDR1, 2, and 3 of the VH domain are also referred to herein as CDR-H1, CDR-H2, and CDR-H3, respectively. When the present application discloses a VL sequence having R as the C-terminal residue, R can alternatively be considered to be the N-terminal residue of the light chain constant region, and therefore the present application should also be understood as disclosing a VL sequence without a C-terminal R.

[0333] The assignment of amino acids to each VL and VH domain follows any conventional definition of CDR, including the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991)), the Chothia definition (Chothia & Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989), the Chothia-Kabat CDR combination, in which CDR-H1 is the Chothia and Kabat CDR combination; The AbM definition used by Molecular's antibody modeling software; and the contact definition (see Table 1) of Martin et al. (bioinfo.org.uk / abs). Kabat provides a widely used numbering scheme (Kabat numbering) that assigns the same number to corresponding residues between different heavy chains or different light chains. When an antibody is said to contain a CDR by a particular definition of CDR (e.g., Kabat), that definition specifies the minimum number of CDR residues (i.e., Kabat CDRs) present in the antibody. This does not exclude the presence of other residues within other conventional CDR definitions but outside the specified definition. For example, antibodies containing CDRs as defined by Kabat include, among other possibilities, antibodies in which the CDR contains Kabat CDR residues but no other CDR residues, and antibodies in which the CDR H1 is a composite Chothia-Kabat CDR H1 and the other CDRs contain Kabat CDR residues but no additional CDR residues based on other definitions. [Table 1] *The Chothia CDR-H1 can end at H32, H33, or H34 (depending on the length of the loop). This is because the Kabat numbering scheme places the insertion of extra residues at 35A and 35B, while Chothia numbering places them at 31A and 31B. If neither H35A nor H35B is present (Kabat numbering), the Chothia CDR-H1 loop ends at H32. If only H35A is present, it ends at H33. If both H35A and H35B are present, it ends at H34.

[0334] The term "antibody" includes intact antibodies and their binding fragments. Typically, fragments compete with the intact antibody from which they are derived for specific binding to a target, including separate heavy and light chains, Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques or by enzymatic or chemical separation of intact immunoglobulins. The term "antibody" also includes bispecific and / or humanized antibodies. Bispecific or bifunctional antibodies are artificial hybrid antibodies having two different heavy / light chain pairs and two different binding sites (e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). In some bispecific antibodies, the two different heavy / light chain pairs include a humanized 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2 heavy / light chain pair and a heavy / light chain pair specific for a different epitope on sortilin than that bound by 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2.

[0335] In some bispecific antibodies, one heavy chain / light chain pair is a humanized 5E20 antibody, a humanized 8H24 antibody, a humanized 11M14 antibody, a humanized 5M13 antibody, a humanized 2F18 antibody, a humanized 2P22 antibody, a humanized 6B15 antibody, a humanized 2C14 antibody, a humanized 9N18 antibody, or a humanized 4N2 antibody, as further disclosed below, and the other heavy chain / light chain pair is derived from an antibody that binds to a receptor expressed on the blood-brain barrier, such as the insulin receptor, insulin-like growth factor (IGF) receptor, leptin receptor, or lipoprotein receptor, or the transferrin receptor (Friden et al., Proc. Natl. Acad. Sci. USA 88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such bispecific antibodies can be transferred across the blood-brain barrier by receptor-mediated transcytosis. Brain uptake of bispecific antibodies can be further enhanced by engineering the bispecific antibody to reduce its affinity for blood-brain barrier receptors. Reduced affinity for receptors results in broader distribution in the brain (see, for example, Atwal et al., Sci.Trans.Med.3, 84ra43, 2011; Yu et al., Sci.Trans.Med.3, 84ra44, 2011).

[0336] Exemplary bispecific antibodies can also be: (1) dual variable domain antibodies (DVD-Igs), in which each light and heavy chain contains two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) Tandabs, which are fusions of two single-chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each of the target antigens; (3) flexibodies, which are combinations of scFvs and diabodies resulting in multivalent molecules; (4) so-called "dock and lock" molecules based on the "dimerization docking domain" of protein kinase A, which, when applied to Fabs, can result in trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; or (5) so-called Scorpion molecules, which, for example, contain two scFvs fused to either end of a human Fc region. Examples of useful platforms for preparing bispecific antibodies include BiTE (Micromet), DART (MacroGenics), Fcab and Mab2 (F-star), Fc-engineered IgG1 (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

[0337] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed from contiguous amino acids or from noncontiguous amino acids juxtaposed by one or more tertiary foldings of a protein. Epitopes formed from adjacent amino acids (also known as linear epitopes) typically are retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, "Epitope Mapping Protocols," in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0338] Antibodies that recognize the same or overlapping epitopes can be identified in simple immunoassays that demonstrate the ability of one antibody to compete with the binding of another antibody to a target antigen. Antibody epitopes can also be defined by X-ray crystallography of the antibody bound to its antigen to identify contact residues. Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other.

[0339] Competition between antibodies is determined by an assay in which the antibody under test inhibits the specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). A test antibody competes with a reference antibody if an excess of the test antibody (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) inhibits binding of the reference antibody by at least 50%, as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by competitive assays (competing antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes sufficiently close to the epitope bound by the reference antibody so that steric hindrance occurs.

[0340] The term "pharmaceutically acceptable" means the carrier, diluent, excipient or adjuvant must be compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

[0341] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0342] An individual is at high risk for a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) and individuals with the risk factor have a statistically significantly higher risk of developing the disease than individuals without the risk factor.

[0343] The term "biological sample" refers to a sample of biological material obtainable from or within a biological source, such as a human or mammalian subject. Such samples can be organs, organelles, tissues, tissue sections, body fluids, peripheral blood, plasma, serum, cells, molecules such as proteins and peptides, and any portion or combination thereof. The term biological sample can also encompass any material derived by processing the sample. Derived materials can include cells or their progeny. Processing of a biological sample can include one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like.

[0344] The term "control sample" refers to a biological sample that is not known or suspected to contain tissue affected by a progranulin-related disease, or at least not known or suspected to contain a given type of diseased tissue. The control sample can be obtained from an individual not affected by a progranulin-related disease. Alternatively, the control sample can be obtained from a patient affected by a progranulin-related disease. Such a sample can be obtained simultaneously with the biological sample suspected of containing a progranulin-related disease, or on a different occasion. Both the biological sample and the control sample can be obtained from the same tissue. Preferably, the control sample consists of essentially or entirely normal healthy tissue and can be used for comparison with the biological sample suspected of containing a region affected by a progranulin-related disease. Preferably, the tissue in the control sample is of the same type as the tissue in the biological sample. Preferably, the cells suspected of being affected by a progranulin-related disease in the biological sample originate from the same cell type (e.g., neurons or glia) as the type of cells in the control sample.

[0345] The term "disease" refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, disorder, pathology, ailment, condition, or symptom in which physiological function is impaired, regardless of the nature of the etiology.

[0346] The term "symptom" refers to subjective evidence of disease, such as a change in gait, as perceived by the subject. "Sign" refers to objective evidence of disease as observed by a physician.

[0347] The term "positive response to treatment" refers to a more favorable response in an individual patient or the average response in a population of patients compared to the average response in a control population not receiving treatment.

[0348] For the purposes of classifying amino acid substitutions as conservative or non-conservative, amino acids are grouped as follows: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for another.

[0349] The percentage of sequence identity is determined by using antibody sequences that are maximally aligned according to the Kabat numbering convention. After alignment, when a subject antibody region (for example, the entire mature variable region of a heavy or light chain) is compared with the same region of a reference antibody, the percentage of sequence identity between the subject and reference antibody regions is calculated by dividing the number of positions occupied by the same amino acid in both the subject and reference antibody regions by the total number of aligned positions in the two regions, not counting gaps, and multiplying this by 100 to convert it into a percentage.

[0350] A composition or method that "comprises" or "comprises" one or more listed elements may include other elements not specifically listed. For example, a composition that "comprises" or "comprises" an antibody may contain the antibody alone or in combination with other components.

[0351] Specifying a range of values ​​includes all integers in the range, or all integers that define the range, and all subranges defined by integers in the range.

[0352] Unless otherwise clear from the context, the term "about" encompasses insubstantial variations, such as values ​​within the standard error of measurement (eg, SEM) of the stated value.

[0353] Statistical significance means p≦0.05.

[0354] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof. Detailed Description I. General

[0355] The present invention provides antibodies that bind to sortilin.

[0356] Exemplary antibodies of the invention are 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2. Some antibodies of the invention are useful for treating, inhibiting, or slowing the progression of diseases or disorders associated with altered levels of Progranulin. Although an understanding of the mechanism is not necessary to the practice of the invention, elevated extracellular Progranulin levels may occur as a result of antibody-binding Sortilin, among other mechanisms. The antibodies of the invention or agents that induce such antibodies can be used in methods for treating or preventing frontotemporal dementia, as well as other diseases and disorders associated with altered progranulin levels, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease (a type of neuronal ceroid lipofuscinosis (NCL)), neurodegenerative disorders, and neurodegenerative disorders associated with aging. Age-related neurodegenerative disorders tend to progress irreversibly and are typically associated with one or more biological hallmarks of aging: genomic instability, telomere shortening, epigenetic changes, loss of proteostasis, mitochondrial dysfunction, cellular senescence, nutrient sensing dysregulation, stem cell depletion, and altered intercellular communication (see, e.g., Hou, Y et al., 2019, Nature Reviews Neurology 15, pages 565-581). II.Target molecule

[0357] Unless otherwise clear from the context, reference to Sortilin refers to the native human form of Sortilin, including all isoforms, including soluble forms, regardless of the presence of post-translational modifications (e.g., phosphorylation, glycosylation, or acetylation). The amino acid sequence of the extracellular domain of Sortilin is shown below. The 33 amino acid signal peptide is shown in bold. [ka]

[0358] The amino acid sequence of the extracellular domain of sortilin, not including the 33 amino acid signal peptide, is shown below. [ka]

[0359] References to sortilin include known spontaneous mutations and their permutations as listed in the Swiss-Prot database, as well as mutations associated with disease states.

[0360] Furthermore, reference to Sortilin includes Sortilin with known post-translational modifications. Examples of known post-translational modifications are listed in the UniProtKB / Swiss-Prot database.

[0361] Unless otherwise clear from the context, reference to sortilin or a fragment thereof includes the naturally occurring human amino acid sequence, including isoforms, mutants and allelic variants thereof. III. Antibodies A. Binding Specificity and Functional Properties

[0362] The present invention provides antibodies that specifically bind to Sortilin. The Examples describe the isolation of eight murine monoclonal antibodies against human Sortilin. The epitope specificity of three of these antibodies has been mapped.

[0363] From the primary screen, the epitope specificity of antibody 5E20 was mapped to be within about residues 555-561 (FTESFLT, SEQ ID NO: 202) of the human sortilin ECD of SEQ ID NO: 215. Further screening mapped the epitope to residues E557, S558, F559, L560 P510, and Y535 of the sortilin ECD of SEQ ID NO: 215.

[0364] From the primary screen, the epitope specificity of antibody 8H24 was mapped to be within about residues 134-143 (RTEFGMAIGP, SEQ ID NO: 213) of the human sortilin ECD of SEQ ID NO: 215. Further screening mapped the epitope to residues D74, R76, F97, K110, Y535, L560, and E557 of the sortilin ECD of SEQ ID NO: 215.

[0365] From the primary screen, the epitope specificity of antibody 11M14 was mapped to be within about residues 553-562 of the human sortilin ECD of SEQ ID NO: 215. Further screening mapped the epitope to residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO: 215.

[0366] Some antibodies specifically bind to an epitope within E(S / Q / D)FL (SEQ ID NO: 206). Some antibodies of the invention specifically bind to a peptide comprising or consisting of residues 588-594 of the sortilin ECD of SEQ ID NO: 1 (corresponding to residues 555-561 of the sortilin ECD of SEQ ID NO: 215), i.e., residues FTESFLT (SEQ ID NO: 202). Some antibodies of the invention specifically bind to a peptide comprising or consisting of residues 590-593 of the sortilin ECD of SEQ ID NO: 1 (corresponding to residues 557-560 of the sortilin ECD of SEQ ID NO: 215), i.e., ESFL (SEQ ID NO: 203). Some antibodies of the invention specifically bind to a peptide comprising or consisting of residues 632-643 of the sortilin ECD of SEQ ID NO: 1 (corresponding to residues 599-610 of SEQ ID NO: 215), i.e., DGCILGYKEQFL) (SEQ ID NO: 204). Some antibodies of the invention specifically bind to a peptide comprising or consisting of residues 663-674 of the sortilin ECD of SEQ ID NO: 1 (corresponding to residues 630-641 of the sortilin ECD of SEQ ID NO: 15), i.e., residues PSICLCSLEDFL (SEQ ID NO: 205). Some antibodies of the invention specifically bind to a peptide comprising or consisting of the consensus motif E(S / Q / D)FL (SEQ ID NO: 206).

[0367] Some antibodies bind within the amino acid sequence HYYTILDSGGIIVAIEHSSRPINVIKFSTDEGQCWQTYTFTRDPIYFTGLASEPGARSMNISIWGFTESFLTSQWVSYTIDFKDILER (SEQ ID NO: 210) (corresponding to residues 523-610 of the sortilin ECD of SEQ ID NO: 1). Some antibodies specifically bind peptides within the preceding sequence. Peptides within this sequence include an amino acid sequence comprising or consisting of TGL, FTESFLTSQW (SEQ ID NO: 211), or LTSQW (SEQ ID NO: 212).

[0368] Some antibodies specifically bind to a peptide comprising or consisting of the amino acid sequence RTEFGMAIGP (SEQ ID NO:213, corresponding to residues 167-176 of the sortilin ECD of SEQ ID NO:1, corresponding to residues 134-143 of the sortilin ECD of SEQ ID NO:215). Some antibodies specifically bind to a peptide comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO:214, corresponding to residues 586-595 of the sortilin ECD of SEQ ID NO:1, corresponding to residues 553-562 of the sortilin ECD of SEQ ID NO:215). Some antibodies specifically bind to residues D74, R76, F97, K110, Y535, L560, and E557 of the sortilin ECD of SEQ ID NO:215. Some antibodies specifically bind to residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO: 215. Some antibodies specifically bind to residues E557, S558, F559, L560, P510 and Y535 of the sortilin ECD of SEQ ID NO: 215. That is, binding of the antibody to the sortilin ECD can be reduced by mutation of any of the residues designated to form the epitope.

[0369] These antibodies can be obtained by immunization with a sortilin polypeptide purified from a natural source or recombinantly expressed. The present invention also provides antibodies that bind to the same epitope as any of the aforementioned antibodies, such as the epitope of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. Also included are antibodies that compete with any of the aforementioned antibodies for binding to sortilin, e.g., compete with 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. In one embodiment, an antibody that binds to the same epitope as a reference antibody such as 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2, or competes with the reference antibody, shares one or more of its functional properties, such as the ability to inhibit Progranulin internalization into cells. Optionally, such properties are possessed to the same extent or to a greater extent than those of the reference antibody within experimental error. Some antibodies that specifically bind to Sortilin increase Progranulin levels without inhibiting the binding of other SORT1 ligands, such as neurotensin, to Sortilin. Some antibodies that specifically bind to Sortilin do so without inducing Sortilin internalization.

[0370] The above antibodies can be generated de novo by immunizing with a sortilin peptide comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), comprising or consisting of the amino acid sequence ESFL (SEQ ID NO: 203), comprising or consisting of the amino acid sequence DGCILGYKEQFL) (SEQ ID NO: 204), comprising or consisting of the amino acid sequence PSICLCSLEDFL (SEQ ID NO: 205), comprising or consisting of the amino acid sequence E(S / Q / D)FL (SEQ ID NO: 206), comprising or consisting of the amino acid sequence RTEFGMAIGP) (SEQ ID NO: 213), comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO: 214), or by immunizing with a full-length sortilin ECD polypeptide or a fragment thereof containing such residues and screening for specific binding to a peptide containing such residues. Antibodies to conformational epitopes, such as epitopes comprising or consisting of residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559, and L560 of the Sortilin ECD of SEQ ID NO: 215, comprising or consisting of residues E557, S558, F559, L560, P510, and Y535 of the Sortilin ECD of SEQ ID NO: 215, can be generated by immunization with a full-length ECD or a fragment thereof comprising the residues of the epitope. Such sortilin peptides are preferably linked to a heterologous conjugate molecule that serves to elicit an antibody response against the peptide. Linkage may be direct or via a spacer peptide or amino acid. Cysteine ​​is used as a spacer amino acid because its free SH group facilitates attachment of a carrier molecule. Polyglycine linkers (e.g., 2-6 glycines) with or without cysteine ​​residues between the glycine and the peptide can also be used. The carrier molecule is used to provide a T cell epitope that helps elicit an antibody response to the peptide.Several carriers are commonly used, notably keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin (BSA). Peptide spacers can be added to peptide immunogens as part of solid-phase peptide synthesis. Carriers are typically added by chemical crosslinking. Some examples of chemical cross-linking agents that can be used include cross-N-maleimido-6-aminocaproyl ester or m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see, e.g., Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1988; Sinigaglia et al., Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47 (1993); Hammer et al., Cell, 74:197-203 (1993); Falk K. et al., Immunogenetics, 39:230-242 (1994); WO 98 / 23635; and Southwood et al., J. Immunology, 160:3363-3373 (1998)). If present, the carrier and spacer may be attached to either end of the immunogen.

