Antibodies that bind to sortilin and inhibit the binding of progranulin

The present invention relates to monoclonal anti-sortilin antibodies useful for modifying inadequate levels of progranulin (PGRN). In particular, these antibodies can be used to treat frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In addition, monoclonal antibodies are also predicted to be useful in treating neurodegenerative diseases such as Alzheimer's disease (AD).

Sequence Listing Reference: This application contains one or more sequence listings (see below) in accordance with Section 1.821 of the US Patent Law Enforcement Regulations (37 CFR), which is a computer-readable medium (file name: 0993_ST25.txt, file name: 0993_ST25.txt, Published June 22, 2016 and having a size of 144 kB), this file is incorporated herein by reference in its entirety.

Sortilin is a receptor that has been reported to mediate the apoptosis-promoting effects of proneurotrophin and to mediate the transport and sorting of neurotrophin receptors (Nykjaer et al, 2012, Trends Neurosci. 2012; 35). (4): 261-70; Glerup et al, Handb Exp Pharmacol, 2014; 220: 165-89, Carlo et al, J Mol Med (Berl). 2014 Sep; 92 (9): 905-11). Several sortilin ligands, including neurotensin, have been identified, and X-ray crystallography has identified a high-affinity binding site for neurotensin inside the β-propeller tunnel in the sortilin molecule (" Quistgaard et al, Nat Struct Mol Biol.2009 Jan; 16 (1): 96-8; Quistgaard et al, Protein Sci.2014, Sep; 23 (9): 1291-300). More recently, sortilin has been shown to function as a high affinity receptor for the growth factor progranulin (PGRN, Hu et al. Neuron. 2010 Nov 18; 68 (4): 654-67.

PGRN ((Proepicerin, Granulin-Episerin precursor, PC cell-induced growth factor, acroglanin)) is a secretory glycosylation protein with anti-inflammatory and neurotrophic effects (for recent reports, Nguyen, Trends Endocrinol). See Metab.2013 Dec; 24 (12): 597-606). Although PGRN is proteolytically cleaved into granulin, much remains unknown regarding the physiological role of PGRN and granulin and the properties of their receptors. PGRN is a cell cycle regulator and cell motility (He, Z. & Bateman, A., J.MoI.Med. 57: 600-612 (2003); Monami, G., et al., Cancer Res. (5). (5: 7103-7110 (2006)), Wound repair, inflammation (Zhu, J., et al., Cell 777: 867-878 (2002)), induction of growth factors such as vascular endothelial growth factor (VEGF) ( Tangkeangsiπsin, W. & Serrero, G, Carcinogenesis 25.1587-1592 (2004)), and Tumor Development (He, Z. & Bateman, A., J.MoI.Med.81: 600-612 (2003), Monami, G., et al., Cancer Res (5) (5: 7103-7110 (2006); Serrero, G., Biochem Biophys.Res.Commun.505-409-413 (2003), Lu, R & Serrero, G., Proc.Natl Acad Sci U.SA 98 142 -147 (2001); involved in several cellular functions including Liau, LM., Et al., Cancer Res. 60: 1353-1360 (2000)). PGRN binds to TNF receptors It has been reported (Tang W et al., Science 2011, 332 (6028): 478-84), but this observation was also attempted by others (Chen et al., J Neurosci. 2013, 33 (21): 9202-9213).

Binding of PGRN to sortilin has been reported to be mapped to the neurotensin site and, like neurotensin, mediated only through the C-terminal domain of PGRN (Zheng et al. PLoS One. 2011; 6). (6): e21023; Lee et al. Hum Mol Genet. 2013), therefore, neurotensin has been shown to block the interaction of sortilin with PGRN and other ligands. Upon binding, sortilin mediates lysosomal clearance of PGRN, thereby regulating extracellular PGRN levels (Hu et al. 2010). Therefore, it has been shown that sortilin knockdown or overexpression regulates extracellular PGRN levels in cell cultures (Carrasquillo et al. Am J Hum Genet. 2010 Dec 10; 87 (6): 890-7). , Sortilin deficiency in mice has been reported to increase PGRN levels and restore plasma and brain PGRN levels in PGRN +/- mice (Hu et. al.2010). Interestingly, single nucleotide polymorphisms (SNPs) close to sortilin were associated with decreased plasma PGRN and increased sortilin mRNA levels (Carrasquillo et al. Am J Hum Genet. 2010 Dec 10; 87 (6)). : 890-7). These observations suggest that sortilin is a major regulator of extracellular PGRN.

PGRN is frontotemporal dementia (FTD), progressive dementia characterized by behavioral and semantic changes, and frontotemporal lobar degeneration (FTLD) and TAR DNA-binding protein-43 (TDP-43) or tau. Associated with nerve inclusions containing inclusion bodies (Baker et al, 2006, Nature. 2006 Aug 24; 442 (7105): 916-9; Cruts et al, Nature 442: 920-924 (2006); Am J Hum Genet.2010 Dec 10; 87 (6): 890-7, M et al, Trends in Genetics 24: 186-194 (2008)). Most cases of sporadic and familial FTD show TDP-43 lesions similar to ALS (about 50%), due to common pathology and some overlap of genetic factors and symptoms. (Ito D Neurology. 2011 Oct 25; 77 (17): 1636-43; Boxer AL et al, Alzheimers Dement.2013 Mar; 9 (2): 176-88; Rademakers et al, Nat Rev Neurol.2012 August; 8 (8): 423-434). There are no disease-modifying treatment options available for FTD. Some patients with frontal temporal dementia with TDP-43 lesions have a loss-of-function mutation in the granulin gene (GRN) that results in PGRN haploinsufficiency. So far, 69 different mutations in the granulin gene (all resulting in decreased PGRN levels and / or function) have been associated with FTD, and increased extracellular PGRN in plasma and brain can suppress disease processes. it is conceivable that.

PGRN mutations have also been associated with Alzheimer's disease (AD) (Sheng et al., 2014, Gene. 2014 Jun 1; 542 (2): 141-5; Brouwers et al., 2008, Neurology. 2008 Aug. 26; 71 (9): 656-64), suggesting that PGRN deficiency may play an important role in the development of AD. In addition, a neuroprotective effect of PGRN has been observed in mouse AD models (Minami et al, 2014, Nat Med. 2014 Oct; 20 (10): 1157-64), which means that enhancement of PGRN is associated with AD and It supports the idea that it may be beneficial for other neurodegenerative diseases.

<p> This application describes the generation and identification of anti-human saltylin antibodies that can regulate PGRN in cell models and mice. These antibodies unexpectedly bind to a region of sortilin that is distant from the previously reported progranulin binding site, the so-called neurotensin site, and further inhibit the sortilin-PGRN interaction, thereby inhibiting extracellular PGRN. Can be increased.</p><p> We defined six sortilin binding regions and surprisingly confirmed that the most effective antibody binds to a region (Region D). Because PGRN has neuroprotective and anti-inflammatory effects, our findings suggest that such an antibody targeting sortilin may have beneficial effects on a variety of neurodegenerative diseases, including FTD / FTLD. Indicates high sex. A subgroup of these patients has a mutation in the gene encoding PGRN that results in haploinsufficiency. Therefore, sortilin antibodies may provide similar therapeutic effects to patients suffering from other TDP-43 proteinosis and disease in which PGRN levels can affect TDP43 function and lesions (including ALS and AD). Is high.</p>

<p> The inventors of the present invention have produced a monoclonal antibody capable of inhibiting the binding of PGRN to sortilin, which unexpectedly binds to a novel sortilin region designated as "D region" defined by SEQ ID NO: 170. .. Some of the antibodies identified by us have similar properties to D-region antibodies, and experimental evidence has shown that they also bind within the D-region, and these antibodies are described in this article. In the specification, it is called a D + antibody. Accordingly, in one aspect, the invention relates to such antibodies, compositions and / or kits comprising such antibodies, and methods and uses thereof.</p><p> The present invention is a method of preventing or treating a disease associated with decreased PGRN levels in a patient's brain, wherein an effective dose of an antibody that binds to the D region of sortilin or an antigen-binding fragment thereof is administered. It also concerns how to include it. These disorders include , among others, FTD, ALS and TDP43 proteinosis (such as AD).</p>