[0371] Peptides with optional spacers and carriers can be used to immunize laboratory animals or B cells, as described in more detail below. Hybridoma supernatants can be tested for the ability to bind to sortilin peptides comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), comprising or consisting of the amino acid sequence ESFL (SEQ ID NO: 203), comprising or consisting of the amino acid sequence DGCILGYKEQFL) (SEQ ID NO: 204), comprising or consisting of the amino acid sequence PSICLCSLEDFL (SEQ ID NO: 205), comprising or consisting of the amino acid sequence E(S / Q / D)FL (SEQ ID NO: 206), comprising or consisting of the amino acid sequence RTEFGMAIGP (SEQ ID NO: 213), or comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO: 214). For conformational epitopes, antibodies that bind to the extracellular domain of sortilin can be screened for competition with a reference antibody, and / or it can be determined that binding is reduced by mutation of some or all residues within the epitope. When several residues of a conformational epitope are clustered within the length of a typical linear epitope (e.g., up to about 15 residues), peptides comprising or consisting of these residues can be used to elicit antibodies. Thus, for example, for an epitope defined by residues D74, R76, F97, K110, Y535, L560, and E557 of the Sortilin ECD of SEQ ID NO: 215, antibodies can be elicited using peptides comprising or consisting of residues 557-560 of the Sortilin ECD of SEQ ID NO: 215. For an epitope defined by residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559, and L560 of the Sortilin ECD of SEQ ID NO: 215, antibodies can be elicited using peptides comprising or consisting of residues 557-561 of the Sortilin ECD of SEQ ID NO: 215.For the epitope defined by residues E557, S558, F559, L560, P510, and Y535 of the sortilin ECD of SEQ ID NO: 215, antibodies can be elicited using a peptide comprising or consisting of residues 557-560 of the sortilin ECD of SEQ ID NO: 215. The peptide can be conjugated to a carrier or other tag to facilitate screening assays. In this case, the carrier or tag is preferentially different from the combination of spacer and carrier molecule used for immunization to eliminate antibodies specific for the spacer or carrier rather than the sortilin peptide.

[0372] The antibody designated 5E20 is an exemplary antibody that specifically binds to sortilin. 5E20 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 4 and SEQ ID NO: 10, respectively. Unless otherwise clear from the context, reference to 5E20 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. The antibody has been deposited under [deposit number]. This antibody specifically binds to a peptide comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), comprising or consisting of the amino acid sequence ESFL (SEQ ID NO: 203), comprising or consisting of the amino acid sequence DGCILGYKEQFL (SEQ ID NO: 204), comprising or consisting of the amino acid sequence PSICLCSLEDFL (SEQ ID NO: 205), or comprising or consisting of the amino acid sequence E(S / Q / D)FL (SEQ ID NO: 206), or specifically binds to residues E557, S558, F559, L560, and P510 and Y535 of the sortilin ECD of SEQ ID NO: 215. The Kabat / Chothia combination CDRs of the 5E20 heavy chain are set forth as SEQ ID NOs: 5-7, respectively, and the Kabat-Chothia combination CDRs of the 5E20 light chain are set forth as SEQ ID NOs: 11-13, respectively. An exemplary signal peptide sequence for the murine 5E20 variable heavy chain is SEQ ID NO: 3. An exemplary signal peptide sequence for the mouse 5E20 variable light chain is SEQ ID NO:9.

[0373] The antibody designated 8H24 is another exemplary antibody that specifically binds to sortilin. 8H24 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO:28 and SEQ ID NO:34, respectively. Unless otherwise clear from the context, reference to 8H24 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 8H24 has been deposited under [deposit number]. This antibody specifically binds to a peptide comprising or consisting of the amino acid sequence RTEFGMAIGP (SEQ ID NO:213), or specifically binds to residues D74, R76, F97, K110, Y535, L560, and E557 of the sortilin ECD of SEQ ID NO:215. The Kabat / Chothia combination CDRs of the heavy chain of 8H24 are set forth as SEQ ID NOs:29-31, respectively, and the Kabat / Chothia combination CDRs of the light chain of 8H24 are set forth as SEQ ID NOs:35-37, respectively. An exemplary signal peptide sequence for the mouse 8H24 variable heavy chain is SEQ ID NO: 27. An exemplary signal peptide sequence for the mouse 8H24 variable light chain is SEQ ID NO: 33.

[0374] The antibody designated 11M14 is another exemplary antibody that specifically binds to sortilin. 11M14 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO:52 and SEQ ID NO:58, respectively. Unless otherwise clear from the context, reference to 11M14 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 11M14 has been deposited under [deposit number]. This antibody specifically binds to a peptide comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO:214), or specifically binds to residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559, and L560 of the sortilin ECD of SEQ ID NO:215. The Kabat / Chothia combined CDRs of the 11M14 heavy chain are set forth as SEQ ID NOs: 53-55, respectively, and the Kabat / Chothia combined CDRs of the 11M14 light chain are set forth as SEQ ID NOs: 59-61, respectively. An exemplary signal peptide sequence for the murine 11M14 variable heavy chain is SEQ ID NO: 51. An exemplary signal peptide sequence for the murine 11M14 variable light chain is SEQ ID NO: 57.

[0375] The antibody designated 5M13 is another exemplary antibody that specifically binds to sortilin. 5M13 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO:78 and SEQ ID NO:84, respectively. Unless otherwise clear from the context, reference to 5M13 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 5M13 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 5M13 heavy chain are set forth as SEQ ID NOs:79-81, respectively, and the Kabat / Chothia combination CDRs of the 5M13 light chain are set forth as SEQ ID NOs:85-87, respectively. An exemplary signal peptide sequence for the murine 5M13 variable heavy chain is SEQ ID NO:77. An exemplary signal peptide sequence for the murine 5M13 variable light chain is SEQ ID NO:83.

[0376] The antibody designated 2F18 is another exemplary antibody that specifically binds to sortilin. 2F18 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO:90 and SEQ ID NO:96, respectively. Unless otherwise clear from the context, reference to 2F18 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 2F18 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 2F18 heavy chain are set forth as SEQ ID NOs:91-93, respectively, and the Kabat / Chothia combination CDRs of the 2F18 light chain are set forth as SEQ ID NOs:97-99, respectively. An exemplary signal peptide sequence for the murine 2F18 variable heavy chain is SEQ ID NO:89. An exemplary signal peptide sequence for the murine 2F18 variable light chain is SEQ ID NO:95.

[0377] The antibody designated 2P22 is another exemplary antibody that specifically binds to sortilin. 2P22 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 102 and SEQ ID NO: 108, respectively. Unless otherwise clear from the context, reference to 2P22 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 2P22 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 2P22 heavy chain are set forth as SEQ ID NOs: 103-105, respectively, and the Kabat / Chothia combination CDRs of the 2P22 light chain are set forth as SEQ ID NOs: 109-111, respectively. An exemplary signal peptide sequence for the murine 2P22 variable heavy chain is SEQ ID NO: 101. An exemplary signal peptide sequence for the murine 2P22 variable light chain is SEQ ID NO: 107.

[0378] The antibody designated 6B15 is another exemplary antibody that specifically binds to sortilin. 6B15 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 114 and SEQ ID NO: 120, respectively. Unless otherwise clear from the context, reference to 6B15 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 6B15 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 6B15 heavy chain are set forth as SEQ ID NOs: 115-117, respectively, and the Kabat / Chothia combination CDRs of the 6B15 light chain are set forth as SEQ ID NOs: 121-123, respectively. An exemplary signal peptide sequence for the murine 6B15 variable heavy chain is SEQ ID NO: 113. An exemplary signal peptide sequence for the murine 6B15 variable light chain is SEQ ID NO: 119.

[0379] The antibody designated 2C14 is another exemplary antibody that specifically binds to sortilin. 2C14 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 126 and SEQ ID NO: 132, respectively. Unless otherwise clear from the context, reference to 2C14 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 2C14 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 2C14 heavy chain are designated SEQ ID NOs: 127-129, respectively, and the Kabat / Chothia combination CDRs of the 2C14 light chain are designated SEQ ID NOs: 133-135, respectively. An exemplary signal peptide sequence for the murine 2C14 variable heavy chain is SEQ ID NO: 125. An exemplary signal peptide sequence for the murine 2C14 variable light chain is SEQ ID NO: 131.

[0380] The antibody designated 9N18 is another exemplary antibody that specifically binds to sortilin. 9N18 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 138 and SEQ ID NO: 144, respectively. Unless otherwise clear from the context, reference to 9N18 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 9N18 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 9N18 heavy chain are set forth as SEQ ID NOs: 139-141, respectively, and the Kabat / Chothia combination CDRs of the 9N18 light chain are set forth as SEQ ID NOs: 145-147, respectively. An exemplary signal peptide sequence for the murine 9N18 variable heavy chain is SEQ ID NO: 137. An exemplary signal peptide sequence for the murine 9N18 variable light chain is SEQ ID NO: 143.

[0381] The antibody designated 4N2 is another exemplary antibody that specifically binds to sortilin. 4N2 has mature variable heavy and light chain regions (after cleavage of the signal peptide) characterized by SEQ ID NO: 150 and SEQ ID NO: 156, respectively. Unless otherwise clear from the context, reference to 4N2 should be understood to refer to any of the murine, chimeric, veneered, and humanized forms of this antibody. 4N2 has been deposited under [deposit number]. The Kabat / Chothia combination CDRs of the 4N2 heavy chain are set forth as SEQ ID NOs: 151-153, respectively, and the Kabat / Chothia combination CDRs of the 4N2 light chain are set forth as SEQ ID NOs: 157-159, respectively. An exemplary signal peptide sequence for the murine 4N2 variable heavy chain is SEQ ID NO: 149. An exemplary signal peptide sequence for the murine 4N2 variable light chain is SEQ ID NO: 155.

[0382] Some antibodies of the invention bind to the same or overlapping epitope as the antibodies designated 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. Other antibodies with such binding specificity can be produced by immunizing mice with a desired epitope (e.g., sortilin or a portion thereof comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), comprising or consisting of the amino acid sequence ESFL (SEQ ID NO: 203), comprising or consisting of the amino acid sequence DGCILGYKEQFL (SEQ ID NO: 204), comprising or consisting of the amino acid sequence PSICLCSLEDFL (SEQ ID NO: 205), or comprising or consisting of the amino acid sequence E(S / Q / D)FL (SEQ ID NO: 206), comprising or consisting of the amino acid sequence RTEFGMAIGP) (SEQ ID NO: 213), or comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO: 214). A full-length ECD or a fragment thereof comprising the residues of the peptide can be used to elicit antibodies that bind to a conformational epitope comprising or consisting of residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the Sortilin ECD of SEQ ID NO:215, a conformational epitope comprising or consisting of residues D74, R76, F97, K110, Y535, L560 and E557 of the Sortilin ECD of SEQ ID NO:215, or an epitope comprising or consisting of residues E557, S558, F559, L560, P510 and Y535 of the Sortilin ECD of SEQ ID NO:215. The resulting antibodies can be screened for binding to Sortilin in competition with antibodies having the variable regions of murine 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2, if desired. Fragments of Sortilin containing a desired epitope can be linked to a carrier useful for eliciting an antibody response against the fragment and / or combined with an adjuvant useful for eliciting such a response.Such antibodies can be screened for differential binding to sortilin or its fragments compared to mutants of specific residues. Screening against such mutants more precisely defines binding specificity, allowing for the identification of antibodies whose binding is inhibited by mutagenesis of specific residues and likely share the functional properties of other exemplified antibodies. Mutations can be systematic substitutions of alanine (or serine, if alanine is already present) one residue at a time, or at more widely spaced intervals, throughout the entire target or portion of it where the epitope is known to reside. If the same set of mutations significantly reduces binding of the two antibodies, then the two antibodies bind to the same epitope.

[0383] Antibodies having the binding specificity of a selected murine antibody (e.g., 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2) can also be produced using a modification of the phage display method. See Winter, WO 92 / 20791. This method is particularly suitable for producing human antibodies. In this method, either the heavy or light chain variable region of a selected murine antibody is used as the starting material. For example, if a light chain variable region is selected as the starting material, a phage library is constructed in which members display the same light chain variable region (i.e., the murine starting material) and a different heavy chain variable region. The heavy chain variable region can be obtained, for example, from a library of rearranged human heavy chain variable regions. Sortilin or a fragment thereof (e.g., at least 10 8 , preferably at least 10 9 M -1) are selected. The heavy chain variable region from this phage is then used as starting material for constructing a further phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region can be obtained, for example, from a library of rearranged human variable light chain regions. Again, phage showing strong specific binding to sortilin or a fragment thereof are selected. The resulting antibodies usually have the same or similar epitope specificity as the murine starting material.

[0384] The Kabat / Chothia combined CDRs of the heavy chain of 5E20 are shown as SEQ ID NOs: 5 to 7, respectively, and the Kabat / Chothia combined CDRs of the light chain of 5E20 are shown as SEQ ID NOs: 11 to 13, respectively.

[0385] Table 2 shows the 5E20 CDRs defined by Kabat, Chothia, the combination of Chothia and Kabat (also referred to herein as the "Kabat / Chothia combination"), AbM, and Contact. [Table 2]

[0386] The combined Kabat / Chothia CDRs of the heavy chain of 8H24 are shown as SEQ ID NOs: 29 to 31, respectively, and the combined Kabat / Chothia CDRs of the light chain of 8H24 are shown as SEQ ID NOs: 35 to 37, respectively.

[0387] Table 3 shows the 8H24 CDRs defined by Kabat, Chothia, the combination of Chothia and Kabat (also referred to herein as the "Kabat / Chothia combination"), AbM, and Contact. [Table 3]

[0388] The Kabat / Chothia combined CDRs of the heavy chain of 11M14 are shown as SEQ ID NOs: 53 to 55, respectively, and the Kabat / Chothia combined CDRs of the light chain of 11M14 are shown as SEQ ID NOs: 59 to 61, respectively.

[0389] Table 4 shows the 11M14 CDRs defined by Kabat, Chothia, the combination of Chothia and Kabat (also referred to herein as the "Kabat / Chothia combination"), AbM, and Contact. [Table 4]

[0390] The Kabat / Chothia combined CDRs of the heavy chain of 5M13 are shown as SEQ ID NOs: 79 to 81, respectively, and the Kabat / Chothia combined CDRs of the light chain of 5M13 are shown as SEQ ID NOs: 85 to 87, respectively.

[0391] The Kabat / Chothia combined CDRs of the heavy chain of 2F18 are shown as SEQ ID NOs: 91 to 93, respectively, and the Kabat / Chothia combined CDRs of the light chain of 2F18 are shown as SEQ ID NOs: 97 to 99, respectively.

[0392] The combined Kabat / Chothia CDRs of the heavy chain of 2P22 are shown as SEQ ID NOs: 103 to 105, respectively, and the combined Kabat / Chothia CDRs of the light chain of 2P22 are shown as SEQ ID NOs: 109 to 111, respectively.

[0393] The Kabat / Chothia combined CDRs of the heavy chain of 6B15 are shown as SEQ ID NOs: 115 to 117, respectively, and the Kabat / Chothia combined CDRs of the light chain of 6B15 are shown as SEQ ID NOs: 121 to 123, respectively.

[0394] The combined Kabat / Chothia CDRs of the heavy chain of 2C14 are designated SEQ ID NOs: 127-129, respectively, and the combined Kabat / Chothia CDRs of the light chain of 2C14 are designated SEQ ID NOs: 133-135, respectively.

[0395] The Kabat / Chothia combined CDRs of the heavy chain of 9N18 are shown as SEQ ID NOs: 139 to 141, respectively, and the Kabat / Chothia combined CDRs of the light chain of 9N18 are shown as SEQ ID NOs: 145 to 147, respectively.

[0396] The Kabat / Chothia combined CDRs of the heavy chain of 4N2 are shown as SEQ ID NOs: 151 to 153, respectively, and the Kabat / Chothia combined CDRs of the light chain of 4N2 are shown as SEQ ID NOs: 157 to 159, respectively.

[0397] Other antibodies, such as 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2, can be obtained by mutagenesis of the cDNA encoding the heavy and light chains of the exemplary antibodies. Also included in the present invention are monoclonal antibodies that are at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 in the mature heavy and / or light chain variable region, retain their functional properties, and / or differ from the respective antibodies by a small number of functionally discrete amino acid substitutions (e.g., conservative substitutions), deletions, or insertions. Also included are monoclonal antibodies having at least one or all six CDRs, as defined by any conventional definition, preferably by Kabat, that are 90%, 95%, 99% or 100% identical to the corresponding CDRs of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2.

[0398] The present invention also provides antibodies that have, completely or substantially, some or all (e.g., 3, 4, 5, and 6) CDRs from 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. Such antibodies may comprise a heavy chain variable region that has, completely or substantially, at least two, and usually all three, CDRs from the heavy chain variable region of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2, and / or a light chain variable region that has, completely or substantially, at least two, and usually all three, CDRs from the light chain variable region of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. The antibody may comprise both a heavy chain and a light chain. The CDRs are substantially derived from the corresponding 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 CDRs if they contain no more than 4, 3, 2, or 1 substitutions, insertions, or deletions, except that CDR-H2 (as defined by Kabat) may have no more than 6, 5, 4, 3, 2, or 1 substitutions, insertions, or deletions. Such antibodies can have at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity in the amino acid sequence of the mature heavy and / or light chain variable regions to 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2 and retain their functional properties, and / or can differ from 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 or 4N2 by small numbers of functionally discontinuous amino acid substitutions (e.g., conservative substitutions), deletions or insertions.

[0399] Some antibodies identified by such assays are capable of binding to sortilin.

[0400] The present invention further provides means for specifically binding to a peptide consisting of residues E(S / Q / D)FL (SEQ ID NO: 206), residues FTESFLT (SEQ ID NO: 202), residues ESFL (SEQ ID NO: 203), residues DGCILGYKEQFL (SEQ ID NO: 204) or residues PSICLCSLEDFL (SEQ ID NO: 205), or means for specifically binding to residues E557, S558, F559, L560, P510 and Y535 of the sortilin ECD of SEQ ID NO: 215. Exemplary means are antibodies comprising heavy chain CDRs of SEQ ID NOs: 5-7 and light chain CDRs of SEQ ID NOs: 11-13.

[0401] The present invention further provides means for specifically binding to a peptide consisting of residues RTEFGMAIGP (SEQ ID NO: 213), or means for specifically binding to residues D74, R76, F97, K110, Y535, L560 and E557 of the sortilin ECD of SEQ ID NO: 215. Exemplary means are antibodies comprising heavy chain CDRs of SEQ ID NOs: 29-31 and light chain CDRs of SEQ ID NOs: 35-37.

[0402] The present invention further provides means for specifically binding to a peptide consisting of residues WGFTESFLTS (SEQ ID NO: 214), or means for specifically binding to residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO: 215. Exemplary means are antibodies comprising heavy chain CDRs of SEQ ID NOs: 53-55 and light chain CDRs of SEQ ID NOs: 59-61. B. Non-human antibodies

[0403] The production of other non-human antibodies, e.g., mice, guinea pigs, primates, rabbits, or rats, against sortilin or a fragment thereof (e.g., a peptide comprising the amino acid sequence of FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), or PSICLCSLEDFL (SEQ ID NO: 205), E(S / Q / D)FL (SEQ ID NO: 206), RTEFGMAIGP) (SEQ ID NO: 213), or WGFTESFLTS (SEQ ID NO: 214)) can be achieved, for example, by immunizing the animal with sortilin or a fragment thereof. See Harlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988) (incorporated by reference for all purposes). Antibodies to conformational epitopes, such as epitopes comprising residues D74, R76, F97, K110, Y535, L560, and E557 of the Sortilin ECD of SEQ ID NO: 215, residues K110, Y535, E557, T561, and Q563, D74, P510, S558, F559, and L560 of the Sortilin ECD of SEQ ID NO: 215, or residues E557, S558, F559, L560, and P510, and Y535 of the Sortilin ECD of SEQ ID NO: 215, can be elicited using the full-length ECD or sufficient of the ECD to span the epitope residues. Optionally, the immunogen can be the extracellular domain (ECD) of recombinant human Sortilin with a C-terminal HIS tag (ECD-huSortilin-HIS). Optionally, animals are immunized with a sortilin fragment comprising a peptide represented by E(S / Q / D)FL (SEQ ID NO: 206) linked to a carrier. Optionally, the peptide is FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), RTEFGMAIGP (SEQ ID NO: 213), or WGFTESFLTS (SEQ ID NO: 214). Such immunogens can be obtained from natural sources, by peptide synthesis, or by recombinant expression. Optionally, the immunogen can be administered fused to a carrier protein or otherwise conjugated. Optionally, the immunogen can be administered with an adjuvant.As described below, several types of adjuvants can be used. Complete Freund's adjuvant followed by incomplete adjuvant can be used to immunize laboratory animals. Rabbits or guinea pigs are typically used to generate polyclonal antibodies. Mice are typically used to generate monoclonal antibodies. Antibodies can be directed to sortilin or epitopes within sortilin (e.g., FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), E(S / Q / D)FL (SEQ ID NO: 206), RTEFGMAIGP (SEQ ID NO: 213), WGFTESFLTS (SEQ ID NO: 214), or to residues D74, R76, F97, K110 of the sortilin ECD of SEQ ID NO: 215). , Y535, L560 and E557, or to the epitope defined by residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO:215, or to the epitope defined by residues E557, S558, F559, L560 and P510, and Y535 of SEQ ID NO:215. Optionally, screening can be performed against a 15 amino acid peptide containing the consensus motif represented by FTESFLT (SEQ ID NO:202), ESFL (SEQ ID NO:203), DGCILGYKEQFL (SEQ ID NO:204), PSICLCSLEDFL (SEQ ID NO:205), RTEFGMAIGP (SEQ ID NO:213), or WGFTESFLTS (SEQ ID NO:214), or E(S / Q / D)FL (SEQ ID NO:206). Optionally, the peptide comprises or consists of FTESFLT (SEQ ID NO:202), ESFL (SEQ ID NO:203), DGCILGYKEQFL (SEQ ID NO:204), PSICLCSLEDFL (SEQ ID NO:205), RTEFGMAIGP (SEQ ID NO:213), or WGFTESFLTS (SEQ ID NO:214).Such screening can be accomplished by determining the binding of an antibody to a collection of sortilin variants, such as the sortilin variants of SEQ ID NOs: 216-243, or sortilin variants comprising or consisting of amino acid residues 588-594, or 590-593, or 632-643, or 663-674, or 167-176, or 586-595 of SEQ ID NO: 1, or mutants within these residues, and determining which sortilin variants bind to the antibody. Binding can be assessed, for example, by Western blot, FACS, or ELISA. C. Humanized Antibodies

[0404] Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody have been grafted onto human "acceptor" antibody sequences (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequences can be, for example, mature human antibody sequences, a combination of such sequences, a consensus sequence of human antibody sequences, or germline region sequences. Thus, a humanized antibody is an antibody having at least three, four, five, or all of the CDRs, completely or substantially, derived from the donor antibody, and variable region framework sequences and constant regions, if present, completely or substantially, derived from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and heavy chain constant region substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs completely or substantially derived from a donor antibody light chain, and, if present, a light chain variable region framework sequence and light chain constant region substantially derived from human light chain variable region framework and constant region sequences. In addition to nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. CDRs in a humanized antibody are substantially derived from corresponding CDRs in a non-human antibody if at least 85%, 90%, 95%, or 100% of the corresponding residues (defined by any conventional definition, but preferably by Kabat) are identical between the respective CDRs. An antibody chain variable region framework sequence or an antibody chain constant region is substantially derived from a human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical.To be classified as humanized under the 2014 World Health Organization (WHO) International Nonproprietary Name (INN) definition for humanized antibodies, an antibody must have at least 85% identity to the human germline antibody sequence (i.e., before somatic hypermutation). A mixed antibody is one in which one antibody chain (e.g., the heavy chain) meets the threshold, but the other chain (e.g., the light chain) does not. If neither chain meets the threshold, the antibody is classified as chimeric, even if the variable framework regions of both chains are substantially human with some mouse back mutations. See Jones et al. (2016) The INN and out of antibody nonproprietary names, mAbs 8:1, 1-9, DOI: 10.1080 / 19420862.2015.1114320. See also "WHO-INN: International nonproprietary names (INN) for biological and biotechnological substances (a review)" (Internet) 2014. The WHO BioRev2014.pdf definition is available at http: / / www.who.int / medicines / services / inn / BioRev2014.pdf and is incorporated herein by reference. For the avoidance of doubt, the term "humanized" as used herein is not intended to be limited to the 2014 WHO INN definition of a humanized antibody. Some of the humanized antibodies provided herein have at least 85% sequence identity to human germline sequences, and some of the humanized antibodies provided herein have less than 85% sequence identity to human germline sequences. Some of the heavy chains of the humanized antibodies provided herein have about 60% to 100% sequence identity to human germline sequences, e.g., about 60% to 69%, 70% to 79%, 80% to 84%, or 85% to 89% sequence identity.Some heavy chains fall below the 2014 WHO INN definition, e.g., have about 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or 82%, 83%, or 84% sequence identity to human germline sequences, while other heavy chains meet the 2014 WHO INN definition, having about 85%, 86%, 87%, 88%, 89% or more sequence identity to human germline sequences. Some of the light chains of the humanized antibodies provided herein have about 60%-100% sequence identity to human germline sequences, e.g., sequence identity in the range of about 80%-84% or 85%-89%. Some light chains fall below the 2014 WHO INN definition, e.g., having about 81%, 82%, 83%, or 84% sequence identity to the human germline sequence, while other light chains meet the 2014 WHO INN definition, having about 85%, 86%, 87%, 88%, 89% or more sequence identity to the human germline sequence. Some humanized antibodies provided herein that are "chimeric" under the 2014 WHO INN definition have a heavy chain that has less than 85% identity to the human germline sequence and a light chain that has less than 85% identity to the matching human germline sequence. Some humanized antibodies provided herein are "mixed" under the 2014 WHO INN definition, e.g., have a heavy chain that has at least 85% sequence identity to the human germline sequence and a light chain that has less than 85% sequence identity to the matching human germline sequence, or vice versa. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "humanized" and have a light chain with at least 85% sequence identity to a human germline sequence and a heavy chain with at least 85% sequence identity to a matching human germline sequence. Some humanized antibodies provided herein meet the 2014 WHO INN definition of "mixed."Exemplary 5E20 antibodies that meet the 2014 WHO INN definition of "mixed" include antibodies having a mature heavy chain having the amino acid sequence of any of SEQ ID NOs: 163-166 and 168 and a mating mature light chain sequence having the amino acid sequence of any of SEQ ID NOs: 173-176. Exemplary 8H24 antibodies that meet the 2014 WHO INN definition of "mixed" include antibodies having a mature light chain sequence having the amino acid sequence of SEQ ID NO: 180 or SEQ ID NO: 181 and a mating mature heavy chain having the amino acid sequence of SEQ ID NO: 185 or SEQ ID NO: 186. Exemplary 11M14 antibodies that meet the 2014 WHO INN definition of "mixed" include antibodies having a mature light chain sequence having the amino acid sequence of any of SEQ ID NOs: 196-199 and a mating mature heavy chain having the amino acid sequence of any of SEQ ID NOs: 190-192. Further humanized 5E20 antibodies of the present invention include antibodies having a mature heavy chain having the amino acid sequence of SEQ ID NO: 167 or SEQ ID NO: 169 and a paired mature light chain having the amino acid sequence of any of SEQ ID NOs: 173 to 176.

[0405] Humanized antibodies often incorporate all six CDRs (by any conventional definition, but preferably by the Kabat definition) from a murine antibody, but can also be made with less than all CDRs from a murine antibody (e.g., at least three, four, or five CDRs) (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., J. of Mol. Biol., 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., J. Immunol., 164:1432-1441, 2000).

[0406] In some antibodies, only a portion of the CDRs, i.e., a subset of CDR residues required for binding, called SDRs, are required in humanized antibodies to retain binding. CDR residues that do not contact antigen and are not in the SDRs can be identified from regions of the Kabat CDRs lying outside the Chothia hypervariable loops based on previous studies (e.g., residues H60-H65 in CDR H2 are often not required), by molecular modeling, and / or empirically, or as described in Gonzales et al., Mol. Immunol. 41:863, 2004. In such humanized antibodies, where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position can be the amino acid occupying the corresponding position (Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor for donor amino acids in the CDRs reflects a balance of competing considerations. Such substitutions can potentially reduce the number of mouse amino acids in a humanized antibody, thereby reducing potential immunogenicity and / or satisfying the WHO INN definition of "humanization." However, substitutions can also cause changes in affinity, and significant decreases in affinity are preferably avoided. The substitution positions within the CDRs and amino acids to be substituted can also be empirically selected.

[0407] The human acceptor antibody sequence can be selected from among many known human antibody sequences, if desired, to provide a high degree of sequence identity (e.g., 65-85% identity) between the human acceptor sequence variable region framework and the corresponding variable region framework of the donor antibody chain.

[0408] Some humanized antibodies have the same (within experimental error) or improved functional properties, e.g., binding affinity for human sortilin as the mouse antibody from which they are derived, as described in the Examples, and minimize the reduction of surface sortilin while increasing extracellular Progranulin in plasma. For example, some humanized antibodies have binding affinity within 3-, 2-, or 1-fold of the mouse antibody from which they are derived, or affinity that is indistinguishable within experimental error. Some humanized antibodies minimize the reduction of surface sortilin within 3-, 2-, or 1-fold of the mouse antibody from which they are derived, or inhibit the reduction within experimental error from the mouse antibody from which they are derived, while increasing extracellular Progranulin in plasma, as described in the Examples.

[0409] An example of an acceptor sequence for the 5E20 heavy chain is the human mature heavy chain variable region of the human antibody AEX29086VH (AEX29086-VH_huFrwk; SEQ ID NO: 161). The heavy chain variable domains of 5E20 and AEX29086VH share the same length for the CDR-H1 and H2 loops. An example of an acceptor sequence for the 5E20 heavy chain is the human mature heavy chain variable region IMGT#IGHV3-21 * 01 (SEQ ID NO: 162). An example of an acceptor sequence for the 5E20 light chain is the human mature light chain variable region human antibody BAH04687VL (BAH04687-VL_huFrwk; SEQ ID NO: 171). The variable light chain domains of the 5E20 and BAH04687 antibodies share identical lengths for the CDR-L1, L2, and L3 loops. An example of an acceptor sequence for the 5E20 light chain is the human mature light chain variable region IGKV1-12 * 01 (sequence number 172).

[0410] An example of an acceptor sequence for the 8H24 heavy chain is the human antibody AAC51714VH (GenBank Acc.#AAC51714-VH_huFrwk; SEQ ID NO: 178). The variable heavy chain domains of 8H24 and AAC51714 also share the same lengths for the CDR-H1, H2 loops. An example of an acceptor sequence for the 8H24 heavy chain is the human mature heavy chain variable region IMGT#IGHV1-69. *08_IGHJ1*01 (SEQ ID NO: 179). An example of an acceptor sequence for the 8H24 light chain is the human mature light chain variable region human antibody ABC66914VL (GenBank Acc.#ABC66914-VL_huFrwk; SEQ ID NO: 183). The variable light chain domains of the 8H24 and ABC66914 antibodies also share identical lengths for the CDR-L1, L2, and L3 loops. An example of an acceptor sequence for the 8H24 light chain is the human mature light chain variable region IMGT#IGKV2-40 * 01 (sequence number 184).

[0411] An example of an acceptor sequence for the 11M14 heavy chain is the human mature heavy chain variable region of human Ig heavy chain ACS96198 (GenBank Acc. #ACS96198-VH_huFrwk (SEQ ID NO: 188). The human Ig heavy chain ACS96198 has the same canonical class as the 11M14 heavy chain variable region CDRs. An example of an acceptor sequence for the 11M14 heavy chain is the human mature heavy chain variable region IMGT #IGHV3-48 * An example of an acceptor sequence for the 11M14 light chain is NCBI accession code CBZ39892 (GenBank Acc.#CBZ39892-VL_huFrwk (SEQ ID NO: 194), which has the same canonical class for CDR-L1 and L2. An example of an acceptor sequence for the 11M14 light chain is IMGT#IGKV1-39 * 01 (SEQ ID NO: 195).

[0412] If more than one human acceptor antibody sequence is selected, a combination or hybrid of those acceptors can be used, and the amino acids used at different positions in the humanized light and heavy chain variable regions can be taken from any of the human acceptor antibody sequences used.

[0413] Certain amino acids from the human variable region framework residues may be selected for substitution based on their potential effect on CDR conformation and / or binding to antigen, which may be investigated by modeling, by examining the characteristics of amino acids at particular positions, or by empirical observation of the effects of substituting or mutagenesing specific amino acids.

[0414] For example, if an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid can be substituted with the equivalent framework amino acid from the murine antibody if the amino acid is reasonably predicted to be: (1) Direct noncovalent binding to antigen; (2) adjacent to or within the CDR region defined by Chothia, but not Kabat; (3) otherwise interacts with a CDR region (e.g., within about 6 Å of the CDR region) (e.g., identified by modeling the light or heavy chain on the solved structure of a homologous known immunoglobulin chain); or (4) Residues involved in the VL-VH interface.

[0415] In one embodiment, humanized sequences are generated using a two-step PCR protocol that allows for the introduction of multiple mutations, deletions, and insertions using QuikChange site-directed mutagenesis [Wang, W and Malcolm, BA (1999) BioTechniques 26:680-682].

[0416] Framework residues of classes (1) to (3) defined by Queen (U.S. Pat. No. 5,530,101) are sometimes referred to as canonical and vernier residues. Framework residues that help define the conformation of the CDR loop are sometimes referred to as canonical residues (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987); Thornton & Martin, J. Mol. Biol. 263:800-815 (1996)). Framework residues that support the conformation of the antigen-binding loop and play a role in fine-tuning the antibody's fit to the antigen are sometimes referred to as vernier residues (Foote & Winter, J. Mol. Biol. 224:487-499 (1992)).

[0417] Other framework residues that are candidates for substitution are those that form potential glycosylation sites. Further candidates for substitution are acceptor human framework amino acids that are unusual in human immunoglobulins at that position. These amino acids can be substituted with amino acids from the equivalent position in the mouse donor antibody or from the equivalent position in a more typical human immunoglobulin.

[0418] Another framework residue that is a candidate for substitution is the N-terminal glutamine residue (Q), which can be substituted with glutamic acid (E) to minimize the possibility of pyroglutamic acid conversion [Y. Diana Liu et al., 2011, J. Biol. Chem., 286:11211-11217]. The conversion of glutamic acid (E) to pyroglutamic acid (pE) occurs more slowly than that from glutamine (Q). Due to the loss of a primary amine in the conversion of glutamine to pE, the antibody becomes more acidic. Incomplete conversion leads to antibody heterogeneity, which can be observed as multiple peaks using charge-based analytical methods. Differences in heterogeneity may indicate a lack of process control.

[0419] Exemplary humanized antibodies include a humanized form of murine 5E20, designated Hu5E20.

[0420] Murine antibody 5E20 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:4 and SEQ ID NO:10, respectively. The present invention provides seven exemplary humanized mature heavy chain variable regions: hu5E20VHv1, hu5E20VHv2, hu5E20VHv3, hu5E20VHv4, hu5E20VHv5, hu5E20VHv6, and hu5E20VHv7. The present invention further provides four exemplary mature light chain variable regions: hu5E20VLv1, hu5E20VLv2, hu5E20VLv3, and hu5E20VLv4. Figures 1 and 2 show alignments of the heavy and light chain variable regions of murine 5E20 and various humanized antibodies, respectively.

[0421] The following 18 variable region framework positions have been identified as being specific to four exemplary human mature light chain variable regions, as further specified in the Examples, for reasons such as their potential influence on CDR conformation and / or binding to antigen, mediating interactions between the heavy and light chains, interactions with the constant region, being sites for desired or undesired post-translational modifications, being unusual residues for their positions in the human variable region sequence and therefore potentially immunogenic, reducing aggregation potential, and other reasons: Variable regions and seven exemplary human mature heavy chain variable region substitutions were considered: L11 (L11V), L36 (Y36L), L44 (P44F), L46 (L46G), L69 (T69A), L85 (T85D), L87 (Y87F), L100 (G100Q), L106 (I106K), H5 (L5V), H40 (A40T), H42 (G42D), H44 (G44R), H49 (S49A), H77 (T77S), H83 (R83K), H93 (A93S), H94 (K94R).

[0422] Here, as elsewhere, the first-mentioned residue is the residue of the humanized antibody formed by grafting a Kabat CDR or combined Chothia-Kabat CDR (in the case of CDR-H1) onto a human acceptor framework, and the second-mentioned residue is the residue being considered to replace such residue. Thus, within the variable region framework, the first-mentioned residue is human, and within the CDRs, the first-mentioned residue is murine.