<figref num="1">Providing an overview of the development of region allocations for human antibodies based on sortilin region binding, PGRN binding and effects on PGRN levels and cross-blocking between antibodies. Sortilin-binding antibodies were selected and assigned to regions A-E based on binding to the shuffle construct whose sequence corresponded to the tetraodon soltilin sequence within the selected region of the protein (Example 1, Figure 2). Twenty antibodies that inhibited sortilin-PGRN binding (as measured by HTRF analysis) were selected (see Example 10, FIGS. 5 and 6). Of these antibodies, 15 were D-region antibodies, while 3 were D + antibodies (Fig. 6). Subsequent cross-blocking analysis showed 18 D-region and D + antibodies (D + antibodies have different binding patterns to shuffle constructs than D-region antibodies. Nevertheless, D + antibodies are bound regions A outlined above. It indicates that all of the functional properties similar to the D region antibody (such as cell assays) that could not be reliably assigned to ~ E (such as cell assays) cross-blocked each other, indicating that they were in the same saltyrin region. Support the interaction (Example 9, Figure 7). When other region-class sortilin antibodies were tested in the HTRF sortilin-PGRN binding assay, only two of the 41 antibodies showed inhibitory effects. One of these two antibodies cross-blocked with D and D +, but with atypical shuffle construct binding pattern (except that it bound to the hB01-05 region). (Similar), the other antibody did not cross-block with the other antibody that inhibited PGRN-sortilin binding, thus supporting the conclusion that it binds to another sortilin region. These observations indicate that antibody binding to a region in sortilin defined by the D region may inhibit sortilin-PGRN binding. Nineteen cross-blocking antibodies, 18 of which are D-region and D + antibodies, increased extracellular PGRN in the cell assay (Example 13, FIG. 10 and FIG. 11). Three of these antibodies were tested in vivo and found to increase plasma PGRN (FIG. 13, Example 15). The box in FIG. 1 shows the steps in antibody selection. A to E refer to regions to which each sortilin binding antibody has been assigned based on the shuffle construct as set forth in Example 1 and SEQ ID NOs: 171-179. Other refers to an antibody that cannot be assigned to one region and can bind at the boundary between regions A and B. Tet refers to an antibody that also binds to tetraodon-sortilin. In addition to the indicated human antibodies, a series of mouse anti-human soltilin antibodies were generated and characterized in the same manner. Two of these antibodies were shown to be assigned to the D region, cross-block with the human D region and D + antibody, inhibit sortilin-PGRN binding, and increase extracellular PGRN (see Figure 4). ).</figref><figref num="2">The region allocation of the antibody based on the binding to the saltylin shuffle construct is shown. Panel A shows a linear diagram of the shuffle construct used for antibody region allocation as described in Example 1. Soltilin shuffle construct based on the human soltilin sequence (SEQ ID NO: 169) (part in gray) in which the amino acid residue was exchanged for the corresponding amino acid from the tetraodone soltilin sequence (shown in black) (SEQ ID NO: 173). Was generated (Examples 1 to 3). Panel B shows the predicted structure of the shuffle construct shown linearly in A. Dark-colored residues indicate residues modified to the corresponding tetraodon sequence in the shuffle construct. Panel C shows the binding patterns of the antibodies assigned to the D and E region classes, respectively. A "+" indicates a bond to a given shuffle construct, and a "-" indicates no bond. Antibodies were assigned to the regions based on binding patterns to different shuffle constructs. The resulting antibody region class is indicated by A ~ E. In the D and E region antibodies shown, both bind to the human sequence as indicated by the "+" (all gray) and none to the tetraodon sequence as indicated by the "-" (all black). The E-region antibody bound to the hB45678 shuffle construct, while the D-region antibody did not, resulting in binding localization as shown in panel A. For D-region antibodies, binding to the following shuffle regions was observed: hsort, hB06-10, B12390. The antibody did not bind to hB01-05, B45678, tet. For D + antibodies, binding to the following shuffle regions was observed: hsort, B12390. The antibody did not bind to hB01-05, hB06-10, B45678, tet. The F-binding pattern was similar to the D-binding pattern, except that no binding to hB06-10 was observed for the D + antibody. The antibody did not bind to the complete tetraodon saltylin protein except for two. Two antibodies capable of binding to the tetraodon sequence were shown as "tet". Other refers to an antibody that could not be assigned to one region.</figref><figref num="3">It shows the binding affinity of human D region and D + antibody. Binding affinity for Soltilin shuffle constructs by biolayer interferometry with Octet 384 RED (EC50, ng / ml) as described in Example 8. No shading indicates an EC50 of 0.1 ~ 10 ng / ml, light gray shading indicates EC50&gt; 10 ng / ml, and gray shading indicates no binding (NB). Area allocation was made based on the join pattern shown in Figure 2. The shuffle construct is shown in FIG. 2 and the sequences are shown in SEQ ID NOs: 171-179. mAb = monoclonal antibody.</figref><figref num="4">The binding affinity of mouse anti-human antibodies to the saltylin shuffle construct obtained by biolayer interferometry with Octet 384 RED (EC50, ng / ml) as described in Example 8 is shown. No shading indicates binding, and gray shading indicates no binding (NB). Area allocation based on the join pattern is shown in Figure 2.</figref><figref num="5">The effect of sortilin antibody on sortilin PGRN binding is shown. D-region sortilin human monoclonal (humAb) antibody 45 (black circles) did not interfere with binding PGRN to sortilin in contrast to control sortilin E-region antibodies (black triangles) and IgG controls, IgG1-b12 (white triangles). Prevented the bond. Antibody binding was determined by measuring the transfer of PGRN binding to sortilin using homogeneous time-resolved fluorescence (HTRF) (Example 10). Dose-response evaluation of the antibody was performed at 10 concentrations covering 50 pM to 1 μM on the 3-fold dilution curve. Half-inhibition concentration (IC50) values were calculated by non-linear regression using an S-shaped concentration reaction (variable slope) in XLfit 4 (IDBS, UK).</figref><figref num="6">A summary of the effect of the antibody on sortilin-PGRN binding as determined by the homogeneous time-resolved fluorescence (HTRF) analysis shown in Figure 5. A total of 62 antibodies were tested-15 D-region antibodies and 3 D + antibodies were found to inhibit sortilin-PGRN binding, and IC50 values were determined. Inhibitory effects were observed for two additional antibodies (E and other regions). All remaining antibodies were negative in the test.^*The antibody is too weak to fit the dose-response curve. 6% inhibition at 1 μM.^**The control (ctrl) antibody is too weak to fit the dose-response curve. 37% inhibition at 1 μM. These observations indicate that sortilin antibodies characterized by their D region or D + assignment can directly inhibit sortilin binding to PGRN and inhibit sortilin-PGRN binding.</figref><figref num="7">Shows cross-blocking between antibodies. All human and mouse antibodies were tested in a single experiment, where each antibody was bound to human wild-type (WT) sortilin (Fig. 7). Subsequently, all other antibodies were tested for binding to the preformed sortilin: antibody complex (Example 9). (Based on their effect in the HTRF PGRN-sortilin assay, selected 15 D regions and 3 D + human antibodies (Figures 5 and 6) and 2 mouse D region antibodies are all human WT. Inhibited each other's binding to sortilin. Antibodies are designated in other region classes (Table) except for one cross-blocking A region antibody, one antibody with an unknown region allocation (Other) and partial block for D + antibody 548. It did not cross-block with (as shown for the A, E and tetraodon regions) that recognize antibodies numbered AbA1-x, AbE1-x and Abtet, respectively. These data support that all D and D + antibodies capable of inhibiting sortilin-PGRN binding in the HTRF assay interact with the same region in sortilin. Cross-blocking between sortilin antibodies from the same or different regions (regions based on binding to the shuffle construct shown in FIG. 2) was determined by analyzing inhibition of antibody-sortilin binding. Binding of antibody to sortilin-ECD-His, Octet It was measured by the biolayer interferometry using 384RED (Example 9). The left column shows the primary (immobilized) antibody, and the ascending line shows the secondary antibody (antibody tested against the immobilized antibody). Binding of both primary and secondary antibodies to sortilin-ECD-His resulted in response values above 0.1, indicating that both antibodies were bound to different regions of the protein. Reaction values below 0.1 indicate the absence of secondary antibody binding and effective cross-blocking with immobilized (primary) antibodies, suggesting that both antibodies bind to the same region of sortilin.</figref><figref num="8">The effects of the D region and D + sortilin antibody on the binding of the selective small molecule ligand AF38469 to sortilin are shown. The AF38469 binding site was shown to be similar to the neurotensin binding site and was characterized by X-ray crystallography (Schroeder et al. Bioorg Med Chem Lett. 2014 Jan 1; 24 (1): 177-80). PGRN has been reported to bind to the same site (Lee et al. Hum Mol Genet. 2013), and antibodies 45 and 68 that bind to the D region and D +, respectively, inhibit the binding of AF38469 to sortilin. There wasn't. This data suggests that these antibodies have a sortilin binding site that is different from the AF38469 binding site. Thus, antibodies 45 and 68 inhibit PGRN-sortilin binding via a binding site different from the previously estimated PGRN binding site in sortilin.</figref><figref num="9">Effects of Antibodies 45 and 68 on Cell Binding and Endocytosis of PGRN (Example 12). Antibodies 45 and 68 inhibited PGRN binding and / or endocytosis by sortilin overexpressing cells. The addition of neurotensin (NT, 10uM) also reduced PGRN binding or endocytosis as expected, as reflected in the reduced fluorescence, while the isotype control antibody B12 affected PGRN fluorescence levels. I didn't give it. The antibody to be tested (100 nM) was added to S18 cells 30 minutes prior to the addition of recombinant PGRN over 4 hours. The cells were then immobilized, stained for PGRN and analyzed by Cellomics. PGRN fluorescence was measured as average fluorescence per cell. The data are shown as mean ± SD. Data were analyzed by one-way Anova followed by Danette's analysis and all groups were compared to PGRN.^*p &lt;0.05;^**p &lt;0.01.</figref><figref num="10">Extracellular PGRN levels estimated by ELISA in medium from cultures of sortilin overexpressing HEK cells (S18). The sortilin D region (45, 811) and D + (68) antibodies increased PGRN levels and similar effects of the sortilin ligand neurotensin were observed, whereas the control antibody B12 had no effect. These observations indicate that the D region and D + sortilin antibody were able to block the sortilin-mediated clearance of PGRN, thereby increasing extracellular PGRN. All antibodies were tested at 100 nM. Neurotensin was tested at 10 uM. The PGRN level was normalized to the control. The data are shown as mean ± SD. Data were analyzed by one-way Anova followed by Danette's analysis and all groups were compared to controls (CTRL).^*p &lt;0.05;^**p &lt;0.01 (Example 13).</figref><figref num="11">The effect of the antibody on extracellular PGRN in human soltilin overexpressing HEK cells as measured by ELISA as described in Example 13 is shown. All selected D region antibodies and 3 selected D + antibodies increased extracellular PGRN. PGRN levels were analyzed in the same manner as above. PGRN levels are normalized to untreated controls and are shown in%. Two antibodies are induced against human soltilin (1F2F4 and 5E1F6) in mice, the rest being human antibodies. Ab = monoclonal antibody.</figref><figref num="12">(Upper and lower panels) show the effect of sortilin antibody on extracellular PGRN in neurally differentiated iPSC cells (Example 14). Sortilin D region antibody 45 and D + antibody 68 increased PGRN levels, while control antibody B12 and anti-HEL had no effect. Neurodifferentiated iPSC cells were plate-cultured into 96-well plates. After 1 week, the antibody was added to the cells. Medium from cells was collected at 48 or 96 hours, analyzed by human PGRN ELISA (Enzo Life sciences), and samples were analyzed according to the manufacturer's instructions. Soltilin human antibodies 45 and 68 increased PGRN levels in the medium at both time points. Control isotype antibody B12 and anti-Hel (negative control) did not alter extracellular PGRN. The data are shown as mean ± SD. The data were analyzed by one-way Anova followed by Danette's analysis.^*p &lt;0.05;^**p &lt;0.01 (Example 14).</figref><figref num="13">(Panels A to C) show plasma PGRN levels in human soltilin-expressing knock-in (KI) mice treated with soltilin human antibody (Example 15). Sortilin antibody 45 increased plasma PGRN levels, while control antibody had no effect. Time-lapse study: Increased plasma levels of PGRN were observed after infusion of antibody 45 (region D). Mice were injected subcutaneously with (sc) 45 (n = 5) or control (n = 3) antibody at a dose of 10 mg / kg. Each group was slaughtered at different times. Mice treated with the control antibody (anti-Hel) had no changes in plasma PGRN, whereas mice treated with 45 had tapering of PGRN levels. The effect appeared to peak between 24 and 48 hours and gradually diminished by day 4-7. B Subchronic study: Mice were treated twice weekly with 45 at 10 mg / kg and a control antibody (anti-Hel). Samples were taken from buccal blood once a week. Plasma PGRN was high at week 1 and remained at about the same level throughout the study compared to animals treated with control antibody (n = 20). C Dose-response study: Different doses (4 doses: 0.1, 0.4, 2 and 10 mg / kg) of sortilin (45) and control antibody (anti-Hel) were infused and mice were slaughtered on day 2. Plasma PGRN was higher in mice treated with 45 (10 and 2 mg / kg). Lower doses (0.4 and 0.1 mg / kg) had no effect on plasma PGRN. The data are shown as mean ± SD. Data were analyzed by two-way Anova followed by Bonferroni analysis.^*p &lt;0.05;^**p &lt;0.01;^***p &lt;0.001 (Example 15).</figref><figref num="14">A diagram of the sortilin region based on the sortilin shuffle construct given to the predicted sortilin structure is provided. This region is part of the saltylin protein in which the change in selected amino acid residues from the human sequence to the tetraodon sequence inhibited binding to antibodies of that region class. Arrows indicate the reported high affinity binding sites for neurotensin and PGRN (Quistgaard Nat Struct Mol Biol. 2009 Jan; 16 (1): 96-8, Lee et al, Hum Mol Genet. 2013).</figref><figref num="15a-15b">Figures 15a (Panels 1-6) and 15b (Panels 1-3) show representative peptides covering the conformational epitopes of antibodies 45, 68 and 811. All of the peptides shown show protection from exchanges greater than 0.5D, with the exception of peptides 115-125. Peptides 115-125 are examples of peptides that are not affected by the presence of antibodies 45, 68 or 811 and are thereby not part of the conformational epitope (Example 16).</figref><figref num="16a-16b">Figures 16a (Panels 1-6) and 16b (Panels 1-3) show representative peptides covering the conformational epitope of antibody 30. All of the peptides shown show protection from exchanges greater than 0.5D, with the exception of peptides 563-572, 646-656 and 704-714. These three peptides are examples of peptides that are not affected by the presence of antibody 30, thereby being not part of the conformational bond (Example 16).</figref><figref num="17">The figure about the procedure of microdialysis is shown.</figref><figref num="18a">Time course: Systemic administration of antibody 45 or PBS (50 mg / kg, 10 ml / kg, subcutaneous (sc)) 24 hours prior to microdialysis experiment to the level of PRGN in the hippocampus of free-moving hSORT1 mice (24) Time) Shows the effect (Example 17).</figref><figref num="18b">Results of pooled 24-hour dialysis: Free-behavior hSORT1 in mice treated with 3.3 ± 0.3 ng / ml and 1.1 ± 0.1 ng / ml mab # 45- and PBS, respectively, as assessed by push-pull microdialysis. The basal PRGN in the hippocampus of mice is shown (Example 17).</figref><figref num="18c">PRGN levels (mean ± SEM) in the hippocampus of free-behavior hSORT1 mice measured every 2 hours for 24 hours one day after animals were treated with mab # 45 (n = 10) or PBS (n = 8). (Example 17).</figref>