[0423] Exemplary antibodies include any permutation or combination of exemplary mature heavy and light chain variable regions: hu5E20VHv1 / hu5E20VLv1, hu5E20VHv1 / hu5E20VLv2, hu5E20VHv1 / hu5E20VLv3, hu5E20VHv1 / hu5E20VLv4, hu5E20VHv2 / hu5E20VLv1, hu5E20VHv2 / hu5E20VLv2, hu5E20VHv2 / hu5E20VLv3, hu5E20VHv2 / hu5E20VLv4, hu5E20VHv3 / hu5E20VLv1, hu5E20VHv3 / hu5E20VLv2, hu5E20VHv3 / hu5E20VLv3 hu5E20VLv3, hu5E20VHv3 / hu5E20VLv4, hu5E20VHv4 / hu5E20VLv1, hu5E20VHv4 / hu5E20VLv2, hu5E20VHv4 / hu5E20VLv3, hu5E20VHv4 / hu5E20VLv4, hu5E20VHv5 / hu5E20VLv1, hu5E20VHv5 / hu5E20VLv2, hu5E20VHv5 / hu5E20VLv3, hu5E20VHv5 / hu5E20VLv4, hu5E20VHv6 / hu5E20VLv1, hu5E20VHv6 / hu5E20VLv2, hu5E20VHv6 / hu5E20VLv3, hu5E20VHv6 / hu5E20VLv4, hu5E20VHv7 / hu5E20VLv1, hu5E20VHv7 / hu5E20VLv2, hu5E20VHv7 / hu5E20VLv3, hu5E20VHv7 / hu5E20VLv4.

[0424] Exemplary antibodies include any permutation or combination of exemplary mature heavy chain variable regions hu5E20VHv1 (SEQ ID NO: 163), hu5E20VHv2 (SEQ ID NO: 164), hu5E20VHv3 (SEQ ID NO: 165), hu5E20VHv4 (SEQ ID NO: 166), hu5E20VHv5 (SEQ ID NO: 167), hu5E20VHv6 (SEQ ID NO: 168), and hu5E20VHv7 (SEQ ID NO: 169) with any of the exemplary mature light chain variable regions hu5E20VLv1 (SEQ ID NO: 173), hu5E20VLv2 (SEQ ID NO: 174), hu5E20VLv3 (SEQ ID NO: 175), and hu5E20VLv4 (SEQ ID NO: 176).

[0425] The present invention relates to a method for producing a humanized mature heavy chain variable region having a nucleotide sequence identical to that of hu5E20VHv1 (SEQ ID NO: 163), hu5E20VHv2 (SEQ ID NO: 164), hu5E20VHv3 (SEQ ID NO: 165), hu5E20VHv4 (SEQ ID NO: 166), hu5E20VHv5 (SEQ ID NO: 167), hu5E20VHv6 (SEQ ID NO: 168), or hu5E20VHv7 (SEQ ID NO: 169). , 97%, 98%, or 99% identity to hu5E20VLv1 (SEQ ID NO: 173), hu5E20VLv2 (SEQ ID NO: 174), hu5E20VLv3 (SEQ ID NO: 175), and hu5E20VLv4 (SEQ ID NO: 176). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of the backmutations or other mutations in SEQ ID NOs: 163-169 and SEQ ID NOs: 173-176 are retained.

[0426] In some humanized 5E20 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H5 is occupied by L or V, H40 is occupied by A or T, H42 is occupied by G or D, H44 is occupied by G or R, H49 is occupied by A, H77 is occupied by T or S, H83 is occupied by R or K, H93 is occupied by S, and H94 is occupied by R.

[0427] In some humanized 5E20 antibodies, positions H49, H93, and H94 of the VH region are occupied by A, S, and R, respectively, as in hu5E20VHv1. In some humanized 5E20 antibodies, positions H5, H49, H77, H93, and H94 of the VH region are occupied by V, A, S, S, and R, respectively, as in hu5E20VHv2. In some humanized 5E20 antibodies, positions H5, H44, H49, H77, H93, and H94 of the VH region are occupied by V, R, A, S, S, and R, respectively, as in hu5E20VHv3. In some humanized 5E20 antibodies, positions H5, H42, H44, H49, H77, H93, and H94 of the VH region are occupied by V, D, R, A, S, S, and R, respectively, as in hu5E20VHv4. In some humanized 5E20 antibodies, positions H5, H42, H44, H49, H77, H83, H93, and H94 of the VH region are occupied by V, D, R, A, S, K, S, and R, respectively, as in hu5E20VHv5. In some humanized 5E20 antibodies, positions H5, H40, H44, H49, H77, H93, and H94 of the VH region are occupied by V, T, R, A, S, S, and R, respectively, as in hu5E20VHv6. In some humanized 5E20 antibodies, positions H5, H40, H42, H44, H49, H77, H93, and H94 of the VH region are occupied by V, T, D, R, A, S, S, and R, respectively, as in hu5E20VHv7.

[0428] In some humanized 5E20 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L11 is L or V, L36 is L, L44 is F, L46 is G, L69 is A, L85 is T or D, L87 is F, L100 is G or Q, and L106 is I or K. In some humanized 5E20 antibodies, positions L36, L44, L46, L69, and L87 of the VL region are occupied by L, F, G, A, and F, respectively, as in hu5E20VLv1. In some humanized 5E20 antibodies, positions L11, L36, L44, L46, L69, and L87 of the VL region are occupied by V, L, F, G, A, and F, respectively, as in 5E20VLv2. In some humanized 5E20 antibodies, positions L11, L36, L44, L46, L69, L87, L100, and L106 in the VL region are occupied by V, L, F, G, A, F, Q, and K, respectively, as in hu5E20VLv3. In some humanized 5E20 antibodies, positions L11, L36, L44, L46, L69, L85, L87, L100, and L106 in the VL region are occupied by V, L, F, G, A, D, F, Q, and K, respectively, as in hu5E20VLv4.

[0429] In some humanized 5E20 antibodies, the variable heavy chain has 85% or more identity to human sequences. In some humanized 5E20 antibodies, the variable light chain has 85% or more identity to human sequences. In some humanized 5E20 antibodies, the variable heavy chain and variable light chain each have 85% or more identity to human germline sequences. In some humanized 5E20 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 5-7) and the three light chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 11-13).

[0430] Exemplary humanized antibodies include a humanized form of murine 8H24, designated Hu8H24.

[0431] Murine antibody 8H24 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:28 and SEQ ID NO:34, respectively. The present invention provides two exemplary humanized mature heavy chain variable regions: hu8H24VHv1 and hu8H24VHv2. The present invention further provides two exemplary mature light chain variable regions: hu8H24VLv1 and hu8H24VLv2. Figures 3 and 4 show alignments of the heavy and light chain variable regions, respectively, of murine 8H24 and various humanized antibodies.

[0432] The following ten variable region framework positions were considered as candidates for substitution in two exemplary human mature light chain variable regions and two exemplary human mature heavy chain variable regions, as further specified in the Examples, for reasons such as possible effects on CDR conformation and / or binding to antigen, mediation of interactions between heavy and light chains, interactions with the constant region, being sites for desired or undesired post-translational modifications, being an unusual residue for that position in the human variable region sequence and therefore potentially immunogenic, reducing aggregation potential, and other reasons: L2(I2V), L9(L9S), L74(K74T), H2(V2A), H12(K12V), H48(M48I), H67(V67A), H71(A71V), H91(Y91F), H108(L108T).

[0433] Here, as elsewhere, the first-mentioned residue is the residue of the humanized antibody formed by grafting a Kabat CDR or combined Chothia-Kabat CDR (in the case of CDR-H1) onto a human acceptor framework, and the second-mentioned residue is the residue being considered to replace such residue. Thus, within the variable region framework, the first-mentioned residue is human, and within the CDRs, the first-mentioned residue is murine.

[0434] Exemplary antibodies include any permutation or combination of the exemplary mature heavy and light chain variable regions hu8H24VHv1 / hu8H24VLv1, hu8H24VHv1 / hu8H24VLv2, hu8H24VHv2 / hu8H24VLv1, hu8H24VHv2 / hu8H24VLv2.

[0435] Exemplary antibodies include any permutation or combination of the exemplary mature heavy chain variable regions hu8H24VHv1 (SEQ ID NO: 180) and hu8H24VHv2 (SEQ ID NO: 181) with any of the exemplary mature light chain variable regions hu8H24VLv1 (SEQ ID NO: 185) and hu8H24VLv2 (SEQ ID NO: 186).

[0436] The present invention provides variants of the 8H24 humanized antibody, in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu8H24VHv1 (SEQ ID NO: 180) or hu8H24VHv2 (SEQ ID NO: 181), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98%, or 99% identity to hu8H24VLv1 (SEQ ID NO: 185) or hu8H24VLv2 (SEQ ID NO: 186). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 backmutations or other mutations in SEQ ID NOs: 180-181 and SEQ ID NOs: 185-186 are retained.

[0437] In some humanized 8H24 antibodies, at least one of the following positions in the VH region is occupied by an amino acid as specified: H2 is occupied by A, H12 is occupied by K or V, H48 is occupied by I, H67 is occupied by A, H71 is occupied by V, H91 is occupied by F, and H108 is occupied by T. In some humanized 8H24 antibodies, positions H2, H48, H67, H71, H91, and H108 of the VH region are occupied by A, I, A, V, F, and T, respectively, as in hu8H24VHv1. In some humanized 8H24 antibodies, positions H2, H12, H48, H67, H71, H91, and H108 of the VH region are occupied by A, V, I, A, V, F, and T, respectively, as in hu8H24VHv2.

[0438] In some humanized 8H24 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L2 is V, L9 is L or S, and L74 is K or T. In some humanized 8H24 antibodies, position L2 of the VL region is occupied by V, as in hu8H24VLv1. In some humanized 8H24 antibodies, positions L2, L9, and L74 of the VL region are occupied by V, S, and T, respectively, as in hu8H24VLv2.

[0439] In some humanized 8H24 antibodies, the variable heavy chain has 85% or more identity to human sequences. In some humanized 8H24 antibodies, the variable light chain has 85% or more identity to human sequences. In some humanized 8H24 antibodies, the variable heavy chain and variable light chain each have 85% or more identity to human germline sequences. In some humanized 8H24 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 29-31) and the three light chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 35-37).

[0440] Exemplary humanized antibodies include a humanized form of murine 11M14, designated Hu11M14.

[0441] Murine antibody 11M14 comprises mature heavy and light chain variable regions having amino acid sequences comprising SEQ ID NO:52 and SEQ ID NO:58, respectively. The present invention provides three exemplary humanized mature heavy chain variable regions: hu11M14VHv1b, hu11M14VHv2b, and hu11M14VHv3b. The present invention further provides four exemplary mature light chain variable regions: hu11M14VLv1b, hu11M14VLv2b, hu11M14VLv3b, and hu11M14VLv4b. Figures 5 and 6 show alignments of the heavy and light chain variable regions, respectively, of murine 11M14 and various humanized antibodies.

[0442] The following seven variable region framework positions were considered as candidates for substitution in four exemplary human mature light chain variable regions and three exemplary human mature heavy chain variable regions, as further specified in the Examples, for reasons such as possible effects on CDR conformation and / or binding to antigen, mediation of interactions between heavy and light chains, interactions with the constant region, being sites for desired or undesired post-translational modifications, being unusual residues for their positions in the human variable region sequence and therefore potentially immunogenic, conferring aggregation potential, and other reasons: L43 (A43S), L48 (I48V), L71 (F71Y), L76 (N76S), H49 (S49A), H80 (L80G), H82c (L82cG).

[0443] The following variable region CDR positions were considered as candidates for substitution in four exemplary human mature light chain variable regions, as further specified in the Examples: L54 (L54G, L54I). In some humanized 11M14 antibodies, the Kabat-Chothia combination CDR-L2 has an amino acid sequence comprising SEQ ID NO: 72. In some humanized 11M14 antibodies, the Kabat-Chothia combination CDR-L2 has an amino acid sequence comprising SEQ ID NO: 73.

[0444] Here, as elsewhere, the first-mentioned residue is the residue of the humanized antibody formed by grafting a Kabat CDR or combined Chothia-Kabat CDR (in the case of CDR-H1) onto a human acceptor framework, and the second-mentioned residue is the residue being considered to replace such residue. Thus, within the variable region framework, the first-mentioned residue is human, and within the CDRs, the first-mentioned residue is murine.

[0445] Exemplary antibodies include any permutation or combination of exemplary mature heavy and light chain variable regions: hu11M14VHv1b / hu11M14VLv1b, hu11M14VHv1b / hu11M14VLv2b, hu11M14VHv1b / hu11M14VLv3b, hu11M14VHv1b / hu11M14VLv4b, hu11M14VHv2b / hu11M14VLv1b, ... M14VHv2b / hu11M14VLv2b, hu11M14VHv2b / hu11M14VLv3b, hu11M14VHv2b / hu11M14VLv4b, hu11M14VHv3b / hu 11M14VLv1b, hu11M14VHv3b / hu11M14VLv2b,, hu11M14VHv3b / hu11M14VLv3b, hu11M14VHv3b / hu11M14VLv4b.

[0446] Exemplary antibodies include any permutation or combination of exemplary mature heavy chain variable regions hu11M14VHv1b (SEQ ID NO: 190), hu11M14VHv2b (SEQ ID NO: 191), and hu11M14VHv3b (SEQ ID NO: 192) with any of the exemplary mature light chain variable regions hu11M14VLv1b (SEQ ID NO: 196), hu11M14VLv2b (SEQ ID NO: 197), hu11M14VLv3b (SEQ ID NO: 198), and hu11M14VLv4b (SEQ ID NO: 199).

[0447] The present invention provides variants of the 11M14 humanized antibody, in which the humanized mature heavy chain variable region exhibits at least 90%, 95%, 96%, 97%, 98% or 99% identity to hu11M14VHv1b (SEQ ID NO: 190), hu11M14VHv2b (SEQ ID NO: 191) or hu11M14VHv3b (SEQ ID NO: 192), and the humanized mature light chain variable region exhibits at least 90%, 95%, 96%, 97%, 98% or 99% identity to hu11M14VLv1b (SEQ ID NO: 196), hu11M14VLv2b (SEQ ID NO: 197), hu11M14VLv3b (SEQ ID NO: 198) or hu11M14VLv4b (SEQ ID NO: 199). In some such antibodies, at least 1, 2, 3, 4, 5, 6, 7 or all 8 of the positions in SEQ ID NOs: 190-192 and SEQ ID NOs: 196-199 that are subject to backmutation or other mutation are similarly backmutated or otherwise mutated.

[0448] In some humanized 11M14 antibodies, at least one of the following positions in the VH region is occupied by the specified amino acid: H49 is occupied by A, H80 is occupied by L or G, and H82c is occupied by L or G. In some humanized 11M14 antibodies, position H49 in the VH region is occupied by A, as in hu11M14VHv1b. In some humanized 11M14 antibodies, positions H49 and H82c in the VH region are occupied by A and G, respectively, as in hu11M14VHv2b. In some humanized 11M14 antibodies, positions H49 and H80 in the VH region are occupied by A and G, respectively, as in hu11M14VHv3b.

[0449] In some humanized 11M14 antibodies, at least one of the following positions in the VL region is occupied by the specified amino acid: L43 is A or S, L48 is V, L54 is L, G or I, L71 is Y, and L76 is N or S. In some humanized 11M14 antibodies, positions L48 and L71 in the VL region are occupied by V and Y, respectively, as in hu11M14VLv1b. In some humanized 11M14 antibodies, positions L43, L48, L71, and L76 in the VL region are occupied by S, V, Y, and S, respectively, as in hu11M14VLv2b. In some humanized 11M14 antibodies, positions L43, L48, L54, L71, and L76 of the VL region are occupied by S, V, G, Y, and S, respectively, as in hu11M14VLv3b. In some humanized 11M14 antibodies, positions L43, L48, L54, L71, and L76 of the VL region are occupied by S, V, I, Y, and S, respectively, as in hu11M14VLv4b.

[0450] The heavy chain variable region of any of the above-mentioned antibodies can be modified to further reduce immunogenicity. For example, in some humanized antibodies, the H80 position of the VH region is occupied by G. For example, in some humanized antibodies, the H82c position of the VH region is occupied by G. The light chain variable region of any of the above-mentioned antibodies can be modified to further reduce immunogenicity. For example, in some humanized antibodies, the L54 position of the VL region is occupied by G or I.

[0451] In some humanized antibodies, position H82c in the VH region is occupied by G, as in hu11M14VHv3b. In some humanized antibodies, position L54 in the VL region is occupied by G, as in hu11M14VLv3b. In some humanized antibodies, position L54 in the VL region is occupied by I, as in hu11M14VLv4b.

[0452] In some humanized 11M14 antibodies, the Kabat-Chothia combination CDR-L2 has an amino acid sequence comprising SEQ ID NO: 72. In some humanized 11M14 antibodies, the Kabat-Chothia combination CDR-L2 has an amino acid sequence comprising SEQ ID NO: 73.

[0453] In some humanized 11M14 antibodies, the variable heavy chain has 85% or more identity to human sequences. In some humanized 11M14 antibodies, the variable light chain has 85% or more identity to human sequences. In some humanized 11M14 antibodies, the variable heavy chain and variable light chain each have 85% or more identity to human germline sequences. In some humanized 11M14 antibodies, the three heavy chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 53-55) and the three light chain CDRs are as defined by the Kabat / Chothia combination (SEQ ID NOs: 59-61), except that position L54 can be L, G, or I.

[0454] The CDR regions of such humanized 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2 antibodies can be identical or substantially identical to the CDR regions of 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2. The CDR regions can be defined by any conventional definition (e.g., Chothia, or a combination of Chothia and Kabat), but are preferably as defined by Kabat.

[0455] Variable region framework positions follow Kabat numbering unless otherwise specified. Other such variants typically differ from the exemplified Hu5E20, Hu8H24, or Hu11M14 heavy and light chain sequences by a small number of substitutions, deletions, or insertions (e.g., typically no more than 1, 2, 3, 5, 10, or 15). Such differences are usually in the framework, but can also occur in the CDRs.

[0456] An additional mutation possibility in the humanized 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2 variants is additional backmutation in the variable region framework. Many of the framework residues not in contact with the CDRs in the humanized mAb are amenable to amino acid substitution from the corresponding positions in the donor mouse mAb or other mouse or human antibodies, and even many potential CDR-contact residues are suitable for substitution. Even amino acids within the CDRs can be modified, for example, by residues found in the corresponding positions in the human acceptor sequence used to provide the variable region framework. Furthermore, alternative human acceptor sequences can be used, for example, for the heavy and / or light chains. If a different acceptor sequence is used, one or more of the backmutations recommended above may not be performed, since the corresponding donor and acceptor residues are already the same without backmutations.

[0457] Preferably, the substitutions or backmutations (whether conservative or not) in the humanized 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 and 4N2 variants do not substantially affect the binding affinity or potency of the humanized mAb, i.e., its ability to bind to sortilin.

[0458] The humanized variants hu11M14 H1bL3b_IgG1 LALA, hu11M14 H1bL3b_IgG1 LALA_YTE, hu8H24 H1L2_IgG1_LALA, and hu5E20 H7L4_IgG1_LALA increase plasma and CSF progranulin levels in non-human primates (Examples 21-22, Figures 15-16 and 19-20). The humanized variants hu11M14 H1bL3b_IgG1 LALA_ and hu11M14 H1bL3b_IgG1 LALA_YTE decrease sortilin levels in PBMCs from non-human primates (Example 23, Figure 21). hu11M14 H1bL3b_IgG1 LALA_-YTE exhibits improved pharmacokinetic and plasma pharmacodynamic profiles compared to hu11M14 H1bL3b_IgG1 LALA (Example 23, Figures 17-18).

[0459] Hu11M14VHv1bVLv3b is also called hu11M14 H1bL3b. Hu5E20VHv7VLv4 is also called hu5E20H7L4. Hu8H24VHv1VLv2 is also called hu8H24H1L2. D. Chimeric and Veneered Antibodies

[0460] The present invention further provides chimeric and veneered forms of non-human antibodies, particularly the exemplary 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18 and 4N2 antibodies.

[0461] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., murine) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the murine antibody and are approximately two-thirds human in sequence.

[0462] A veneered antibody is a type of humanized antibody that retains some, and usually all, of the CDRs and some of the nonhuman variable region framework residues of a nonhuman antibody, but replaces other variable region framework residues that may contribute to B- or T-cell epitopes, such as exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues from the corresponding positions in a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially derived from a nonhuman antibody and the variable region framework of the nonhuman antibody is made more human-like by the substitutions. Veneered forms of the 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2 antibodies are included in the present invention. E. Human antibodies

[0463] Sortilin or a fragment thereof (e.g., a peptide comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), E(S / Q / D)FL (SEQ ID NO: 206), RTEFGMAIGP) (SEQ ID NO: 213), WGFTESFLTS (SEQ ID NO: 214), or residues D74, R76, F97, K110, Y53 of the sortilin ECD of SEQ ID NO: 215). Human antibodies that specifically bind to the epitope defined by residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO: 215 are provided by various techniques described below. Some human antibodies are selected by competitive binding experiments, by the phage display method of Winter, supra, or otherwise to have the same epitope specificity as a particular murine antibody, such as one of the murine monoclonal antibodies described in the Examples. Human antibodies can also be screened for particular epitope specificity by using only a fragment of Sortilin, such as a Sortilin fragment comprising or consisting of the amino acid sequence of FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), E(S / Q / D)FL (SEQ ID NO: 206), RTEFGMAIGP (SEQ ID NO: 213) or WGFTESFLTS (SEQ ID NO: 214) as the target antigen, and / or by screening antibodies against a collection of Sortilin variants, such as the Sortilin variants of SEQ ID NOs: 216-243, or Sortilin variants comprising various mutations within amino acid residues 588-594, or 590-593, or 632-643, or 663-674, or 167-176, or 586-595 of SEQ ID NO: 1.

[0464] Methods for producing human antibodies include those described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and Engleman et al., U.S. Pat. No. 4,634,666, the use of transgenic mice containing human immunoglobulin genes (e.g., Lonberg et al., WO 93 / 12227 (1993); U.S. Pat. No. 5,877,397; U.S. Pat. No. 5,874,299; U.S. Pat. No. 5,814,318; U.S. Pat. No. 5,789,650; U.S. Pat. No. 5,770,429; U.S. Pat. No. 5,661,016; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,625,126; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,545,806; Neuberger, Nat. Biotechnol. 14:826 (1996) and Kucherlapati, WO 91 / 10741 (1991)), phage display methods (see, e.g., Dower et al., WO 91 / 17271; McCafferty et al., WO 92 / 01047; U.S. Pat. Nos. 5,877,218; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and 5,565,332); and the methods described in WO 2008 / 081008 (e.g., immortalizing memory B cells isolated from humans, e.g., with EBV, screening for desired properties, and cloning and expressing recombinant forms). F. Constant Region Selection

[0465] The heavy and light chain variable regions of a chimeric, veneered, or humanized antibody can be linked to at least a portion of a human constant region. The choice of constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgG1 and IgG3 possess complement-dependent cytotoxicity, while human isotypes IgG2 and IgG4 do not. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. Numbering rules for constant regions include EU numbering (Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)), Kabat numbering (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991)), IMGT unique numbering (Lefranc M.-P. et al., IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains, Dev. Comp. Immunol., 29, 185-203 (2005)), and IMGT exon numbering (Lefranc, supra).

[0466] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be partially or completely deleted or derivatized. Substitutions can be made in the constant region to reduce or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, for example, Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005,2006), or to extend half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213,2004). Exemplary substitutions include Gln at position 250 and / or Leu at position 428 (EU numbering is used in this paragraph for the constant region) to increase the half-life of the antibody. Substitutions at any or all of positions 234, 235, 236, and / or 237 reduce affinity for Fcγ receptors, particularly FcγRI receptors (see, e.g., U.S. Pat. No. 6,624,821). Alanine substitutions at positions 234, 235, and 237 of human IgG1 can be used to reduce effector function. Some antibodies have alanine substitutions at positions 234, 235, and 237 of human IgG1 to reduce effector function. Optionally, positions 234, 236, and / or 237 of human IgG2 are substituted with alanine, and position 235 is substituted with glutamine (see, e.g., U.S. Pat. No. 5,624,821). Some antibodies utilize mutations at one or more of positions 241, 264, 265, 270, 296, 297, 322, 329, and 331 (EU numbering) of human IgG1. Some antibodies utilize mutations at one or more of positions 318, 320, and 322 (EU numbering) of human IgG1. Some antibodies utilize substitutions at positions 234 and / or 235 with alanine and / or 329 with glycine. Some antibodies utilize substitutions at positions 234 and 235 with alanine.In some antibodies, the isotype is human IgG2 or IgG4.

[0467] In some antibodies, the "LALA" double mutation (Leu234Ala and Leu235Ala) is used for reduced effector function (Lund, J. et al. (1992) Mol. Immunol., 29, 53-59; Tamm and Schmidt, 1997, Int Rev Immunol 16(1-2):57-85). The Fc variant L234A / L235A (LALA) (numbering according to EU nomenclature) mutant has been shown to eliminate or reduce interaction with FcγRs (FcγRIa, FcγRIIa, FcγRIIa, and FcγRIIIa) and exhibit significantly reduced effector function. Furthermore, the Fc LALA mutant has also been shown to have reduced interaction with complement proteins and reduced complement-dependent cytotoxicity (CDC). Exemplary antibody heavy chain sequences incorporating the LALA mutation are SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:251 and SEQ ID NO:252.

[0468] Some antibodies use the "YTE" (M252T / S254T / T256E) (numbering according to Eu nomenclature) mutation in the Fc region (WFDall'Acqua et al., 2006 J. Biol. Chem. 281:23514-23). ​​The YTE variant has been shown to have enhanced binding / interaction with the neonatal Fc receptor (FcRn), thereby resulting in an extended circulating half-life of the antibody. Some antibodies use the "LALA" double mutation and the YTE mutation. Exemplary antibody heavy chain sequences incorporating the LALA and YTE mutations are SEQ ID NO:244, SEQ ID NO:246, and SEQ ID NO:248.

[0469] Antibodies can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab')2 and Fv, or as single chain antibodies in which the heavy and light chain mature variable domains are linked via a spacer.

[0470] Human constant regions exhibit allotypic and isoallotypic variations between different individuals, i.e., constant regions may differ in different individuals at one or more polymorphic positions.Isoallotypes differ from allotypes in that the serum that recognizes the isoallotype binds to one or more non-polymorphic regions of other isotypes.Thus, for example, another heavy chain constant region is that of IgG1 G1m3, with or without C-terminal lysine.Reference to human constant region includes constant regions with any natural allotype or any permutation of residues that occupy positions in natural allotypes. G. Expression of Recombinant Antibodies

[0471] Several methods are known for producing chimeric and humanized antibodies using antibody-expressing cell lines (e.g., hybridomas). For example, the immunoglobulin variable region of an antibody can be cloned and sequenced using well-known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. A consensus primer is used for the VH region leader peptide, encompassing the translation initiation codon as the 5' primer and the g2b constant region specific as the 3' primer. Exemplary primers are described in U.S. Patent Application Publication No. 2005 / 0009150 by Schenk et al. (hereinafter "Schenk"). Sequences from multiple independently derived clones can be compared to ensure that no changes were introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing the VH fragment obtained by 5' RACE RT-PCR and 3' g2b-specific primers.

[0472] The light chain variable VL region can be cloned in a similar manner. In one approach, a consensus primer set is designed to amplify the VL region using a 5' primer designed to hybridize to the VL region encompassing the translation initiation codon and a 3' primer specific to the Ck region downstream of the VJ junction region. In the second approach, the VL encoding cDNA is cloned using 5' RACE RT-PCR. Exemplary primers are described in Schenk, supra. The cloned sequence is then combined with the sequence encoding the human (or other non-human) constant region.

[0473] In one approach, the heavy and light chain variable regions are reengineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions and cloned into mammalian expression vectors, such as pCMV-hγ1 for the heavy chain and pCMV-Mcl for the light chain. These vectors encode the human γ1 and Ck constant regions as exon fragments downstream of the inserted variable region cassettes. After sequence verification, the heavy and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned medium is collected 48 hours after transfection and assayed for antibody production by Western blot analysis and antigen binding by ELISA. The chimeric antibodies are then humanized as described above.

[0474] Chimeric, veneered, humanized, and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operably linked to the coding sequences of antibody chains, including naturally associated or heterologous expression control elements such as promoters. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector is incorporated into an appropriate host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the recovery and purification of cross-reactive antibodies.

[0475] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Expression vectors generally contain selection markers, such as ampicillin resistance or hygromycin resistance, to permit detection of cells transformed with the desired DNA sequence.

[0476] E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microorganisms such as yeast are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, etc., as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[0477] Mammalian cells can be used to express nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones (VCH Publishers, NY, 1987). Several suitable host cell lines capable of secreting intact heterologous proteins have been developed, including CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas, including Sp2 / 0 and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences, such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0478] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. Nos. 5,741,957; 5,304,489; and 5,849,992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer derived from a mammary gland-specific gene such as casein or beta-lactoglobulin.

[0479] Vectors containing DNA segments of interest can be introduced into host cells by methods appropriate for the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cell hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To generate transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells or induced pluripotent stem cells (iPSCs), and the nuclei of such cells can be transferred into enucleated oocytes.

[0480] Once vectors encoding the antibody heavy and light chains are introduced into cell cultures, cell pools can be screened for growth productivity and product quality in serum-free medium. Top-producing cell pools can then be subjected to FACS-based single-cell cloning to generate monoclonal lines. Specific productivity of greater than 50 pg or 100 pg per cell per day, corresponding to product titers of greater than 7.5 g / L culture, can be used. Antibodies produced by single-cell clones can also be tested for turbidity, filtration characteristics, PAGE, IEF, UV scanning, HP-SEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assays such as ELISA or Biacore. Selected clones can then be placed in multiple vials and stored frozen for future use.

[0481] Once expressed, antibodies can be purified according to standard procedures in the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis, etc. (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0482] Methodologies for commercial production of antibodies can be used, including codon optimization, promoter selection, transcription element selection, terminator selection, serum-free single cell cloning, cell banking, use of selectable markers for copy number amplification, CHO terminators, or improved protein titer (e.g., U.S. Pat. Nos. 5,786,464; 6,114,148; 6,063,598; 7,569,339; WO 2004 / 050884; WO 2008 / 012142; WO 2008 / 012142; WO 2005 / 019442; WO 2008 / 107388; WO 2009 / 027471; and U.S. Pat. No. 5,888,809). IV.Active immunogen

[0483] Agents used for active immunization serve to induce the same types of antibodies in patients as described above in connection with passive immunization. Agents used for active immunization can be the same types of immunogens used to generate monoclonal antibodies in laboratory animals, e.g., peptides of 3-15, 3-12, 5-12, or 5-8 consecutive amino acids from the region of sortilin corresponding to residues 588-594, or 590-593, or 632-643, or 663-674, or 167-176, or 586-595 of SEQ ID NO: 1 ... Sortilin peptides comprising or consisting of 663 to 674, 167 to 176, or 586 to 595, or sortilin peptides comprising or consisting of the amino acid sequence FTESFLT (SEQ ID NO: 202), sortilin peptides comprising or consisting of the amino acid sequence ESFL (SEQ ID NO: 203), sortilin peptides comprising or consisting of the amino acid sequence DGCILGYKEQFL (SEQ ID NO: 204), or sortilin peptides comprising the amino acid sequence PSICLCSLEDFL (SEQ ID NO: 205). a sortilin peptide comprising or consisting of the amino acid sequence E(S / Q / D)FL (SEQ ID NO: 206); a sortilin peptide comprising or consisting of the amino acid sequence RTEFGMAIGP) (SEQ ID NO: 213); a sortilin peptide comprising or consisting of the amino acid sequence WGFTESFLTS (SEQ ID NO: 214); a portion of amino acid residues D74, R76, F97, K110, Y535, L560, and E557 of the sortilin ECD of SEQ ID NO: 215 or a sortilin peptide comprising or consisting of some or all of amino acid residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559, and L560 of the sortilin ECD of SEQ ID NO:215; or a sortilin peptide comprising or consisting of some or all of amino acid residues E557, S558, F559, L560, P510, and Y535 of the sortilin ECD of SEQ ID NO:215.In the case of conformational epitopes with widely separated residues, peptides can be selected for some residues that are close enough to each other to form a linear epitope. To induce antibodies that bind to the same or overlapping epitopes as 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2, the epitope specificity of these antibodies can be mapped (e.g., by testing binding to a series of overlapping peptides spanning Sortilin). Fragments of Sortilin that consist of, contain, or overlap with the epitope can then be used as immunogens.

[0484] When used, heterologous carriers and adjuvants can be the same as those used to produce monoclonal antibodies, but can also be selected for better pharmaceutical compatibility for human use.Suitable carriers include serum albumin, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, or toxoids or attenuated toxin derivatives from other pathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli, cholera, or Helicobacter pylori.T cell epitopes are also suitable carrier molecules. Some conjugates can be formed by linking the agents of the invention to immunostimulatory polymer molecules (e.g., tripalmitoyl-S-glyceryl cysteine ​​(Pam3Cys), mannan (mannose polymer), or glucan (β1→2 polymer)), cytokines (e.g., IL-1, IL-1α and β peptides, IL-2, γ-INF, IL-10, GM-CSF), and chemokines (e.g., MIP1-α and β, and RANTES). Immunogens can be linked to carriers with or without spacer amino acids (e.g., gly-gly). Additional carriers include virus-like particles. Virus-like particles (VLPs), also called pseudovirions or virus-derived particles, represent subunit structures composed of multiple copies of viral capsid and / or envelope proteins that can self-assemble in vivo into VLPs of defined spherical symmetry. (Powilleit et al., (2007) PLoS ONE 2(5):e415.) Alternatively, the peptide immunogen can be linked to at least one artificial T cell epitope capable of binding to most MHC class II molecules, such as a pan DR epitope ("PADRE"). PADRE is described in U.S. Pat. No. 5,736,142, WO 95 / 07707, and Alexander J et al., Immunity, 1:751-761 (1994). Active immunogens can be presented in multimeric form, in which multiple copies of the immunogen and / or its carrier are presented as a single covalently linked molecule.

[0485] Fragments are often administered with a pharmaceutically acceptable adjuvant. The adjuvant increases the titer and / or binding affinity of the induced antibodies compared to when the peptide is used alone. Various adjuvants can be used in combination with immunogenic fragments of sortilin to elicit an immune response. Some adjuvants enhance the intrinsic response to the immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Some adjuvants include aluminum salts, such as aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL™) (e.g., UK Patent Application Publication No. 2220211 (RIBI ImmunoChem Research Inc., Hamilton, Montana, now part of Corixa)). Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from the bark of the South American Quillaja Saponaria Molina tree (see Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540) (Aquila BioPharmaceuticals, Framingham, MA; now Antigenics, Inc., New York, NY). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally combined with immunostimulants such as monophosphoryl lipid A (see, e.g., Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)), Pluronic® polymers, and killed mycobacteria. Ribi adjuvant is an oil-in-water emulsion. Ribi contains a metabolizable oil (squalene) emulsified in saline containing Tween® 80. Ribi also contains a purified mycobacterial product that acts as an immunostimulant and bacterial monophosphoryl lipid A.Another adjuvant is CpG (WO 98 / 40100). The adjuvant can be administered as a component of a therapeutic composition together with the active agent, or can be administered separately, before, simultaneously with, or after administration of the therapeutic agent.

[0486] Analogs of natural fragments of Sortilin that induce antibodies against Sortilin can also be used. For example, in such peptides, one or more or all L-amino acids can be replaced with D-amino acids. The order of the amino acids can also be reversed (retro peptides). Optionally, the peptide contains all D-amino acids in reverse order (retro-inverso peptides). Peptides and other compounds that do not necessarily have significant amino acid sequence similarity to Sortilin peptides, but still act as mimics of Sortilin peptides and induce a similar immune response, can also be used. Anti-idiotypic antibodies against monoclonal antibodies against Sortilin, such as those described above, can also be used. Such anti-Id antibodies mimic antigens and generate an immune response against them (see Essential Immunology, Roit ed., Blackwell Scientific Publications, Palo Alto, CA 6th ed., p. 181).

[0487] The peptide (and optionally a carrier fused to the peptide) can also be administered in the form of a nucleic acid encoding the peptide and expressed in situ in the patient. The nucleic acid segment encoding the immunogen is typically linked to regulatory elements, such as a promoter and enhancer, that allow expression of the DNA segment in the intended target cells of the patient. For expression in blood cells, as desired for the induction of an immune response, promoter and enhancer elements from light or heavy chain immunoglobulin genes, or the CMV major intermediate-early promoter and enhancer, are suitable for directing expression. The linked regulatory elements and coding sequence are often cloned into a vector. Antibodies can also be administered in the form of nucleic acids encoding the heavy and / or light chains of the antibody. When both heavy and light chains are present, the chains are preferably linked as a single-chain antibody. Antibodies for passive administration can also be prepared, for example, by affinity chromatography from the serum of patients treated with the peptide immunogen.