As used herein, the term "sortilin" is synonymous with sortilin proteins (eg, identified in UniProt as Q99523, 1 and 2). The amino acid numbering of sortilin is shown for SEQ ID NO: 169 as shown below, where Met is amino acid 1.<chemistry num="1"><img file="JP6979397B2_D0001.tif" /></chemistry>

As used herein, the term "D region" is used in SEQ ID NO: 170: set forth below.<chemistry num="2"><img file="JP6979397B2_D0002.tif" /></chemistry>It is intended to refer to the region of sortilin consisting of amino acids in (corresponding to residues 523-610 of SEQ ID NO: 169).

For D-region antibodies, binding to the following shuffle regions was observed: hsort, hB06-10, B12390. The antibody did not bind to hB01-05, B45678, tet. For D + antibodies, binding to the following shuffle regions was observed: hsort, B12390. The antibody did not bind to hB01-05, hB06-10, B45678, tet. For the antibody called "D +", a binding pattern similar to that of the "D" region antibody was observed, except that binding to hB06-10 was not observed with the D + antibody. Despite the different binding patterns to the shuffle construct, the D + antibody shared functional properties (such as cell assays) with the D region antibody.

Specific D and D + binding antibodies bind to specific regions as defined in SEQ ID NO: 185, 186 or 187. Accordingly, the present invention relates to an antibody or antigen-binding fragment thereof that binds to the D region sequence of SEQ ID NO: 170 and within the region No. 185, 186 or 187 thereof, in certain embodiments. By binding, these antibody or antigen binding fragments affect PGRN levels and can therefore be used to treat PGRN-related diseases such as FTD, ALS and AD.

Further analysis of the binding of the D and D + antibodies of the invention identified partial binding to adjacent regions (region A) for some of the antibodies. This area contains SEQ ID NO: 180 and below:<chemistry num="3"><img file="JP6979397B2_D0003.tif" /></chemistry>Corresponds to amino acids 78-254 of SEQ ID NO: 169, as shown in. This area was given the term "A area".

Thus, in certain embodiments, the invention is an antibody or antibody that binds to the D region defined above and to SEQ ID NOs: 170, 185, 186 or 187 and further has an affinity for the A region identified in SEQ ID NO: 180. Regarding the antigen-binding fragment. Within the A region, a particular antibody or antigen-binding fragment thereof has an affinity for the A region amino acid identified in SEQ ID NO: 181, 182, 183 or 184.

PGRN (proepicerin, granulin-epicerin precursor, PC cell-induced growth factor, aclogranin) has 7.5 repetitions of smaller granulin motifs in the range of 6-25 kDa that can be proteolytically cleaved from the precursor PGRN 68.5. It encodes a kDa secretory glycoprotein (He, Z. &amp; Bateman, A., J.MoI.Med. 81: 600-6X2 (2003)). In non-neuronal cells, PGRN is a cell cycle regulator and cell motility (He, Z. &amp; Bateman, A., J.MoI.Med. 57: 600-612 (2003); Monami, G., et ah, Cancer. Growth factors such as Res. (5 (5: 7103-7110 (2006)), wound repair, inflammation (Zhu, J., et ah, Cell 777: 867-878 (2002)), vascular endothelial growth factor (VEGF)) (Tangkeangsiπsin, W. &amp; Serrero, G, Carcinogenesis 25.1587-1592 (2004)), and tumorigenesis (He, Z. &amp; Bateman, A., J.MoI.Med.81: 600-612 (2003), Monami, G., et al., Cancer Res (5 (5: 7103-7110 (2006); Serrero, G., Biochem Biophys) .Res.Commun.505-409-413 (2003), Lu, R &amp; Serrero, G., Proc.Natl Acad Sa U.SA 98 142-147 (2001); Liau, L M., et al., Cancer It is associated with various events such as Res.60: 1353-1360 (2000)).

PGRN mutations result in haploinsufficiency (Baker, M., et ah, Nature 442: 916-919 (2006); Cruts, M., et ah, Nature 442: 920-924 (2006)), familial FTD It is known to be present in almost 50% of cases of FTD, and the PGRN mutation is considered to be the main genetic factor of FTD (Cruts, M. &amp; Van Broeckhoven, C, Trends Genet. 24: 186- 194 (2008); Le Ber, I., et ah, Brain 129: 3051-3065 (2006)). The loss-of-function heterozygous properties of PGRN mutations suggest that PGRN expression plays an important dose-dependent role in protecting healthy individuals from the development of FTD in healthy individuals.

The term "antibody" (Ab) in the context of the invention is an immunoglobulin molecule capable of binding to an epitope of a molecule ("antibody"), or, according to certain embodiments of the invention, a fragment of an immunoglobulin molecule. Point to. Native antibodies typically include a tetramer usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH in the present specification) and a heavy chain constant region composed of three regions (CH1, CH2 and CH3). The heavy chain can be of any isotype including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM and IgE. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). The light chain contains a κ chain and a λ chain. Heavy and light chain variable regions are typically involved in antigen recognition, while heavy and light chain constant regions are the first of various cells of the immune system (eg, effector cells) and classical complement systems. It can mediate the binding of immunoglobulins to host tissues or factors, including component (C1q). The VH and VL regions can be further subdivided into hypervariable regions called "complementarity determining regions", which are interspersed with regions of more conserved sequences called "framework regions" (FRs). Each VH and VL consists of three CDR regions and four FR regions located in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, from amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain binding regions that interact with the antigen. Of particular relevance are antibodies that are "isolated" to exist in a physical environment different from those that may exist in nature, or that are modified to differ from the natural antibodies in the amino acid sequence and those thereof. It is an antigen-binding fragment.