[0488] DNA can be delivered in naked form (i.e., without colloidal or encapsulating material). Alternatively, retroviral systems, including vectors derived from retroviruses such as MMLV, HIV-1, and ALV (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol. 67, 5911 (1993)); adeno-associated viral vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)); lentiviral vectors, such as HIV or FIV. Several viral vector systems can also be used, including those based on the gag sequence, viral vectors from the pox family, including vaccinia virus and avian poxvirus, viral vectors derived from the alphavirus genus, e.g., Sindbis and Semliki Forest viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Pat. No. 5,643,576), and rhabdoviruses, e.g., vesicular stomatitis virus (see WO 96 / 34625) and papillomaviruses (Ohe et al., Human Gene Therapy, 6, 325-333 (1995); Woo et al., WO 94 / 12629 and Xiao & Brandsma, Nucleic Acids. Res. 24, 2630-2622 (1996)).

[0489] The DNA encoding immunogen, or the DNA encoding antibody heavy and / or light chain, or the vector containing them can be packaged in liposome.Suitable lipids and related analogues are described in US Patent No. 5,208,036, US Patent No. 5,264,618, US Patent No. 5,279,833 and US Patent No. 5,283,185.The vector and DNA encoding immunogen, or the heavy and / or light chain of antibody can also be adsorbed or associated with particulate carrier, examples of which include polymethyl methacrylate polymer and polylactide and poly(lactide-co-glycolide) (see, for example, McGee et al., J. Micro Encap. 1996). Vectors or segments thereof encoding antibody heavy and / or light chains can be incorporated ex vivo into, for example, cells removed from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells, which can then be reimplanted into the patient, typically after selection of cells that have incorporated the transgene. (See, e.g., WO 2017 / 091512.) Exemplary patient-derived cells include patient-derived induced pluripotent stem cells (iPSCs) or other types of stem cells (embryonic, hematopoietic, neural, or mesenchymal). A vector or a segment thereof encoding the heavy and / or light chain of an antibody can be introduced ex vivo into any desired region of cells, such as the albumin gene or other safe harbor genes. Cells incorporating the vector can be transplanted with or without prior differentiation. The cells can be transplanted into a specific tissue, such as a secretory tissue or the location of a lesion, or systemically, such as by injection into the blood. For example, the cells can be transplanted into a patient's secretory tissue, such as the liver, and, if necessary, can be pre-differentiated into cells present in that tissue, such as hepatocytes in the case of the liver. Expression of the antibody in the liver results in the secretion of the antibody into the blood. H. Antibody Screening Assays

[0490] Antibodies can be initially screened for the intended binding specificity as described above. Active immunogens can similarly be screened for the ability to induce antibodies with such binding specificity. In this case, the active immunogen is used to immunize laboratory animals and the resulting sera are tested for the appropriate binding specificity.

[0491] Antibodies with the desired binding specificity can then be tested in cell and animal models. An exemplary cell model is HEK293 cells expressing recombinant huSortilin (human sortilin) ​​on their surface. Another exemplary cell model is U251MG cells (a human glioblastoma cell line) that endogenously express sortilin on their cell surface. Human cell models include IPSC-derived cortical neurons or microglia expressing the FTD-GRN mutation. An exemplary mouse cell model is primary neurons derived from GRN+ / - and / or GRN- / - transgenic mice with AAV-TDP43CT-GFP (Chang, MC et al., J. Exp. Med 2017;214(9):2611-2628). TDP43CT formed cytoplasmic clusters, and GRN- / - neurons accumulated approximately 40% higher levels of TDP43CT than WT neurons. This phenotype was rescued by the addition of recombinant PRGN (10 μM).

[0492] The activity of antibodies or active agents can be assessed by a variety of criteria, including the increase and inhibition or delay of extracellular progranulin or behavioral impairment. Active immunogens can also be tested for the induction of antibodies in serum. Both passive and active immunogens can be tested for passage of antibodies across the blood-brain barrier to the brain of wild-type or transgenic animals. Antibody-inducing antibodies or fragments can also be tested in disease-free non-human primates or in non-human primates that naturally or inducedly develop symptoms of a disease characterized by altered progranulin levels (e.g., as in Examples 21-22 and Figures 14-21). Testing of antibodies or active agents is typically performed in conjunction with a control experiment, where the antibody or active agent is absent (e.g., replaced by vehicle). The reduction, delay, or inhibition of signs or symptoms of the disease attributable to the antibody or active agent under test can then be assessed compared to the control. V. Patients suitable for treatment

[0493] Altered progranulin levels have been found in several diseases, including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, and aging-related neurodegenerative disorders. The present regimen can also be used to treat or prevent any of these diseases. Due to the widespread association between neurological diseases and symptoms and altered progranulin levels, the present regimen can be used to treat or prevent any subject who exhibits reduced progranulin (e.g., plasma or CSF) levels compared to the average value in individuals without neurological disease. The present regimen can also be used to treat or prevent neurological diseases in individuals with a mutation in progranulin associated with the neurological disease. The present method is particularly suitable for treating or preventing frontotemporal dementia. In some embodiments, the method further comprises detecting, measuring, and / or monitoring progranulin levels, e.g., as described in Example 8.

[0494] Patients suitable for treatment include individuals at risk for the disease but who do not show symptoms, as well as those who currently show symptoms. Patients at risk for the disease include those with a known genetic risk for the disease. Such individuals include those who have relatives who have experienced the disease and those whose risk is determined by analysis of genetic or biochemical markers. Genetic markers of risk include mutations in progranulin, such as the FTD-GRN mutation described above. Individuals currently suffering from frontotemporal dementia can be recognized by PET imaging based on the presence of characteristic dementia and the risk factors described above. Furthermore, several diagnostic tests are available to identify individuals with frontotemporal dementia. These include measuring CSF and plasma progranulin levels.

[0495] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, or 30 years of age). However, it is usually not necessary to begin treatment until the patient is 40, 50, 60, or 70 years of age. Treatment typically requires multiple doses over a period of time. Treatment can be monitored by assaying antibody levels over time. If response declines, booster doses are indicated. I. Nucleic acids

[0496] The present invention further provides nucleic acids encoding any of the above heavy and light chains (e.g., SEQ ID NOS: 4, 10, 24-25, 28, 34, 48-49, 52, 58, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 163-169, 173-176, 180-181, 185-186, 190-192, 196-199). Exemplary nucleotide sequences include SEQ ID NOS: 2, 8, 26, 32, 50, 56, 76, 82, 88, 94, 100, 106, 112, 118, 124, 130, 136, 142, 148, and 154. Optionally, such nucleic acids may further encode a signal peptide and be expressed with the signal peptide linked to the variable region. The coding sequence of the nucleic acid may be operably linked to regulatory sequences to ensure expression of the coding sequence, such as a promoter, enhancer, ribosome binding site, or transcription termination signal. Regulatory sequences may include a promoter, such as a prokaryotic or eukaryotic promoter. The nucleic acids encoding the heavy or light chains may be codon-optimized for expression in a host cell. The nucleic acids encoding the heavy and light chains may encode selectable genes. The nucleic acids encoding the heavy and light chains may exist in isolated form or may be cloned into one or more vectors. The nucleic acids may be synthesized, for example, by solid-phase synthesis or PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy and light chains may be linked as a single contiguous nucleic acid, for example, within an expression vector, or may be separate, for example, each cloned into its own expression vector. J. Conjugated Antibodies

[0497] Conjugated antibodies that specifically bind to an antigen such as sortilin are useful for detecting the presence of sortilin, monitoring and evaluating the effectiveness of therapeutic agents used to treat patients diagnosed with a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders, increasing extracellular progranulin levels, or treating or preventing a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders in patients. For example, such antibodies can be conjugated to other therapeutic moieties, other proteins, other antibodies, and / or detectable labels. See WO 03 / 057838; U.S. Pat. No. 8,455,622. Such a therapeutic moiety can be any agent that can be used to treat, counter, ameliorate, prevent or ameliorate an undesirable symptom or disease in a patient, such as a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, a neurodegenerative disorder, or an age-related neurodegenerative disorder. In some embodiments, the method further comprises detecting, measuring and / or monitoring progranulin levels, e.g., as in Example 8.

[0498] Conjugated therapeutic moieties may include neurotrophic agents, neuroprotective agents, radiotherapeutic agents, radioactive (radiopharmaceutical), fluorescent, paramagnetic tracers, ultrasound contrast agents, immunomodulatory agents, or any biologically active agent that promotes or enhances the activity of an antibody or modifies its bioavailability and distribution within the body or organ. Neurotrophic agents can be any agent, including chemical or proteinaceous substances, that promotes the maintenance, growth, or differentiation of neurons. Neuroprotective agents can be agents, including chemical or proteinaceous agents, that protect neurons from acute injury or degenerative processes. Immunomodulatory agents can be any agent that stimulates or inhibits the development or maintenance of an immunological response. Radiotherapeutic agents can be any molecule or compound that emits radiation. When such therapeutic moieties are coupled to a sortilin-specific antibody, such as the antibody described herein, the coupled therapeutic moiety will have specific affinity for cells affected by sortilin and progranulin-related diseases relative to normal cells. Furthermore, smaller amounts of the therapeutic moiety can be used.

[0499] Some such antibodies can be modified to act as immunotoxins. See, e.g., U.S. Patent No. 5,194,594. For example, ricin, a plant-derived cytotoxin, can be conjugated to an antibody by using the bifunctional reagents S-acetylmercaptosuccinic anhydride for the antibody and succinimidyl 3-(2-pyridyldithio)propionate for the ricin. See Pietersz et al., Cancer Res. 48(16):4469-4476 (1998). Coupling results in loss of ricin's B-chain binding activity but does not impair the toxic potential of ricin's A-chain or the activity of the antibody. Similarly, saporin, an inhibitor of ribosome assembly, can be conjugated to an antibody via a disulfide bond between chemically inserted sulfhydryl groups. See Polito et al., Leukemia 18:1215-1222 (2004).

[0500] Some such antibodies can be linked to radioisotopes, such as yttrium. 90 (90Y), Indium 111 (111In), 131 I, 99 mTc, radioactive silver-111, radioactive silver-199 and bismuth 213 Radioisotopes can be conjugated to antibodies using conventional bifunctional chelates. Sulfur-based linkers can be used to conjugate radioactive silver-111 and radioactive silver-199. Hazra et al., Cell Biophys. 24-25:1-7 (1994). Conjugation of silver radioisotopes can involve reducing immunoglobulins with ascorbic acid. For radioisotopes such as In and Y, ibritumomab tiuxetan can be used, reacting with such isotopes to form In-ibritumomab tiuxetan and Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0501] Some such antibodies may be linked to other therapeutic moieties. Such therapeutic moieties may be, for example, cytotoxic, cytostatic, neurotrophic, or neuroprotective. For example, the antibody may be conjugated to a toxic chemotherapeutic agent such as a tubulin inhibitor, such as maytansine, geldanamycin, a tubulin-binding agent (e.g., auristatin), or a minor groove binder, such as calicheamicin. Other representative therapeutic moieties include agents known to be useful in the treatment, management, or amelioration of diseases or disorders associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders.

[0502] Antibodies can also be coupled to other proteins. For example, antibodies can be coupled to Fynomers. Fynomers are small binding proteins (e.g., 7 kDa) derived from the human Fyn SH3 domain. They can be stable and soluble and lack cysteine ​​residues and disulfide bonds. Fynomers can be engineered to bind to target molecules with the same affinity and specificity as antibodies. They are suitable for creating antibody-based multispecific fusion proteins. For example, Fynomers can be fused to the N- and / or C-termini of antibodies to create bispecific and trispecific FynomAbs with different architectures. Fynomers can be selected using Fynomer libraries via screening techniques using FACS, Biacore, and cell-based assays, which allow for the efficient selection of Fynomers with optimal properties. Examples of finomers are disclosed in Grabulovski et al., J. Biol. Chem. 282:3196-3204 (2007); Bertschinger et al., Protein Eng. Des. Sel. 20:57-68 (2007); Schlatter et al., MAbs. 4:497-508 (2011); Banner et al., Acta. Crystallogr. D. Biol. Crystallogr. 69(Pt6):1124-1137 (2013); and Brack et al., Mol. Cancer Ther. 13:2030-2039 (2014).

[0503] The antibodies disclosed herein may also be coupled or conjugated to one or more other antibodies (e.g., to form antibody heteroconjugates), which may bind to a different epitope within Sortilin or which may bind to a different target antigen.

[0504] The antibody can also be coupled with a detectable label.Such an antibody can be used for detecting sortilin in subjects who have or are being treated for diseases or disorders associated with changes in progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders, and / or for assessing the effectiveness of treatment.Such an antibody is particularly useful for making such determinations in subjects who have or are susceptible to diseases or disorders associated with changes in progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders, or in suitable biological samples obtained from such subjects.In some embodiments, the method further comprises detecting, measuring, and / or monitoring progranulin levels, for example, as in Example 8. Representative detectable labels that can be coupled or linked to an antibody include various enzymes, such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, aequorin; radioactive materials, such as radioactive silver-111, radioactive silver-199, bismuth, 213 , iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 5 S), tritium ( 3 H), indium ( 115 In, 113 In, 112 In, 111 In,), technetium ( 99 Tc), thallium ( 201Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn and 117 positron-emitting metals using various positron emission tomography techniques; non-radioactive paramagnetic metal ions; and molecules that are radiolabeled or conjugated to specific radioisotopes.

[0505] Radioisotopes can be attached to antibodies using conventional bifunctional chelates. Sulfur-based linkers can be used to attach radioactive silver-111 and radioactive silver-199. Hazra et al., Cell Biophys. 24-25:1-7 (1994). Attaching silver radioisotopes can involve reducing immunoglobulins with ascorbic acid. For radioisotopes such as In and Y, ibritumomab tiuxetan can be used, reacting with such isotopes to form In-ibritumomab tiuxetan and Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48 Suppl 1:S91-S95 (2001).

[0506] Therapeutic moieties, other proteins, other antibodies, and / or detectable labels can be coupled or conjugated to antibodies of the invention directly or indirectly through intermediates (e.g., linkers). See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospect Of The Therapeutic Use Of Radiolabeled Antibodies In Cancer," in Monoclonal Antibodies For Cancer Detection And Therapy. See, "Therapy," Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985); and Thorpe et al., Immunol. Rev., 62:119-58 (1982). Suitable linkers include, for example, cleavable and non-cleavable linkers. Different linkers can be used that release the coupled therapeutic moiety, protein, antibody, and / or detectable label under acidic or reducing conditions, upon exposure to specific proteases, or other defined conditions. VI. Pharmaceutical Compositions and Methods of Use

[0507] In preventive applications, the antibody or the agent for inducing the antibody or its pharmaceutical composition is administered to a patient who is susceptible to or otherwise at risk of a disease (e.g., frontotemporal dementia) in a regimen (dosage, frequency, and route of administration) effective for reducing the risk of the disease, reducing the severity, or delaying the onset of at least one sign or symptom of the disease. In particular, the regimen is preferably effective for increasing the extracellular progranulin level in the brain or plasma, and / or inhibiting and / or delaying the onset of behavioral disorders. In therapeutic applications, the antibody or the agent for inducing the antibody is administered to a patient who is suspected of having or already has a disease (e.g., frontotemporal dementia) in a regimen (dosage, frequency, and route of administration) effective for improving at least one sign or symptom of the disease or at least suppressing further deterioration. In particular, the regimen is preferably effective for increasing or at least normalizing the level of progranulin and / or behavioral disorders.

[0508] A regimen is considered to be therapeutically or prophylactically effective if an individual treated patient achieves better outcomes than the average outcomes in a control population of comparable patients not treated by the methods of the invention, or if better results are demonstrated in treated patients versus control patients in a controlled clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial) at the p<0.05 or 0.01 or even 0.001 level.

[0509] The effective dose will vary depending on many different factors, such as the means of administration, the target site, the physiological condition of the patient, whether the patient is a carrier of FTD-GRN, whether the patient is human or an animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic.

[0510] Exemplary dosage ranges for antibodies are about 0.01-60 mg / kg, or about 0.1-3 mg / kg, or 0.15-2 mg / kg, or 0.15-1.5 mg / kg of patient body weight. Antibodies can be administered at such doses daily, on alternate days, weekly, biweekly, monthly, quarterly, or according to any other schedule determined by empirical analysis. Exemplary treatments involve administration in multiple doses over an extended period of, for example, at least six months. Further exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months.

[0511] The amount of drug for active administration varies from 0.1 to 500 μg per patient, more typically 1 to 100 or 1 to 10 μg per injection for human administration. The timing of injections can vary significantly, from once daily to once a year to once every 10 years. A typical regimen consists of an immunization followed by booster injections at time intervals, such as 6-week or 2-month intervals. Another regimen involves an immunization followed by booster injections at 1, 2, and 12 months. Another regimen requires lifelong injections every 2 months. Alternatively, booster injections can be administered irregularly as indicated by monitoring the immune response. Exemplary dosage and administration regimens for non-human primates are provided in Examples 21-22 and Figures 14-21.

[0512] Antibodies or agents for inducing antibodies are preferably administered via peripheral routes (i.e., the administered or induced antibodies cross the blood-brain barrier to reach the intended site in the brain). Administration routes include topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, intranasal, intraocular, intradermal, or intramuscular. Some routes for administering antibodies are intravenous and subcutaneous. Some routes for active immunization are subcutaneous and intramuscular. This type of injection is most typically performed in the arm or leg muscles. In some methods, the agent is injected directly into a specific tissue where deposits have accumulated, such as intracranial injection.

[0513] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, pharmaceutical excipients, or agents. The formulation depends on the selected route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, or saline or acetate buffer (to reduce discomfort at the injection site). The solution can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0514] This regimen can be administered in combination with another drug that is effective in treating or preventing the disease being treated. For example, in the case of frontotemporal dementia, this regimen can be combined with antidepressants such as trazodone and selective serotonin reuptake inhibitors (e.g., citalopram (Celexa), paroxetine (Paxil) or sertraline (Zoloft)), and / or antipsychotic sedatives (e.g., olanzapine (Zyprexa) or quetiapine (Seroquel), Alector's AL001, or the antibodies described in published U.S. Patent Application Publication No. 20170267761). In some embodiments, the method further comprises detecting, measuring and / or monitoring progranulin levels, for example, as described in Example 8.