The term "epitope" means an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of surface grouping of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural properties as well as specific charge properties. The conformational epitopes and linear epitopes are distinguished by the fact that their binding to the former, rather than the latter, is always lost in the presence of denaturing solvents. Epitopes are amino acid residues directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, the amino acid residues are within the footprint of the specific antigen-binding peptide). It may contain other amino acid residues that are not directly involved in binding.

As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment, portion, region or domain of an antibody capable of binding to an epitope (how it is produced). It means whether or not it is done (eg, by cleavage, by recombination, synthetically, etc.), and thus the term "antigen binding" means, for example, that "antigen binding fragment of antibody" is "epitope binding of antibody". Just as it is intended to be the same as "fragment", it is intended to mean the same as "epitope binding". Antigen binding fragments may contain all 1, 2, 3, 4, 5 or 6 of the CDR regions of such antibodies and are capable of binding to such epitopes, but such It may exhibit specificity, affinity or selectivity for such epitopes that differ from those of antibodies. However, preferably, the antigen binding fragment will contain all six of the CDR regions of such an antibody. Antigen-binding fragments of an antibody may be, or may include, a single polypeptide chain (eg, scFv), or amino-terminal and carboxyl-terminal (eg, bispecific antibodies, Fab fragments, etc.). Fab.₂It may be part of, or may contain, two or more polypeptide chains, each having (such as a fragment). Fragments of antibodies exhibiting antigen-binding ability are obtained, for example, by protease cleavage of intact antibodies. More preferably, two regions of the Fv fragment, VL and VH, are naturally encoded by separate genes, or polynucleotides encoding such gene sequences (eg, their coding cDNAs) are VL and A single protein chain (known as a single chain Fv (scFv)) to which the VH region binds to form a monovalent antigen binding molecule; for example, Bird et al., (1988) Science 242: 423-426; And Huston et Al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879-5883) can be linked using a recombinant method by a flexible linker that allows them to be made. Alternatively, bispecific antibodies by using flexible linkers (eg, less than about 9 residues) that are too short to allow the VL and VH regions of a single polypeptide chain to bind together. It is possible to form a (bispecific antibody), a bispecific antibody (diabody), or a similar molecule (two such polypeptide chains bind together to form a divalent antigen-binding molecule) ( For a description of bispecific antibodies, see, for example, PNAS USA See 90 (14), 6444-8 (1993)). Examples of antigen-binding fragments included within the scope of the invention are described in (i) Fab'or Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 regions, or WO 200709782. Monovalent antibody; (ii) F (ab') 2 fragment, bivalent fragment containing two Fab fragments linked by disulfide cross-linking in the hinge region; (iii) Fd fragment essentially consisting of VH and CH1 regions (Iv) Fv fragment essentially from the VL and VH regions, (v) essentially from the VH region and with domain antibodies (Holt et al; Trends Biotechnol.2003 Nov; 2i (ll): 484-90) Called dAb fragment (Ward et al., Nature 341,544-546 (1989)); (vi) Camelid or Nanobody (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5_ (l): l ll-24) and (vii) isolated complementarity determining regions (CDRs). In addition, two regions of the Fv fragment, VL and VH, are encoded by separate genes, which are a single protein chain (single chain) in which the VL and VH regions are combined to form a monovalent molecule. Known as antibodies or single chain Fv (scFv), eg Bird et al., Science 242,423-426 (1988) and Huston et al., PNAS USA (See 85,5879-5883 (1988)) can be linked using recombinant methods by synthetic linkers that allow them to be made. These and other useful antibody fragments in the context of the present invention are further described herein. The term antibody is provided by antibody-like polypeptides such as chimeric and humanized antibodies, as well as any known technique such as enzymatic cleavage, peptide synthesis, and recombination techniques, unless otherwise specified. It should also be understood that it also includes antibody fragments that retain the ability to bind to the fragment). The antibody produced can have any isotype. As used herein, "isotype" refers to an immunoglobulin class encoded by a heavy chain constant region gene (eg, IgG1, IgG2, IgG3 or IgG4). Such antibody fragments are obtained using conventional techniques known to those of skill in the art; suitable fragments capable of binding to the desired epitope are easily screened for usefulness as well as intact antibodies. obtain.

The term "bispecific antibody" refers to an antibody containing two independent antigen-binding fragments, each targeting an independent target. These targets can be epitopes present on different proteins, or different epitopes present on the same target. Bispecific antibody molecules can be made using compensatory amino acid modifications in the constant region of HC of the parent monospecific bivalent antibody molecule. The resulting heterodimer antibody contains one Fab given by two different parental monospecific antibodies. Amino acid modifications in the Fc region result in improved stability of heterodimeric antibodies with stable bispecificity over time (Ridgway et al., Protein Engineering 9,617-621 (1996), Gunasekaran et al. , JBC 285,19637-1 (2010), Moore et al., MAbs 3: 6 546-557 (2011), Strop et al., JMB 420,204-219 (2012), Metz et al., Protein Engineering 25:10 571-580 (2012), Labrijn et al., PNAS 110: 113,5145-5150 (2013), Spreter Von Kreudenstein et al., MAbs 5: 5 646-654 (2013)). Bispecific antibodies can also include molecules produced using ScFv fusion. The two unispecific scfvs are then independently bound to the Fc region, which is capable of forming a stable heterodimer to produce a single bispecific molecule (Mabry). et al., PEDS 23: 3 115-127 (2010). Bispecific molecules have double binding ability.

"Anti-sortilin antibody" or "sortilin antibody" (as used herein synonymously, depending on the context described) is an antibody that specifically binds to sortilin, in particular the sortilin D region, SEQ ID NO: 170. It is the antigen-binding fragment. Anti-soltilin antibodies that bind to the saltylin D region are usually within the D region (eg, SEQ ID NO: 185, 186 or 187) having affinity (IC50) at 22 nM or less, such as 22nM to 1nM, 10nM to 1nM or 5nM to 1nM. It will bind to a conformational epitope or linear epitope of 3, 4, 5, 6 or 7 consecutive amino acids. According to certain embodiments, anti-sortilin antibodies can also bind to region A (SEQ ID NO: 180, 181, 182, 183 or 184), but their primary biological function is by binding to region D. It is emphasized that it is believed to be achieved.

The binding sites identified are quite unique, as shown, for example, with the binding of the selective small molecule ligand AF38469 to sortilin. The AF38469 binding site was shown to be similar to the neurotensin binding site and was characterized by X-ray crystallography (Schroeder et al. Bioorg Med Chem Lett. 2014 Jan 1; 24 (1): 177-80). PGRN has been reported to bind to the same site (Lee et al. Hum Mol) Genet. 2013). Antibodies 45 and 68 that bind to the D region and D +, respectively, did not inhibit the binding of AF38469 to sortilin. This data suggests that these antibodies have a sortilin binding site that is different from the AF38469 and neurotensin binding sites. Thus, in certain embodiments, the invention is capable of specifically binding to sortilin and inhibiting the binding of PGRN to sortilin, but the binding inhibits the binding of neurotensin or AF38469 to sortilin. With respect to an antibody, or an antigen-binding fragment thereof, which does not or substantially does not inhibit. This can be demonstrated using, for example, a scintillation proximity assay (SPA) and the transfer of binding to sortilin (Example 11). One way to explain this finding is that the antibody, or its antigen-binding fragment, binds to the surface region of sortilin, while small molecules such as neurotensin bind to the inside of the binding pocket. possible.

As used herein, the term "human antibody" (which may be abbreviated as "humAb" or "HuMab") includes antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Is intended. Human antibodies of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (eg, by random or site-specific mutations in vitro, or during gene rearrangements, or somatic mutations. Mutations induced by).

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to the preparation of antibody molecules of a single molecular composition. Traditional monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. In certain embodiments, the monoclonal antibody can be composed of more than one Fab region, thereby increasing its specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not intended to be limited by any particular production method (eg, recombinant, transgenic, hybridoma, etc.).

The antibodies of the invention, and their sortilin antigen-binding fragments thereof, are preferably human or, for example, mouse antibodies (denoted as 1F2, 5E1) and are "humanized", especially when used for therapeutic purposes. The term "humanization" refers to antigen binding sites obtained from non-human species-derived immunoglobulins, commonly prepared using recombinant techniques, and the remaining immunoglobulins based on the structure and / or sequence of human immunoglobulins. Refers to a molecule with a structure. The antigen binding site is only the complete non-human antibody variable region fused to the human constant region, or the complementarity determining regions (CDRs) of such variable regions transplanted into the appropriate human framework regions of the human variable region. Can include either. Framework residues of such humanized molecules may be wild-type (eg, fully human), or they are one not found in human antibodies whose sequences functioned as the basis for humanization. Alternatively, it can be modified to include multiple amino acid substitutions. Humanization reduces or eliminates the likelihood that the constant region of the molecule acts as an immunogen in a human individual, but the potential for an immune response to the foreign variable region remains (Lo Buglio, AF et al. (1989) Mouse / Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response , Proc.Natl.Acad.Sci. (USA) 86: 4220-4224). Another approach focuses not only on providing constant regions of human origin, but also on modifying variable regions to reshape them as close as possible to human form. Both heavy and light chain variable regions contain three complementarity determining regions (CDRs) that differ in their response to the antigen of interest and determine their ability to bind, and these complementarity determining regions (CDRs) are located. It is known that four framework regions (FRs) that are relatively conserved in a given species and are thought to provide a scaffolding for CDRs are adjacent. When a non-human antibody is prepared for a particular antigen, the variable region is "reformed" or by transplanting the CDRs derived from the non-human antibody into the FR present in the modified human antibody. Can be "humanized". The application of this technique to various antibodies is Sato, K. et al. (1993) Cancer Res 53: 851-856. Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332: 323. -327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239: 1534-1536; Kettleborough, CA et al. (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting : The Importance Of Framework Residues On Loop Conformation , Protein Engineering 4: 773-3783; Maeda, H. et al. Reported by 1149-1154. In certain embodiments, the humanized antibody preserves all CDR sequences (eg, a humanized mouse antibody comprising all 6 CDRs derived from the mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (1, 2, 3, 4, 5, 6, 6) modified relative to the original antibody. Is also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanize an antigen is well known (eg, US Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; See No. 6,881,557).

The term "antibody" XX "" refers to the light chain, light chain variable region, or light chain variable region CDR1-3 defined by their respective SEQ ID NOs, and the heavy chain, heavy chain defined by their respective SEQ ID NOs. It is intended to indicate an antibody comprising or consisting of variable regions, or heavy chain variable regions CDR1-3, or an antigen-binding fragment thereof (eg, antibody "5E1"). In certain embodiments, antibodies or antigen-binding fragments thereof are defined by their full heavy chain variable regions defined by their SEQ ID NOs and their light chain variable regions defined by their SEQ ID NOs. ..

The numbering of amino acid residues in this region is IMGT®, International ImMunoGeneTics Information System® or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS &amp; Foeller, C. (1991). .Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no.91-3242, USD department of Health and Human Services; Chothia, C. &amp; Lesk, AM (1987). Canonical structures For The Hypervariable Performed according to domains Of Immunogloblins.J.Mol.Biol.196,901-917.

As used herein, an antibody or antigen-binding fragment thereof is associated with that epitope more frequently, more rapidly, for a longer period of time and / or with higher affinity or binding activity compared to another epitope. When reacting or associating, it is said to "specifically" bind to the region of another molecule (ie, an epitope). In one embodiment, the antibody of the invention, or antigen-binding fragment thereof, binds at least 10-fold stronger to its target (sortilin) than another molecule; preferably at least 50-fold stronger, more preferably at least 100-fold stronger. .. Preferably, the antibody, or antigen-binding fragment thereof, binds under physiological conditions, eg, in vivo. Thus, "specifically binding to sortilin" includes the ability of an antibody, or antigen-binding fragment thereof, to bind to sortilin with such specificity and / or under such conditions. Suitable methods for determining such binding are known to those of skill in the art, and exemplary methods are described in the accompanying examples. As used herein, the term "binding" in the context of binding an antibody to a given antigen is typically BIAcore® 3000 or T200 using the antigen as a ligand and the antibody as a sample. Approximately 10 as determined by, for example, surface plasmon resonance (SPR) technology in any of the instruments^-7M or less, for example about 10^-8M or less, for example about 10^-9Refers to binding with an affinity corresponding to a KD of M or less, at least 10 times greater than its affinity for binding to a non-specific antigen (eg, BSA, casein) other than a given antigen or a closely related antigen. It binds to a given antigen with an affinity corresponding to a low, eg, at least 100-fold lower, eg, at least 1,000-fold lower, eg, at least 10,000-fold lower, eg, at least 100,000-fold lower KD. The amount of lower affinity depends on the KD of the antibody, so if the KD of the antibody is very low (ie, the antibody is very specific), the affinity for the antigen will be the affinity for the non-specific antigen. The amount that is lower than sex can be at least 10,000 times. In particular, the present invention provides binding affinities corresponding to 22 nM or less, such as 22 nM to 1 nM, 10 nM to 1 nM or 5 nM to 1 nM, when determined, for example, by biolayer interferometry with Octet 384RED (Example 8). Regarding the anti-sortilin antibody shown.

In certain embodiments of the invention, the invention relates to an antibody capable of competing with humAb antibody 45 or humAb antibody 68 for binding to sortilin, or an antigen-binding fragment thereof. In another embodiment, the invention relates to an antibody capable of competing with antibody 45 for binding of sortilin to the D region as defined in SEQ ID NO: 170, or an antigen binding fragment thereof. Inhibition of such competitive binding is performed using assays and methods well known in the art, eg, with immobilized human sorbylin, with the BIAcore® chip, with and with antibody polypeptides tested. It can be determined by incubating with a control antibody (such as antibody "45" or "68") without. Alternatively, a pairwise mapping technique can be used, in which a control antibody (such as antibody "45" or "68") is immobilized on the surface of a BIAcore® chip and the human sorbylin antigen is bound to the immobilized antibody. , Next, the secondary antibody is tested for its ability to bind simultaneously to human soltilin (disclosure is incorporated herein by reference in the'BIAcore® Assay. See Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012).

As used herein, the term "kd" (seconds-1 or 1 / second) refers to the dissociation rate constant of a particular antibody-antigen interaction. The value is also called a koff value.

As used herein, the term "ka" (M-1 x s-1 or 1 / M s) refers to the binding rate constant of a particular antibody-antigen interaction.

As used herein, the term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing kd by ka.

As used herein, the term "KA" (M-1 or 1 / M) refers to the binding equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing ka by kd.

In one embodiment, the invention has the following properties: (i) Binding affinity of 0.5-10 nM, eg 1-5 nM or 1-2 nM, for sortilin (K)._D); (ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis; (v) Antibodies that exhibit one or more of the ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice. Regarding antigen-binding fragments.

The term "ability to reduce and / or inhibit PGRN binding to sortilin" is less than 50 nM, preferably 10 nM ~, using the time-resolved fluorescence assay (HTFR) disclosed in Example 10. Contains antibodies capable of inhibiting binding to PGRN at 0.2 nM IC50.

The term "ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells" is at least 25%, eg 25% to 500%, 25 as measured by the ELISA assay disclosed in Example 13. Includes the ability to increase the concentration of PGRN in the medium by% ~ 400% or 25% ~ 200%.

"The ability of sortilin-expressing cells to reduce and / or inhibit endocytosis of PGRN" is at least 10% as measured by the cellomic-based assay disclosed in Example 12, but is preferably preferred. Includes the ability to reduce the intracellular concentration of PGRN by 20-100%.

"The ability to increase the amount and / or concentration of plasma PGRN in human-sortilin-expressing knock-in mice" is at least 25 percent as measured by the ELISA assay disclosed in Example 15, preferably 50. Includes the ability to increase the concentration of PGRN in plasma by ~ 500 percent.

It is envisioned that the ability to increase PGRN in the brain can also be measured, for example by microdialysis. Therefore, "the ability to increase the amount and / or concentration of PGRN in the brain" is at least 25 percent as measured by microdialysis, but preferably 50-500 percent of the concentration of PGRN in the brain. Includes the ability to increase.

In some antibodies, a small portion of the CDR, a subset of the CDR residues required for binding, called SDR, is required to retain binding in the humanized antibody. CDR residues that do not contact the relevant epitope and are not in the SDR are outside the Chothia hypervariable loop, based on previous studies (eg, residues H60-H65 in CDR H2 are often not needed). From the Kabat CDR region at (Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes of Health Publication No. 91-3242; Chothia, C. et al. (1987) Canonical Structures For The Hypervariable Regions Of Immunoglobulins , J.Mol.Biol.196: 901-917), by molecular modeling and / or by experiment, or by Gonzales, NRet It can be specified as described in al. (2004) SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity, Mol. Immunol. 41: 863-872. In such humanized antibodies at positions where one or more donor CDR residues are absent or all donor CDRs are omitted, the amino acids occupying this position correspond (by Kabat numbering) in the receptor antibody sequence. It can be an amino acid that occupies a position. The number of such substitutions of the receptor for the donor amino acid in the included CDRs reflects a balance of competing considerations. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in humanized antibodies and, as a result, reducing their potential immunogenicity. However, substitutions can also cause changes in affinity, and it is preferable to avoid a significant decrease in affinity. The location of the substitution in the CDR and the amino acid to be substituted can also be selected experimentally.

A single amino acid modification of a CDR residue can result in loss of functional binding (Rudikoff, S.etc. (1982) Single Amino Acid Substitution Altering Antigen-binding. Specificity , Proc.Natl.Acad.Sci. (USA) 79 (6): 1979-1983) provides a means for systematically identifying another functional CDR sequence. Such mutant CDRs. In one preferred method for obtaining the CDR, the polynucleotide encoding the CDR is mutated (eg, by random mutation or by a site-specific method (eg, polymerase chain mediation by a primer encoding the mutation locus). By sex amplification), a CDR with substituted amino acid residues is produced. The identity of the relevant residues in the original (functional) CDR sequence is replaced by the identity of the substituted (non-functional) mutant CDR sequence. By comparing to sex, the BLOSUM62.iij substitution score for that substitution can be identified. The BLOSUM system provides a matrix of amino acid substitutions created by analyzing a database of sequences for reliable alignment (Eddy, SR (2004) Where Did The BLOSUM62 Alignment Score Matrix Come From?, Nature Biotech.22 (8): 1035-1036; Henikoff, JG (1992) Amino acid substitution matrices from protein blocks, Proc.Natl.Acad.Sci. (USA) 89: 10915-10919; Karlin, S. et al (1990) Methods For Assessing The Statistical Significance Of Molecular Sequence Features By Using General Scoring Schemes, Proc.Natl.Acad.Sci. (USA) 87: 2264-2268; Altschul, SF (1991) Amino Acid Substitution Matrices From An Information Theoretic Perspective , J.Mol.Biol.219,555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 shows the BLOSUM62.iij replacement score (the higher the score, the more the replacement). Is more conservative, and thus the substitution is more likely to have no effect on function). If the resulting CDR-containing antigen-binding fragment is unable to bind to saltillin, for example, the BLOSUM62.iij substitution score is inadequate. New substitution candidates are selected and generated that are considered conservative to and have a higher substitution score. Thus, for example, the original residue is glutamate (E) and a non-functional substitution residue. If is histidine (H), the BLOSUM62.iij substitution score is 0, with more conservative changes (to aspartate, asparagine, glutamine, or lysine) preferred.

<tables num="1"><img file="JP6979397B2_D0004.tif" /></tables>

Therefore, the present invention envisions the use of random mutations to identify improved CDRs. In the context of the present invention, conservative substitutions can be defined by substitutions within the range of amino acid types reflected in one or more of the three tables below.

<tables num="2"><img file="JP6979397B2_D0005.tif" /></tables>

<tables num="3"><img file="JP6979397B2_D0006.tif" /></tables>

<tables num="4"><img file="JP6979397B2_D0007.tif" /></tables>

More conservative substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

Further groups of amino acids can be formulated, for example, using the principles described in Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), WH Freeman and Company.