[0515] The antibody is administered in an effective regimen, which refers to a dosage, route of administration, and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and / or improves at least one sign or symptom of the disorder being treated. If the patient already suffers from the disorder, the regimen can be referred to as a therapeutically effective regimen. If the patient is at high risk for the disorder compared to the general population but has not yet experienced symptoms, the regimen can be referred to as a prophylactically effective regimen. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient compared to historical controls or previous experience of the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials in a population of treated patients compared to a control population of untreated patients.

[0516] Exemplary dosages of antibodies are 0.1-60 mg / kg (e.g., 0.5, 3, 10, 30, or 60 mg / kg), or 0.5-5 mg / kg body weight (e.g., 0.5, 1, 2, 3, 4, or 5 mg / kg), or 10-4000 mg or 10-1500 mg as a fixed dose. Dosage will depend, among other factors, on the patient's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0517] Administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Some antibodies can be administered intravenously or subcutaneously into the systemic circulation. Intravenous administration can be by infusion over a period of time, such as 30 to 90 minutes.

[0518] The administration frequency depends, among other things, on the circulating half-life of the antibody, the patient's condition, and the administration route.The administration frequency can be daily, weekly, monthly, quarterly, or at irregular intervals, depending on the change or progression of the patient's condition of the disorder being treated.An exemplary frequency for intravenous administration is between weekly and every three months over the course of continuous treatment, although more or less frequent administration is also possible.For subcutaneous administration, an exemplary administration frequency is daily to monthly, although more or less frequent administration is also possible.

[0519] The number of doses administered depends on whether the disorder is acute or chronic and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, one to ten doses are often sufficient. In some cases, a single bolus dose, in divided doses as needed, is sufficient for acute disorders or acute exacerbations of chronic disorders. Treatment can be repeated for recurrence or acute exacerbations of acute disorders. For chronic disorders, the antibody can be administered at regular intervals, for example, weekly, every two weeks, monthly, every three months, or every six months, for at least 1, 5, or 10 years, or for the life of the patient. A. Diagnostic and Monitoring Methods

[0520] Also provided are methods for detecting sortilin in a subject, for example, by measuring sortilin in a sample from the subject or by in vivo imaging of sortilin in the subject. Such methods are useful for diagnosing or confirming diseases associated with sortilin or progranulin or sensitivity thereto. The methods can also be used in asymptomatic subjects. The methods are also useful for monitoring disease progression and / or response to treatment in subjects previously diagnosed with a disease associated with sortilin and progranulin, such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or aging-related neurodegenerative disorders. In some embodiments, the methods further comprise detecting, measuring, and / or monitoring progranulin levels, e.g., as described in Example 8.

[0521] The method works by administering a reagent to a subject, such as any antibody that binds to sortilin described in this application (e.g., murine, humanized, chimeric, or veneered 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, and 4N2 antibodies), and then detecting the agent after it binds. The method may be performed at an epitope within amino acid residues FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), or E(S / Q / D)FL (SEQ ID NO: 207), RTEFGMAIGP (SEQ ID NO: 213), or WGFTESFLTS (SEQ ID NO: 214), or at residues D74, R76, F97, K110, Y535, L ... An antibody that specifically binds to the epitope defined by residues K110, Y535, E557, T561, Q563, D74, P510, S558, F559 and L560 of the sortilin ECD of SEQ ID NO:215 can be used. In some methods, the antibody binds to a peptide consisting of an epitope within amino acid residues FTESFLT (SEQ ID NO: 202), ESFL (SEQ ID NO: 203), DGCILGYKEQFL) (SEQ ID NO: 204), PSICLCSLEDFL (SEQ ID NO: 205), E(S / Q / D)FL (SEQ ID NO: 207), RTEFGMAIGP (SEQ ID NO: 213), or WGFTESFLTS (SEQ ID NO: 214). The antibody typically binds to an epitope on sortilin. If desired, a clearing response can be avoided by using an antibody fragment lacking the full-length constant region, such as a Fab. In some methods, the same antibody can function as both a treatment and a diagnostic reagent.

[0522] Diagnostic reagents can be administered intravenously to the patient's body or directly to the brain by intracranial injection or by drilling a hole in the skull. The dosage of the reagent should be within the same range as the treatment method. Typically, the reagent is labeled, but in some methods, the primary reagent with affinity for sortilin is unlabeled and a secondary labeling agent is used to bind to the primary reagent. The choice of label depends on the detection means. For example, fluorescent labels are suitable for optical detection. The use of paramagnetic labels is suitable for tomographic detection without surgical intervention. Radioactive labels can also be detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT).

[0523] Diagnosis is made by comparing the number, size and / or intensity of labeled loci with corresponding baseline values.Baseline values ​​can represent the average level in a group of unaffected individuals.Baseline values ​​can also represent previous levels determined in the same subject.For example, baseline values ​​can be determined in a subject before treatment is initiated, and subsequent measurements can be compared with the baseline value.If a patient has a decrease in cell surface sortilin, treatment can be adjusted to increase cell surface sortilin to normal levels.

[0524] The method of in vivo imaging of sortilin is useful for monitoring cell-surface sortilin in patients being treated with an agent that increases extracellular progranulin, for example, patients being treated for a disease or disorder associated with altered progranulin levels, such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, a neurodegenerative disorder, or an age-related neurodegenerative disorder, or a susceptibility to such a disease. For example, the method can be used in patients being treated for frontotemporal dementia. If the patient has decreased cell-surface sortilin, treatment can be adjusted to increase cell-surface sortilin to normal levels. In some embodiments, the method further comprises detecting, measuring, and / or monitoring progranulin levels, e.g., as in Example 8.

[0525] In some patients, the diagnosis of the disease or disorder associated with changes in progranulin level can be aided by carrying out PET scan.PET scan can be carried out, for example, by using conventional PET imager and auxiliary equipment.Scanning typically includes one or more brain regions that are generally known to be associated with the disease or disorder associated with changes in progranulin level, and one or more regions that are used as control.

[0526] The signals detected in a PET scan can be represented as a multidimensional image. Multidimensional images can be two-dimensional, representing a cross-section through the brain; three-dimensional, representing the brain in three dimensions; or four-dimensional, representing changes in the brain in three dimensions over time. A color scale can be used, with different colors indicating different amounts of label, and essentially the amount of sortilin protein detected. Scan results can also be presented numerically, with numbers relating to the amount of label detected and the resulting amount of sortilin. Label present in a brain region known to be associated with a particular disease or disorder associated with changes in progranulin levels (e.g., frontotemporal dementia), can be compared with label present in a region not known to be associated with the disease or disorder to provide a ratio indicating the extent of deposits in the former region. For the same radiolabeled ligand, such a ratio provides comparable measurements of sortilin and its changes across different patients.

[0527] In some methods, a PET scan is performed simultaneously with an MRI or CAT scan or during the same patient visit. MRI or CAT scans provide more anatomical detail of the brain than PET scans. However, PET scan images can be overlaid with MRI or CAT scan images to more accurately show the location of the PET ligand and the predicted sortilin to the brain anatomy. Some machines can perform both a PET scan and an MRI or CAT scan without the patient having to change position between scans, facilitating image overlay.

[0528] Suitable PET ligands include radiolabeled antibodies of the invention (e.g., murine, humanized, chimeric, or veneered 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 antibodies). The radioisotope used can be, for example, C 11 , N 13 , O 15 , F 18 or I 123The interval between administering the PET ligand and performing the scan may depend on the PET ligand, particularly its rate of uptake and clearance from the brain, and the half-life of the radiolabel.

[0529] PET scans can also be performed as a preventative measure in asymptomatic patients or patients with mild cognitive impairment who have not yet been diagnosed with a disease or disorder associated with altered progranulin levels, but who are at high risk for developing such a disease or disorder. For asymptomatic patients, scans are particularly useful for individuals considered at high risk for a disease or disorder associated with altered progranulin levels due to family history, genetic or biochemical risk factors, or maturity. Prophylactic scans can begin, for example, in patients between the ages of 45 and 75. In some patients, the first scan is performed at age 50.

[0530] Preventive scans can be performed, for example, at intervals of 6 months to 10 years, preferably 1 to 5 years. In some patients, preventive scans are performed annually.

[0531] The foregoing description of diagnosing, monitoring, and adjusting treatment for Sortilin and Progranulin-related diseases and disorders has primarily focused on the use of PET scans. However, to practice such methods, any other technique for visualizing and / or measuring Sortilin that is suitable for use with the Sortilin antibodies of the present invention (e.g., murine, humanized, chimeric, or veneered 5E20, 8H24, 11M14, 5M13, 2F18, 2P22, 6B15, 2C14, 9N18, or 4N2 antibodies) can be used instead of PET scans.

[0532] Also provided are methods for detecting immune responses to sortilin in patients suffering from or susceptible to diseases and disorders associated with sortilin and progranulin. The methods can be used to monitor a course of therapeutic and prophylactic treatment with the agents provided herein. Antibody profiles following passive immunization typically show an immediate peak in antibody concentration followed by an exponential decay. Without further administration, the decay approaches pretreatment levels within days to months, depending on the half-life of the administered antibody. For example, the half-life of some human antibodies is approximately 20 days.

[0533] In some methods, a baseline measurement of antibodies to sortilin in the subject is taken before administration, followed by a second measurement shortly thereafter to determine the peak antibody level, and one or more additional measurements are taken at intervals to monitor the decay of the antibody level. When the antibody level falls to baseline or a predetermined percentage (e.g., 50%, 25%, or 10%) of the peak below baseline, a further dose of antibody is administered. In some methods, the peak level or a subsequently measured lower background level is compared to a previously determined reference level to constitute a beneficial prophylactic or therapeutic treatment regimen in another subject. If the measured antibody level is significantly lower than the reference level (e.g., less than the mean minus one or preferably two standard deviations of the reference value in a population of subjects who would benefit from treatment), administration of an additional dose of antibody is indicated.

[0534] Biological samples obtained from subjects with, suspected of, or at risk of having a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders can be contacted with the antibodies disclosed herein to assess the presence of sortilin. For example, the sortilin level in such subjects can be compared with that present in healthy subjects. Alternatively, the sortilin level of such subjects undergoing disease treatment can be compared with that of subjects not undergoing treatment for a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders. Some such tests involve biopsies of tissues obtained from such subjects. ELISA assays can also be useful methods for assessing sortilin in, for example, fluid samples. VII. Kit

[0535] The present invention further provides kits (e.g., containers) containing the antibodies disclosed herein and associated materials, such as instructions for use (e.g., package inserts). The instructions can include, for example, instructions for administering the antibody and, optionally, one or more additional agents. The antibody containers can be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.

[0536] Package insert refers to instructions customarily included in commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, contraindications and / or warnings regarding the use of such therapeutic product.

[0537] The kit may also comprise a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. VIII. Other Uses

[0538] Antibodies can be used to detect sortilin or its fragments in clinical diagnostic or treatment settings or in research. For example, antibodies can be used to detect the presence of sortilin in a biological sample. Binding of the antibody to a biological sample can be compared to binding of the antibody to a control sample. The control sample and the biological sample can comprise cells of the same tissue origin. The control sample and the biological sample can be obtained from the same individual or different individuals, on the same or different occasions. If necessary, multiple biological samples and multiple control samples can be evaluated on multiple occasions to protect against random variations regardless of differences between samples. A direct comparison can then be made between the biological sample and the control sample to determine whether antibody binding to the biological sample (i.e., the presence of sortilin) ​​is increased, decreased, or the same as that of the antibody binding to the control sample. Increased binding of the antibody to the biological sample compared to the control sample indicates the presence of sortilin in the biological sample. In some cases, the increase in antibody binding is statistically significant. Optionally, antibody binding to the biological sample is at least 1.5, 2, 3, 4, 5, 10, 20 or 100 times greater than antibody binding to the control sample.

[0539] Furthermore, antibodies can be used to detect the presence of sortilin in biological samples to monitor and evaluate the effectiveness of therapeutic agents used to treat patients diagnosed with a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders. In some embodiments, the method further comprises detecting, measuring, and / or monitoring progranulin levels, as in Example 8. A biological sample from a patient diagnosed with a disease or disorder associated with altered progranulin levels, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, neurodegenerative disorders, or age-related neurodegenerative disorders is evaluated to establish a baseline of antibody binding to the sample (i.e., a baseline for the presence of sortilin in the sample) before initiating treatment with the therapeutic agent. In some cases, multiple biological samples from the patient are evaluated multiple times to establish both a baseline and a measure of random variation independent of treatment. The therapeutic agent is then administered according to a regimen. The regimen may include multiple administrations of the agent over a period of time. If necessary, antibody binding (i.e., the presence of sortilin) ​​is assessed multiple times in multiple biological samples from the patient to both establish a measure of random variation and indicate a tendency for response to immunotherapy. The various assessments of antibody binding to the biological samples are then compared. If only two assessments are performed, a direct comparison can be made between the two assessments to determine whether antibody binding (i.e., the presence of sortilin) ​​increased, decreased, or remained the same between the two assessments. If three or more measurements are performed, the measurements can begin before treatment with the therapeutic agent and be analyzed over time during the treatment period. In patients with decreased antibody binding to the biological sample (i.e., decreased cell-surface sortilin), treatment can be adjusted to increase cell-surface sortilin to normal levels.Assessment of antibody binding can be performed in conjunction with assessment of other signs and symptoms of a disease or disorder associated with altered progranulin levels, such as frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Batten disease, a neurodegenerative disorder, or an age-related neurodegenerative disorder. In some embodiments, the method further comprises detecting, measuring, and / or monitoring progranulin levels, e.g., as in Example 8.

[0540] Antibodies can also be used as research reagents for laboratory studies in detecting Sortilin or its fragments. For such use, antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in the form of a kit with all the reagents necessary to carry out a detection assay. Antibodies can also be used to purify Sortilin or its binding partners, for example, by affinity chromatography.

[0541] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Where different versions of a sequence are associated with an accession number at different times, the version associated with the accession number as of the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or the filing date of the priority application, if applicable, that references the accession number. Similarly, where different versions of a publication, website, etc. are published at different times, the most recently published version as of the effective filing date of this application is meant unless otherwise specified. Any feature, step, element, embodiment, or aspect of the present invention can be used in combination with any other, unless otherwise specified. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims. [Example]

[0542] Example 1. Production of mouse anti-human sortilin antibody

[0543] A. Immunity

[0544] Eight approximately 6-week-old female mice of various ancestry (2x Swiss Webster, 2x NZB / w, 2x AJ, 1x SJL, and 1x Balb / C) were immunized subcutaneously at the hock (lateral tarsus just above the ankle) with recombinant human sortilin extracellular domain (ECD) bearing a C-terminal HIS tag (ECD-huSortilin-HIS) as shown in Table 5 below. The immunogen in PBS was mixed 1:1 with the appropriate adjuvant (see table below) for the first eight injections, then received a final injection without adjuvant. Mice were then titered against ECD-huSortilin-HIS (R&D Systems) and an unrelated HIS-tagged protein to ensure specificity for sortilin on days 17 and 24 of a 28-day immunization schedule.

[0545] [Table 5]

[0546] B. Fusion & Screening

[0547] All mice showed antigen-specific titers and were sacrificed. Popliteal, inguinal, and mesenteric lymph nodes, as well as spleens, were harvested (spleens from all mice were pooled, and lymph nodes from all mice were pooled) and processed. Splenocytes were cryopreserved, and B cell enrichment (Stem Cell Technologies) was performed on pooled lymphocytes according to the manufacturer's protocol. Approximately 80 million B cells were fused at a 1:1 ratio with SP2 / 0 mouse myeloma cells (ATCC) by electrofusion. One-third of the fused cells were seeded into 11 x 384-well plates in fusion medium (80 μl / well) and incubated at 37°C with 5% CO2. The remaining cells were cryopreserved.

[0548] After 8–10 days, the supernatants were screened by ELISA. For the primary screening, 384-well ELISA plates (chemiluminescence-compatible) were coated with 1 μg / mL recombinant ECD-huSortilin-HIS (20 μL / well) and incubated overnight at 4°C. All plates were blocked with 3% BSA in PBS for 1 h at room temperature. The plates were then washed three times with Tris-buffered saline containing 0.1% Tween 20 (TBST). 20 μL of supernatant was added to similar wells of the ELISA plate, incubated, and then washed as described above. 20 μL / well of goat anti-mouse IgG-specific HRP (Jackson Immunoresearch) diluted 1:7000 in 1% BSA in TBST was then added to react with the bound antibody, incubated for 1 h, and washed as described above. Super signal Pico chemiluminescent ELISA substrate (ThermoFisher) was used for detection. Twenty microliters of substrate was added to each well, and relative light units (RLU) were immediately measured using a SpectraMax Paradigm (Molecular Devices). The top 576 wells (based on the RLU signal) were transferred to a 96-well plate and cultured for two days. Confirmatory screening was then performed using ECD-huSortilin-HIS and an unrelated His protein to determine whether the antibodies were sortilin-specific. 105 nonclonal hybridomas were selected from the confirmation screening and further cultured for both cryopreservation and generation of saturated supernatants for further testing. 105 saturated supernatants were tested for blocking of progranulin (sortilin's ligand) binding to sortilin by ELISA (as in Example 4). The results are shown in Figure 7. 29 antibodies blocked progranulin binding to sortilin. 29 blocking hybridomas and two nonblocking (negative control) hybridomas were thawed and cloned to generate monoclonal antibodies.

[0549] Example 2: Cloning and purification of monoclonal antibodies

[0550] Hybridoma cloning was performed by serial dilution or using a Clonepix 2 (Molecular Devices). Hybridomas cloned using the Clonepix 2 were performed according to the manufacturer's protocol. For serial dilution cloning, cells were counted and seeded into half of a 384-well plate in cloning medium at a density of 0.5 cells / well and incubated at 37°C with 5% CO2. The next day, each well was visually inspected under a microscope, and wells containing single colonies of cells were marked as monoclonal. After 7–8 days of culture, supernatants were screened by ELISA for binding to ECD-huSortilin-HIS. 28 monoclonal antibodies were successfully cloned. 28 hybridomas were expanded for cryopreservation and antibody purification. Antibodies were purified using protein A chromatography using standard methods. After purification, the antibodies were exchanged into 1x PBS, and protein concentrations were determined by absorbance at 280 nm. The purified monoclonal antibodies (mAbs) were then transferred for further screening.