Phage display technology can be used instead to increase (or decrease) CDR affinity. This technique, called affinity maturation, uses mutations or "CDR walking" and re-selection using the target antigen or its antigenic antigen-binding fragment to the initial antibody or parent. Identify antibodies with a CDR that binds with a higher (or lower) affinity for the antigen when compared to the antibody (eg, Glasser et al. (1992) J. Immunology). See 149: 3903). Mutagenesis of whole codons rather than a single nucleotide results in a semi-randomized repertoire of amino acid mutations. A library consisting of a pool of mutant clones can be constructed, each of which is dependent on a single amino acid modification in a single CDR and presents each possible amino acid substitution for each CDR residue. Includes mutants. Mutants with increased (or decreased) binding affinity for the antigen can be screened by contacting the immobilized mutant with the labeled antigen. Any screening method known in the art can be used to identify mutant antibodies with increased or decreased affinity for the antigen (eg, ELISA) (Wu et al. 1998, Proc. Natl). .Acad.Sci. (USA) 95:6037; see Yelton et al., 1995, J.Immunology 155: 1994). CDR walking that randomizes the light chain can be used (see Schier et al., 1996, J. Mol. Bio. 263: 551).

Methods for achieving such affinity maturation include: Krause, JC et al. (2011) An Insertion Mutation That Distorts Antibody Binding Site Architecture Enhances Function Of A Human Antibody, MBio.2 (1) pii: e00345-10.doi: 10.1128 / mBio.00345-10; Kuan, CT et al. (2010) Affinity-Matured Anti-Glycoprotein NMB Recombinant Immunotoxins Targeting Malignant Gliomas And Melanomas, Int.J.Cancer 10.1002 / ijc.25645; Hackel, BJet al. (2010) Stability And CDR Composition Biases Enrich Binder Functionality Landscapes , J.Mol.Biol.401 (1): 84-96; Montgomery, DLet al. (2009) Affinity Maturation and characterization Of A Human Monoclonal Antibody Against HIV-1 gp41 , MAbs 1 (5): 462 -474; Gustchina, E. et al. (2009) Affinity Maturation By Targeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab Derived From A Synthetic Naive Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With Improved HIV-1 Neutralization Potency And Breadth , Virology 393 (1): 112-119; Finally, W Jet al. (2009) Affinity Maturation Of A Humanized Rat Antibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A High Level Of Mutational Plasticity Both Inside And Outside The Complementarity-Determining Regions , J. Mol.Biol.388 (3): 541-558; Bostrom, J. et al. (2009) Improving Antibody Binding Affinity And Specificity For Therapeutic Development , Methods Mol.Biol .525: 353-376; Steidl, S.et al. (2008) In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification, Mol.Immunol.46 (1): 135-144; and Barderas, R. et al. (2008) Affinity Maturation Of Antibodies Assisted By In Silico Modeling , Proc.Natl.Acad.Sci. (USA) 105 (26): 9029-9034.

Thus, the sequence of CDR variants of the included antibodies or their antigen-binding fragments is by substitution; for example, 4 amino acid residues, 3 amino acid residues, 2 amino acid residues or 1 amino acid residue to be substituted. Can differ from the CDR sequence of the parent antibody. According to one embodiment of the invention, it is further envisioned that the amino acids in the CDR regions can be replaced by conservative substitutions, as defined in the following three tables.

The term "transgenic non-human animal" refers to one or more human heavy and / or light chain transgenes or trans-chromosomes (integrated or non-integrated into the animal's natural genomic DNA). Refers to a non-human animal having a genome capable of expressing a fully human antibody, including any). For example, transgenic mice are human light chain transgenes and human heavy chain transgenes or human weights such that mice produce human anti-sortilin antibodies when immunized with cells expressing sortilin antigen and / or sortilin. It may have any of the chain transgenes. The human heavy chain transgene can be integrated into the chromosomal DNA of mice as in the case of transgenic mice such as HuMAb mice (such as HCo7 or HCo12 mice), or the human heavy chain transgene can be published internationally 02 / As described in the 43478 pamphlet, it can be maintained extra-chromosomally, as in the case of transgene KM mice. Such transgenic and introduced chromosomal mice (collectively referred to herein as "transgenic mice") are subjected to VDJ recombination and isotype switching to give a given antigen (IgG, IgA, IgM, IgD and / /. Or it is possible to produce multiple isotypes of human monoclonal antibodies against (eg IgE).

Transgenic, non-human animals are also used to produce antibodies against specific antigens by introducing genes encoding specific antibodies, eg, by operably linking genes expressed in animal milk. Can be done.

As used herein, the term "treatment" or "treating" is used to improve, delay, alleviate, or suppress the progression or severity of a disease or disorder. Or it means ameliorating, delaying, alleviating, or suppressing one or more of the symptoms or side effects of such a disease or disorder. In the spirit of the present invention, "treatment" or "treating" further means a method for obtaining a beneficial or desired clinical result, wherein the "beneficial or desired clinical result" is limited. Not done, but partial or global, detectable or undetectable, alleviated symptoms, reduced degree of disability or illness, stable (ie, not exacerbated) These include delaying or slowing the progression of the disease or disorder, amelioration or alleviation of the disease or disorder, and remission of the disease or disorder.

An "effective amount", when applied to an antibody of the invention or an antigen-binding fragment thereof, is required to achieve the intended biological effect or desired therapeutic outcome (including, but not limited to, clinical outcome). Refers to a sufficient amount over a reasonable dose and duration. The phrase "therapeutically effective amount", when applied to an antibody of the invention or an antigen-binding fragment thereof, improves or alleviates the progression of a disorder or disease state, or the progression of a disorder or disease symptom. It is intended to represent the amount of antibody, or antigen-binding fragment thereof, sufficient to stabilize, suppress, delay, alleviate, or delay. In one embodiment, the method of the invention provides administration of an antibody, or antigen-binding fragment thereof, in combination with another compound. In such cases, the "effective amount" is the amount of combination sufficient to cause the intended biological effect.

Therapeutically effective amounts of the anti-sortilin antibody or antigen-binding fragment thereof of the present invention include the condition, age, sex, and body weight of the individual, and the ability of the anti-sortilin antibody or antigen-binding fragment thereof to elicit the desired response in the individual. It can change depending on the factors of. A therapeutically effective amount is also an amount that outweighs any toxic or adverse effects of the antibody or antibody portion by a therapeutically beneficial effect.

The antibody is preferably a human or humanized antibody.

The numbering of amino acid residues in this region is IMGT®, International ImMunoGeneTics Information System® or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS &amp; Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no.91-3242, USD department of Health and Human Services; Chothia, C. &amp; Lesk, AM (1987). Canonical structures For The Hyperrvariable domains Follow Of Immunogloblins.J.Mol.Biol.196,901-917.

Antibody 5E1: Therefore, the present invention has (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 1; (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 2; (c) having the amino acid sequence of SEQ ID NO: 3. Light chain CDR3; (d) Heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 4; (e) Heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and (f) Heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 6. With respect to an antibody comprising or consisting of it, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 7.

Antibodies 1F2: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 9; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 10; (c) SEQ ID NO: Light chain CDR3 with 11 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 12; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 13; and (f) Amino acid sequence of SEQ ID NO: 14 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 16 and a light chain variable region of SEQ ID NO: 15.

Antibodies 068: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 17; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 18; (c) SEQ ID NO: Light chain CDR3 with 19 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 20; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 21; and (f) Amino acid sequence of SEQ ID NO: 22 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 24 and a light chain variable region of SEQ ID NO: 23.

Antibodies 1320: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 25; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 26; (c) SEQ ID NO: Light chain CDR3 with 27 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 28; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 29; and (f) Amino acid sequence of SEQ ID NO: 30 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 32 and a light chain variable region of SEQ ID NO: 31.

Antibodies 93-05: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 33; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 34; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 35; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 36; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 37; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 38. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 40 and a light chain variable region of SEQ ID NO: 39.

Antibodies 93-01: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 41; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 42; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 43; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 44; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 45; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 46. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 48 and a light chain variable region of SEQ ID NO: 47.

Antibodies 924: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 49; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 50; (c) SEQ ID NO: Light chain CDR3 with 51 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 52; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 53; and (f) Amino acid sequence of SEQ ID NO: 54 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 56 and a light chain variable region of SEQ ID NO: 55.

Antibodies 1276: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 57; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 58; (c) SEQ ID NO: Light chain CDR3 with 59 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 60; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 61; and (f) Amino acid sequence of SEQ ID NO: 62 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 64 and a light chain variable region of SEQ ID NO: 63.

Antibodies 849: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 65; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 66; (c) SEQ ID NO: Light chain CDR3 with 67 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 68; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 69; and (f) Amino acid sequence of SEQ ID NO: 70 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 72 and a light chain variable region of SEQ ID NO: 71.

Antibodies 531-02: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 73; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 74; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 75; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 76; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 77; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 78; With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 80 and a light chain variable region of SEQ ID NO: 79.

Antibodies 548-01: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 81; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 82; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 83; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 84; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 85; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 86. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 88 and a light chain variable region of SEQ ID NO: 87.

Antibodies 548-02: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 89; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 90; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 91; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 92; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 93; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 94. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 96 and a light chain variable region of SEQ ID NO: 95.

Antibodies 1289-02: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 97; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 98; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 99; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 100; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 101; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 102. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 104 and a light chain variable region of SEQ ID NO: 103.

Antibodies 811-02: According to another embodiment, the invention is: (a) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 105; (b) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 106; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 107; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 108; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 109; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 110. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 112 and a light chain variable region of SEQ ID NO: 111.

Antibodies 566-01: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 113; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 114; (c). Light chain CDR3 with the amino acid sequence of SEQ ID NO: 115; (d) Heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 116; (e) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 117; and (f) Heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 118. With respect to an antibody comprising or consisting of heavy chain CDR3 having an amino acid sequence, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 120 and a light chain variable region of SEQ ID NO: 119.