[0551] Example 3: Sortilin Binding ELISA

[0552] Purified anti-huSortilin mAbs binding to huSortilin were characterized by ELISA (ELISA protocol as in Example 1) using a 12-point titration of mAb concentration. mAbs were diluted 2-fold from a starting concentration of 67 nM and then added to ECD-huSortilin-HIS coated plates. Data were analyzed and graphed using Prism. 50 The activity (nM) was calculated. A commercially available goat anti-huSortilin antibody (R&D Biosystems) was used as a positive control. The results are shown in Figure 8.

[0553] Example 4 Progranulin Blocking ELISA

[0554] Blocking ELISA using non-clonal saturated supernatant

[0555] To select which hybridomas to clone, we examined whether the saturated supernatants blocked the binding of progranulin to sortilin. Untagged human progranulin (huPGRN, Adipogen) was first biotinylated at a 5:1 ratio using EZ-Link NHS-PEG4-biotin (Thermo Fisher) and ZebaSpin columns according to the manufacturer's protocol. The results are shown in Figure 7.

[0556] 384-well ELISA plates were coated with 2 μg / mL recombinant ECD-huSortilin-HIS (20 μL / well) and incubated overnight at 4°C. All plates were blocked with 3% BSA in PBS for 1 hour at room temperature. Plates were then washed three times with TBST. Saturated supernatants were diluted 4-fold with 1% BSA in TBST. 20 μL of diluted supernatant was added to wells and incubated for 1 hour at room temperature. Plates were then washed with TBST, and 20 μL of 2 μg / mL huPGRN-biotin was added to each well, incubated for 1 hour at room temperature, and then washed as above. Streptavidin-HRP (GE Life Sciences) diluted 1:10,000 in 1% BSA in TBST was then added at 20 μL / well to react with the bound huPGRN-biotin and incubated for 1 h, followed by washing and detection using Super signal Pico chemiluminescent ELISA substrate (ThermoFisher) as described above. Hybridomas that blocked huPGRN binding to huSortilin by at least 30% (corresponding supernatants) compared to the no Ab control were selected for cloning.

[0557] Blocking ELISA using purified mAb

[0558] Twenty-eight purified anti-huSortilin mAbs were further characterized for their ability to block PGRN binding to Sortilin. Blocking ELISAs were performed as described above using six 3-fold dilutions of mAb in 1% BSA in TBST starting at 134 nM. Data were analyzed and % blocking calculated using Microsoft Excel and GraphPad Prism. Results are shown in Figure 8.

[0559] Example 5: Neurotensin competitive Biacore assay

[0560] PGRN and neurotensin (NT) are known to bind to similar regions on sortilin. Surface plasmon resonance (SPR) (Biacore T200 instrument) was used to investigate whether anti-huSortilin mAb blocks NT binding to sortilin. Briefly, 25 nM anti-huSortilin mAb was captured onto a CM5 anti-mouse Fc chip (60 s association). Then, 100 nM recombinant ECD-huSortilin-HIS (60 s association, 30 s dissociation) preincubated with 0 nM, 100 nM, 500 nM, or 1000 nM NT (Tocris) was flowed over the captured mAb surface. The chip surface was regenerated between cycles using 10 nM glycine pH 1.5. Data were analyzed and graphed using Biacore T200 Evaluation Software 2.0.15.12933. Eighteen anti-Sortilin mAbs did not compete with NT for binding to Sortilin, six showed some competition, and three clearly competed with NT for binding to Sortilin. The results are shown in Figure 9.

[0561] Example 6: Sortilin Binding Cell-Based Assay

[0562] We then examined the ability of purified anti-Sortilin mAbs to bind recombinant huSortilin expressed on the surface of HEK293 cells and U251MG cells (a human glioblastoma cell line) that endogenously express Sortilin on the cell surface. Parental HEK293 cells were used as a negative control. All 28 purified anti-Sortilin mAbs were tested at a single concentration (67 nM). Cells were harvested and seeded at 20K cells per well in a 96-well plate. mAbs diluted in PBS + 2% FBS were added to the wells (50 μL / well), and the cells were incubated on ice for 1 hour. The cells were then washed twice with PBS + 2% FBS and incubated with 50 μL / well of 5 μg / mL Alexa-Fluor 647 goat anti-mouse IgG (Jackson Immunoresearch) in PBS + 2% FBS for 30 minutes on ice. The cells were then washed twice as described above and resuspended in 100 μL of PBS + 2% FBS. Data were acquired using a BD LSR II and analyzed using FlowJo software version 10.6. Calculation of median fluorescence intensity (MFI) values ​​showed that all 28 anti-Sortilin mAbs bound to huSortilin-expressing HEK293 cells. Three of them also showed background binding to parental HEK cells. Twenty-two mAbs showed varying degrees of binding to Sortilin on the surface of U251MG cells. Six mAbs showed no binding to U251 cells. The results are shown in Figure 10.

[0563] Example 7: Progranulin Blocking Titration Cell-Based Assay

[0564] All 28 purified anti-Sortilin mAbs were tested at a single concentration (67 nM) to determine their ability to block huPGRN-biotin binding to cell surface huSortilin-expressing HEK293 cells. Cells were cultured using standard methods. Cells were harvested and seeded at 20,000 cells per well in a 96-well plate. mAbs diluted in PBS + 2% FBS containing 30 nM huPGRN-biotin were added to the wells (50 μL / well), and the cells were incubated on ice for 1 hour. Cells were then washed twice with PBS + 2% FBS and incubated with 50 μL / well of 1:200 steptavidin-APC (BD Biosciences) in PBS + 2% FBS for 30 minutes on ice. Cells were then washed as above and resuspended in 100 μL of PBS + 2% FBS. Data were acquired using a BD LSR II and analyzed using FlowJo software version 10.6 and Microsoft Excel. Twelve anti-Sortilin mAbs showed % blocking of huPGRN greater than 30% and were selected for further characterization. The results are shown in Figure 11.

[0565] The cell-based blocking assay was repeated with the top 12 blocking mAbs using dose escalation from 67 nM to 0.4 nM. Blocking assays were performed as described above. Data were analyzed using FlowJo software version 10.6 and GraphPad Prism software. The results are shown in Figure 12.

[0566] The 12 mAbs were divided into three tiers. Tier 1 contained mAbs that showed high potency for huPGRN blocking (8H24, 5E20, 2C14), and seven mAbs (tier 2) showed moderate blocking potency (2F18, 6B15, 11M14, 11H24, 6M23, 2P22, and 9N18). The final two mAbs (7A22 and 5L16) showed low huPGRN blocking potency.

[0567] Example 8: Extracellular Progranulin Levels and Surface Sortilin Levels

[0568] U251MG cells were seeded into 96-well plates and incubated overnight at 37°C with 5% CO2. The next day, the medium was removed, and fresh medium containing 50 nM of either an anti-Sortilin mAb, a commercially available goat anti-hSortilin (positive blocking antibody, R&D Biosystems), or an isotype control antibody was added to the wells. The cells were then cultured for 72 hours. The supernatant was then used to determine extracellular huPGRN levels, and the cells were harvested to examine cell surface Sortilin levels.

[0569] Increased PGRN levels were measured by plate-based electrochemiluminescence using a Meso Scale Discovery (MSD). Standard binding 96-well MSD plates were coated overnight at 4°C with 1 μg / mL mouse anti-huPGRN (R&D Biosystems) in PBS. The plates were blocked with 3% MSD Buffer A in PBS for 1 hour at room temperature. The plates were then washed three times with TBST. 50 μL of cell supernatant was added to the wells and incubated for 1 hour at room temperature. The plates were then washed as above, and 50 μL of 1 μg / mL biotinylated goat anti-huPGRN (R&D Biosystems) in 1% MSD Buffer A in PBS was added to the wells, and the plates were incubated for 1 hour at room temperature. The plates were washed as above, and 0.5 μg / mL SULFO-TAG-streptavidin (MSD) in 1% MSD Buffer A in PBS was added to the wells. The plates were incubated for 1 hour at room temperature and then washed as above. 150 μL of 2x MSD Read Buffer T was added to the wells, and the electrochemiluminescence signal was read using a Meso Sector S 600. Data were analyzed using Discovery Workbench software (MSD) and quantified against a standard curve using 3-fold serial dilutions starting with 100 ng / mL recombinant huPGRN (R&D Biosystems). Fold PGRN levels relative to the control were calculated using Microsoft Excel. Nineteen of the 25 mouse anti-huSortilin mAbs tested showed a >1.5-fold increase in huPGRN without mAb control or isotype control wells. The results are shown in Figure 13.

[0570] The percent reduction in surface sortilin levels was determined by flow cytometry. U251MG cells were harvested and seeded into 96-well untreated plates. 2.5 μg / mL goat anti-human sortilin antibody (R&D Biosystems) was added to the wells (50 μL / well), and the cells were incubated on ice for 1 hour. The cells were then washed twice with PBS + 2% FBS and incubated with 50 μL / well 5 μg / mL Alexa-fluor 647-donkey anti-goat (Jackson Immunoresearch) secondary antibody in PBS + 2% FBS for 30 minutes on ice. The cells were then washed twice as described above and resuspended in 100 μL of PBS + 2% FBS. Data were acquired using a BD LSR II and analyzed to determine median fluorescence intensity (MFI) using FlowJo software version 10.6. The MFI values ​​were used to calculate the percent cell surface huSortilin compared to the no mAb control wells using Microsoft Excel. Most mAbs reduced cell surface sortilin levels in U251 cells to varying degrees. 13 / 25 mouse anti-huSortilin mAbs increased extracellular huPGRN levels by more than 1.5-fold and reduced cell surface sortilin by up to 60%. The results are shown in Figure 13.

[0571] Example 9: Characterization of mouse mAbs by BIAcore

[0572] To compare the binding affinity of the mouse anti-huSortilin antibody with recombinant human sortilin, analysis was performed using a Biacore T200. The anti-mouse antibody was immobilized on a SensorChip CM5 (GE Healthcare Life Sciences) via amine coupling, and the mouse antibody (ligand) was captured to a level ensuring maximum binding of 50 RU (approximately 100 RU of ligand binding). Various concentrations of sortilin (ranging from 0.1 nM to 300 nM) were passed over the captured ligand at 50 μL / min in running buffer (HBS + 0.05% P-20, 1 mg / mL BSA) for association times of 180 to 300 seconds and dissociation times of 300 to 900 seconds. Regeneration of the chip surface was achieved by two short injections of 10 mM glycine-HCl, pH 1.7. Data were blank-subtracted against both the sensor without ligand and a 0 nM analyte concentration. The analysis was performed using a global 1:1 fit with Biacore Insight Evaluation software (v2.0) with the bulk refractive index set to 0 RU. Binding data are shown in Table 6.

[0573] [Table 6]

[0574] Example 10: Epitope mapping of mouse 5E20

[0575] PEPperMAP linear epitope mapping was performed by PEPperPrint GmbH. Epitope analysis of 5E20 was performed by peptide microarray analysis. The sequence of the extracellular domain of human sortilin (756 amino acids, SEQ ID NO: 1) was extended with neutral GSGSGSGSG (SEQ ID NO: 207) linkers at the C- and N-termini to avoid truncated peptides. The concatenated extended antigen sequence was translated into linear 15-amino acid peptides with 14-amino acid peptide overlaps. The resulting human sortilin peptide microarray contained 756 different peptides printed in duplicate (1,512 spots) and was framed by additional HA (YPYDVPDYAG, SEQ ID NO: 200, 46 spots) and c-Myc (EQKLISEEDL, SEQ ID NO: 201, 46 spots) control peptides.

[0576] After synthesis, the microarrays were blocked (Rockland catalog number MB-070) to prevent nonspecific binding. Mouse 5E20 was then applied to the microarrays at a concentration of 1 μg / mL along with a positive control mouse monoclonal anti-HA (12CA5) DyLight 800 (0.5 μg / mL) at 4°C for 16 hours with shaking at 140 rpm. The microarrays were washed, and a secondary antibody (goat anti-mouse IgG (H+L) DyLight 680 (0.2 μg / mL)) was applied for 45 minutes at room temperature. After further washing, the microarrays were imaged using a Licor Odyssey Imaging System.

[0577] Spot intensity quantification and peptide annotation were based on 24-bit grayscale tiff files with a scan intensity of 7 / 7, which exhibits a higher dynamic range than 16-bit colorized tiff files. Microarray image analysis was performed using the PepSlide® Analyzer. A software algorithm separated the fluorescence intensity of each spot into raw, foreground, and background signals and calculated the average median foreground intensity and inter-spot deviation of spot replicates (see the "Raw Data" tab). Based on the median averaged foreground intensities, an intensity map was generated, and interactions in the peptide map were highlighted by an intensity color code, with red for high spot intensities and white for low spot intensities. A maximum inter-spot deviation of 40% was allowed; otherwise, the corresponding intensity value was set to zero.

[0578] A very strong monoclonal antibody response to an epitope-like spot pattern formed by adjacent peptides with the consensus motif FTESFLT was observed for mouse 5E20 (SEQ ID NO: 202). [ka] Two very weak additional responses to peptides with were observed, likely due to cross-reactivity based on slight sequence similarity (see underlined amino acid positions).

[0579] Example 11: Characterization of humanized mAbs: Fusortilin binding ELISA

[0580] 384-well ELISA plates were coated with 1 μg / mL recombinant ECD-huSortilin-HIS (20 μL / well) and incubated overnight at 4°C. All plates were blocked with 3% BSA in PBS for 1 hour at room temperature. Plates were then washed three times with Tris-buffered saline containing 0.1% Tween 20 (TBST). 20 μL of a 3-fold dilution of humanized anti-Sortilin mAb from 20 μg / mL to 0.3 ng / mL in 1% BSA in TBST was added to the wells, incubated for 1 hour at room temperature, and then washed as described above. Goat anti-human IgG-specific HRP (Jackson Immunoresearch) diluted 1:7000 in 1% BSA in TBST was then added at 20 μL / well to react with the bound antibody. Plates were incubated for 1 hour and then washed as described above. Super signal Pico chemiluminescent ELISA substrate (ThermoFisher) was used for detection. 20 μL of substrate was added to each well, and relative light units (RLU) were immediately measured using a SpectraMax Paradigm (Molecular Devices). Data were analyzed and g...

Claims

1. a humanized light chain variable region comprising the CDRs of SEQ ID NO:59, SEQ ID NO:72, and SEQ ID NO:61; a humanized heavy chain variable region comprising the CDRs of SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55; a humanized light chain constant region fused to the humanized light chain variable region; and a humanized heavy chain constant region, with or without a C-terminal lysine, fused to the humanized heavy chain variable region, the humanized heavy chain constant region comprising Y at position 252, T at position 254, and E at position 256 according to EU nomenclature; A humanized anti-human sortilin antibody comprising:

2. 2. The humanized anti-human sortilin antibody of claim 1, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 190, and the humanized light chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO:

198.

3. 2. The humanized anti-human sortilin antibody of claim 1, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 190 and the humanized light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

198.

4. 2. The humanized anti-human sortilin antibody of claim 1, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 98% identical to SEQ ID NO: 190, and the humanized light chain variable region comprises an amino acid sequence at least 98% identical to SEQ ID NO:

198.

5. 2. The humanized anti-human sortilin antibody of claim 1, wherein the humanized heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 190 and the humanized light chain variable region comprises the amino acid sequence of SEQ ID NO:

198.

6. The humanized anti-human sortilin antibody of claim 5, wherein the humanized anti-human sortilin antibody has a human IgG1 isotype.

7. The humanized anti-human sortilin antibody of claim 6, wherein the humanized heavy chain constant region comprises A at position 234 and A at position 235 according to the EU nomenclature.

8. The humanized anti-human sortilin antibody of claim 7, wherein the humanized anti-human sortilin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 244, with or without a C-terminal lysine, and a light chain comprising the amino acid sequence of SEQ ID NO:

245.

9. a humanized light chain variable region comprising the CDRs of SEQ ID NO:59, SEQ ID NO:72, and SEQ ID NO:61; a humanized heavy chain variable region comprising the CDRs of SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:55; a humanized light chain constant region fused to the humanized light chain variable region; and a humanized heavy chain constant region, with or without a C-terminal lysine, fused to the humanized heavy chain variable region, the humanized heavy chain constant region comprising A at position 234, A at position 235, M at position 252, S at position 254, and T at position 256 according to EU nomenclature; A humanized anti-human sortilin antibody comprising:

10. 10. The humanized anti-human sortilin antibody of claim 9, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO: 190 and the humanized light chain variable region comprises an amino acid sequence at least 90% identical to SEQ ID NO:

198.

11. 10. The humanized anti-human sortilin antibody of claim 9, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 190 and the humanized light chain variable region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

198.

12. 10. The humanized anti-human sortilin antibody of claim 9, wherein the humanized heavy chain variable region comprises an amino acid sequence at least 98% identical to SEQ ID NO: 190 and the humanized light chain variable region comprises an amino acid sequence at least 98% identical to SEQ ID NO:

198.

13. 10. The humanized anti-human sortilin antibody of claim 9, wherein the humanized heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 190 and the humanized light chain variable region comprises the amino acid sequence of SEQ ID NO:

198.

14. The humanized anti-human sortilin antibody of claim 13, wherein the humanized anti-human sortilin antibody has a human IgG1 isotype.

15. The humanized anti-human Sortilin antibody of claim 14, wherein the humanized anti-human Sortilin antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 250, with or without a C-terminal lysine, and a light chain comprising the amino acid sequence of SEQ ID NO:

245.

16. A pharmaceutical composition comprising the humanized anti-human sortilin antibody of any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. A nucleic acid encoding the heavy and light chains of a humanized anti-human sortilin antibody according to any one of claims 1 to 15.

18. A composition for increasing progranulin levels in a subject having or at risk of developing a disease or disorder associated with altered progranulin levels, the composition comprising a humanized anti-human sortilin antibody described in any one of claims 1 to 15.

19. A composition for treating or preventing a disease or disorder associated with altered progranulin levels in a subject, the composition comprising a humanized anti-human sortilin antibody described in any one of claims 1 to 15.

20. 20. The composition of claim 19, 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.

21. 16. A composition for detecting sortilin in a subject having or at risk of a disease associated with a disease or disorder associated with altered progranulin levels, the composition comprising a humanized anti-human sortilin antibody described in any one of claims 1 to 15.

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