Antibodies 562: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 121; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 122; (c) SEQ ID NO: Light chain CDR3 with 123 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 124; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 125; and (f) Amino acid sequence of SEQ ID NO: 126 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 128 and a light chain variable region of SEQ ID NO: 127.

Antibodies 193: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 129; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 130; (c) SEQ ID NO: Light chain CDR3 with 131 amino acid sequence; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 132; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 133; and (f) Amino acid sequence of SEQ ID NO: 134 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 136 and a light chain variable region of SEQ ID NO: 135.

Antibodies 88: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 137; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 138; (c) SEQ ID NO: Light chain CDR3 with 139 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 140; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 141; and (f) Amino acid sequence of SEQ ID NO: 142 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 144 and a light chain variable region of SEQ ID NO: 143.

Antibodies 045: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 145; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 146; (c) SEQ ID NO: Light chain CDR3 with 147 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 148; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 149; and (f) Amino acid sequence of SEQ ID NO: 150 With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 152 and a light chain variable region of SEQ ID NO: 151.

Antibodies 044: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 153; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 154; (c) SEQ ID NO: Light chain CDR3 with 155 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 156; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 157; and (f) Amino acid sequence of SEQ ID NO: 158. With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 160 and a light chain variable region of SEQ ID NO: 159.

Antibodies 002: According to another embodiment, the invention is: (a) light chain CDR1 having the amino acid sequence of SEQ ID NO: 161; (b) light chain CDR2 having the amino acid sequence of SEQ ID NO: 162; (c) SEQ ID NO: Light chain CDR3 with 163 amino acid sequences; (d) Heavy chain CDR1 with amino acid sequence of SEQ ID NO: 164; (e) Heavy chain CDR2 with amino acid sequence of SEQ ID NO: 165; and (f) Amino acid sequence of SEQ ID NO: 166. With respect to an antibody comprising or consisting of heavy chain CDR3 having, or an antigen-binding fragment thereof.

Preferably, the monoclonal antibody comprises or may consist of a heavy chain variable region of SEQ ID NO: 168 and a light chain variable region of SEQ ID NO: 167.

The antibodies described above are, according to one embodiment, 4 or less amino acid differences, or 3 or less amino acid differences, or 2 from the CDR1, CDR2, and / or CDR3 (VH and / or VL) sequences. It may further include variants having one or less amino acid differences, or one or less amino acid differences.

In addition, the antibody can be in the composition with a pharmaceutically acceptable carrier. The antibodies of the invention can be used therapeutically. In particular, the antibodies of the invention can be used to treat FTD or ALS or TDP43 proteinosis (such as Alzheimer's disease (AD)).

The treatment envisioned by the present invention may be long term and the patient may be treated for at least 2 weeks, eg at least 1 month, 6 months, 1 year or more.

The antibodies of the invention may be, for example, monoclonal antibodies produced by the hybridoma method first described by Kohler et al., Nature 256, 495 (1975), or by recombinant DNA or other methods. It may be a monoclonal antibody. Monoclonal antibodies are also described, for example, in Clackson et al., Nature 352, 624-628 (1991) and Marks et. It can be isolated from a phage antibody library using the technique described in al., J.MoI.Biol.222,581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies are prepared from mouse spleen B lymphocytes obtained from mice immunized with the antigen of interest, for example, in the form of cells expressing the antigen on the surface or nucleic acids encoding the antigen of interest. Obtained from the hybridoma. Monoclonal antibodies are also obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals (rats, rabbits, dogs, sheep, goats, primates, etc.).

In one embodiment, the antibody of the invention is a human antibody. Human monoclonal antibodies to sortilin can be produced using transgenic or chromosome-introduced mice that have a portion of the human immune system rather than the mouse system. Such transgenic and chromosome-introduced mice include mice referred to herein as HuMAb mice and KM mice, respectively.

HuMAb mice have unrearranged human heavy and constant (μ and Y) and light chain variable and constant (κ) chains, with targeted mutations that inactivate the endogenous μ and K chain loci. It contains a minilocus of a human immunoglobulin gene encoding an immunoglobulin sequence (Lonberg, N. et al., Nature 368, 856-859 (1994)). Thus, mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transduction genes undergo class switching and somatic mutations, resulting in high affinity. Produces human IgG, k monoclonal antibody (Lonberg, N. et al. (1994), see above; Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994), Lonberg, N. and Huszar, D. ., Intern.Rev.Immunol.Vol.13 65-93 (1995) and Harding, F.and Lonberg, N., Ann.NYAcad.Sci (Outlined in 764 536-546 (1995)). HuMAb mice were produced by Taylor, L. et al., Nucleic Acids Research 20,6287-6295 (1992), Chen, J. et al., International Immunology 5,647-656 (1993), Tuaillon et al., J. et al. Immunol.152,2912-2920 (1994), Taylor, L. et al., International Immunology 6,579-591 (1994), Fishwild, D. et al., Nature Biotechnology It is described in detail in 14,845-851 (1996). U.S. Patent No. 5,545,806, U.S. Patent No. 5,569,825, U.S. Patent No. 5,625,126, U.S. Patent No. 5,633,425, U.S. Patent No. 5,789,650, U.S. Patent No. 5,877,397, U.S. Patent 5,661,016, U.S. Patent No. 5,814,318, U.S. Patent No. 5,874,299, U.S. Patent No. 5,770,429, U.S. Patent No. 5,545,807, International Publication No. 98/24884, International Publication No. See also the 94/25585, International Publication 93/1227, International Publication 92/22645, International Publication 92/03918 and International Publication 01/09187.

HCo7, HCo12, HCo17 and HCo20 mice have JKD disruption in their endogenous light chain (κ) gene (as described in Chen et al., EMBO J. 12, 811-820 (1993)), their endogenous causes. CMD disruption in sex heavy chain genes (as described in Example 1 of WO 01/14424), and KCo5 human κ light chain transgene (Fishwild et al., Nature Biotechnology) As described in 14,845-851 (1996)). In addition, HCo7 mice carry the HCo7 human heavy chain transgene (as described in US Pat. No. 5,770,429), and HCo12 mice have the HCo12 human heavy chain transgene (International Publication No. 01/14424). HCo17 mice have the HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187). However, HCo20 mice carry the HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and κ light chain transgenes in background homozygotes due to disruption of the endogenous mouse heavy chain and κ light chain loci.

In the KM mouse strain, the endogenous mouse κ light chain gene is homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and is an endogenous mouse heavy chain gene. Is homozygously destroyed as described in Example 1 of WO 01/09187. This mouse strain carries the human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14,845-851 (1996). This mouse strain also carries a human heavy chain introduction chromosome composed of chromosome 14 fragment hCF (SC20), as described in WO 02/43478. HCo12-Balb / c, HCo17-Balb / c and HCo20-Balb / c mice are described in International Publication No. 09/097006 Pamphlet, HCo12, HCo17 and HCo20, KCo5 [J / K] ( Can be generated by crossing Balb).

In the KM mouse strain, the endogenous mouse κ light chain gene is homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) and is an endogenous mouse heavy chain gene. Is homozygously destroyed as described in Example 1 of WO 01/09187. This mouse strain carries the human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14,845-851 (1996). This mouse strain also carries a human heavy chain introduction chromosome composed of the chromosome 14 antigen binding fragment hCF (SC20), as described in WO 02/43478.

Spleen cells derived from these transgenic mice can be used to produce hybridomas that secrete human monoclonal antibodies according to well-known techniques. The human monoclonal or polyclonal antibody of the present invention, or an antibody of the present invention derived from another species, also produces and recovers another non-human mammalian or plant transgenic for the relevant immunoglobulin heavy and light chain sequences. It can be gene-introduced by the production of the antibody in a possible form. In connection with transgenic production in mammals, antibodies can be produced and recovered from goat, bovine, or other mammalian milk. See, for example, US Pat. No. 5,827,690, US Pat. No. 5,756,687, US Pat. No. 5,750,172 and US Pat. No. 5,741,957.

The antibodies of the invention can be of any isotype. Isotype selection is typically guided by the desired effector function, such as ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. The human light chain constant region, either κ or λ, can be used. If desired, the class of anti-sortilin antibodies of the invention can be switched by known methods. For example, the antibody of the present invention, which was originally IgM, can be class-switched to the IgG antibody of the present invention. In addition, class switching techniques can be used to convert one IgG subclass to another IgG subclass, eg, IgGl to IgG2. Thus, the effector function of the antibodies of the invention can be altered for a variety of therapeutic uses, for example, by isotype switching to IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies. In one embodiment, the antibody of the invention is an IgG1 antibody, such as IgG1, k. An antibody is said to be of a particular isotype if its amino acid sequence is most homologous to that isotype compared to other isotypes.

In one embodiment, the antibody of the invention is a full length antibody, preferably an IgG antibody, in particular an IgG1, k antibody. In another embodiment, the antibody of the invention is an antibody antigen binding fragment or single chain antibody.

Antibodies and antigen-binding fragments thereof are obtained, for example, by antigen-binding fragmentation using conventional techniques, and antigen-binding fragments can be screened for usefulness as described herein for all antibodies. For example, an F (ab') 2 antigen binding fragment can be produced by treating the antibody with pepsin. The resulting F (ab') 2 antigen-binding fragment can be treated to reduce the disulfide bridge to produce a Fab'antigen-binding fragment. Fab antigen-binding fragments are obtained by treating IgG antibodies with papain; Fab'antigen binding fragments are obtained by pepsin digestion of IgG antibodies. The F (ab') antigen binding fragment can also be produced by binding the Fab'described below by a thioether bond or a disulfide bond. The Fab'antigen binding fragment is an antibody antigen binding fragment obtained by cleaving the disulfide bond in the hinge region of F (ab') 2. The Fab'-antigen binding fragment is obtained by treating the F (ab') 2 antigen binding fragment with a reducing agent such as dithiothreitol. Antibody-antigen-binding fragments can also be produced by expression of nucleic acids encoding such antigen-binding fragments in recombinant cells (see, eg, Evans et al., J. Immunol. Meth. 184, 123-38 (1995)). .. For example, a chimeric gene encoding a portion of an F (ab') 2 antigen-binding fragment is a DNA encoding the CH1 and hinge regions of the H chain to produce such a cleaved antibody antigen-binding fragment molecule. It may contain a sequence followed by a translation stop codon.

In one embodiment, the anti-sortilin antibody is described in a monovalent antibody, preferably Pamphlet 2007059782 with a deletion of the hinge region, which is incorporated herein by reference in its entirety. It is a valent antibody. Thus, in one embodiment, the antibody is a monovalent antibody and the anti-saltylin antibody is i) a step of providing a nucleic acid construct encoding the light chain of the monovalent antibody, wherein the construct is selected. Contains the nucleotide sequence encoding the VL region of the antigen-specific anti-saltylin antibody and the nucleotide sequence encoding the constant CL region of Ig, wherein the nucleotide sequence and Ig encode the VL region of the selected antigen-specific antibody. The nucleotide sequence encoding the CL region is operably linked together and in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region is administered in the presence of polyclonal human IgG or when administered to an animal or human. With the step of modifying the CL region to include amino acids capable of forming disulfide or covalent bonds with other peptides containing the same amino acid sequence in the CL region;

Similarly, in one embodiment, the anti-saltylin antibody is (i) the variable region or antigen-binding portion of the antibody of the invention described herein, and (ii) the CH region or CH2 of an immunoglobulin and A monovalent antibody comprising that region, including the CH3 region, where the CH region or its region is the region corresponding to the hinge region and, if the immunoglobulin is not an IgG4 subtype, other CH regions such as the CH3 region. Amino acids in which a region can form a disulfide bond with the same CH region in the presence of polyclonal human IgG, or another covalent or stable non-covalent heavy chain bond with the same CH region. It is modified to contain no residues.

In a further embodiment, the heavy chain of the monovalent antibody is modified so that the entire hinge region is deleted.

In another further embodiment, the sequence of the monovalent antibody is modified so that it does not contain a receptor site for N-linked glycosylation.

The present invention "doubles" where the anti-soltilin binding region (eg, the soltilin binding region of an anti-Soltilin monoclonal antibody) is part of a bivalent or polyvalent bispecific backbone that targets two or more epitopes. Also includes "specific antibodies" (eg, the second epitope is an active transport receptor such that bispecific antibodies can exhibit improved transcytosis across biological barriers such as blood-brain barriers. Can contain epitopes of). Thus, in another further embodiment, a monovalent Fab of an anti-sortilin antibody can be bound to an additional Fab or scfv that targets a different protein to produce a bispecific antibody. Bispecific antibodies have dual functions, such as therapeutic functions provided by anti-sortilin binding regions, and transport functions that can bind to receptor molecules to facilitate transport across biological barriers such as the blood-brain barrier. May have.

The antibody of the present invention and its antigen-binding fragment also include a single-chain antibody. Single-chain antibodies are peptides to which the heavy and light chain Fv regions are bound. In one embodiment, the invention presents that the heavy and light chains in the Fv of the anti-soltilin antibody of the invention are in a peptide single chain (typically about 10, 12, 15 or more amino acid residues). ) Provides a single chain Fv (scFv) bound to a flexible peptide linker. Methods for producing such antibodies are described, for example, in US Pat. No. 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds.Springer-Verlag, New York, pp.269-315 (1994). ), Bird et al., Science 242,423-426 (1988), Huston et al., PNAS USA 85,5879-5883 (1988) and McCafferty et al., Nature It is described in 348,552-554 (1990). Single-chain antibodies are monovalent when only a single VH and VL are used, bivalent when two VHs and VLs are used, or used by three or more VHs and VLs. If so, it can be multivalued.

The antibodies and antigen-binding fragments thereof described herein can be modified by inclusion of any suitable number of modified amino acids and / or binding to such conjugated substituents. Compatibility in this regard is generally determined by the ability to at least substantially retain sortilin selectivity and / or sortilin specificity associated with non-derivatized pro-sortilin antibodies. Inclusion of one or more modified amino acids may be advantageous, for example, to increase polypeptide serum half-life, reduce polypeptide antigenicity, or increase polypeptide storage stability. Amino acids are, for example, co-translationally or post-translationally modified during recombinant production (eg, N-linked glycosylation in the NXS / T motif upon expression in mammalian cells). ) Or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, prenylated (eg, farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated. Examples include amino acids and phosphorylated amino acids. There are sufficient references throughout the literature to guide those skilled in the art of amino acid modifications. An example protocol can be found in Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, NJ. The modified amino acid can be selected from, for example, glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to lipid moieties, or amino acids conjugated to organic inducers. ..

Antibodies of the invention and antigen-binding fragments thereof can also be chemically modified by covalent binding to polymers, eg, to increase their blood half-life. Exemplary polymers and methods for binding them to peptides are described, for example, in US Pat. No. 4,766,106, US Pat. No. 4,179,337, US Pat. No. 4,495,285 and US Pat. No. 4,609,546. It is shown. Further exemplary polymers include polyoxyethylated polyols and polyethylene glycols (PEGs) (eg, PEGs with a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000, such as about 3,000 to 12,000 g / mol). Can be mentioned.

The antibodies of the invention and antigen-binding fragments thereof can be further used in diagnostic methods or as ligands for diagnostic imaging.

In one embodiment, an antibody of the invention and an antigen-binding fragment thereof comprising one or more radiolabeled amino acids are provided. Radiolabeled anti-sortilin antibodies can be used for both diagnostic and therapeutic purposes (binding to radiolabeled molecules is another possible feature). A non-limiting example of such a sign is, but not limited to, bismuth (²¹³Bi), carbon (¹¹C,¹³C,¹⁴C), chrome (⁵¹Cr), cobalt (⁵⁷Co,⁶⁰Co), copper (⁶⁴Cu), dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er), Fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga,⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), Holmium (¹⁶⁶Ho), hydrogen (³H), indium (¹¹¹In,¹¹²In,¹¹³In,¹¹⁵In), iodine (¹²¹I,¹²³I,¹²⁵I,¹³¹I), iridium (¹⁹²Ir), iron (⁵⁹Fe), krypton (^81mKr), lantern (¹⁴⁰La), lutetium (¹⁷⁷Lu), manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), nitrogen (¹³N,¹⁵N), oxygen (¹⁵O), palladium (¹⁰³Pd), Rin (Pd)³²P), potassium (⁴²K), Praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re,¹⁸⁸Re), rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb,⁸²Rb), ruthenium (⁸²Ru,⁹⁷Ru), Samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium (⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr,⁸⁹Sr,⁹²Sr), sulfur (³⁵S), technetium (⁹⁹Tc), thallium (²⁰¹Tl), tin (¹¹³Sn,¹¹⁷Sn), xenon (¹³³Xe), Ytterbium (¹⁶⁹Yb,¹⁷⁵Yb,¹⁷⁷Yb), yttrium (⁹⁰Y) and zinc (⁶⁵Zn) can be mentioned. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (eg, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo, eds., Lippincott Raven)). (1996)) and US Pat. No. 4,681,581, US Pat. No. 4,735,210, US Pat. No. 5,101,827, US Pat. No. 5,102,990 (US Reissue Patent No. 35,500), US Patent. See 5,648,471 and US Pat. No. 5,697,902. For example, radioactive isotopes can be bound by the chloramine T method (Lindegren, S. et al. (1998) Chloramine-T In High-Specific-. Activity Radioiodination Of Antibodies Using N-Succinimidyl-3- (Trimethylstannyl) Benzoate As An Intermediate , Nucl.Med.Biol.25 (7): 659-665; Kurth, M. et al. (1993) Site-Specific Conjugation Of A Radioiodinated Phenethylamine Derivative To A Monoclonal Antibody Results In Increased Radioactivity Localization In Tumor , J. Med. Chem.36 (9): 1255-1261; Rea, D Wet al. (1990) Site-specifically radioiodinated antibody for targeting tumors , Cancer Res. 50 (3 Suppl): 857s-861s).

The present invention relates to fluorescent labels (such as rare earth chelates (eg, europium chelate)), fluorescein-type labels (eg, fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), rhodamine-type labels. (For example, ALEXA FLUOR® 568 (Invitrogen), TAMRA® or Dansil Lorid), VIVOTAG 680 XL FLUOROCHROME® (Perkin Elmer), Fluorescein; , BODIPY Also provided are anti-sortilin antibodies and antigen-binding fragments thereof that are detectable and labeled using FL-SE® (Invitrogen) or analogs thereof, all of which are suitable for optical detection. Chemiluminescent labels may be used (eg, luminol, luciferase, luciferin, and aequorin). Such diagnostics and detections also include, but are not limited to, the diagnostic molecules of the invention, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or enzymes including acetylcholinesterase. It can be done by binding to a detectable substance, or, but not limited to, a prosthetic group complex such as streptavidin / biotin and avidin / biotin.

Chemiluminescent labels may be used (eg, luminol, luciferase, luciferin, and aequorin). Such diagnostics and detections also include, but are not limited to, the diagnostic molecules of the invention, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or enzymes including acetylcholinesterase. It can be done by binding to a detectable substance, or, but not limited to, a prosthetic group complex such as streptavidin / biotin and avidin / biotin. Paramagnetic labels can also be used and are preferably detected using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels are, but are not limited to, aluminum (Al), barium (Ba), calcium (Ca), terbium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), gadolinium. (Gd), holmium (Ho), iridium (Ir), lithium (Li), magnesium (Mg), manganese (Mn), molybdenum (M), neodymium (Nd), osmium (Os), oxygen (O), palladium (Pd), Platinum (Pt), Rodium (Rh), Luthenium (Ru), Samalium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm), Tin (Sn), Titanium (Ti), Tungsten (W), and Thulium (Zi), especially Co⁺², CR⁺², Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺², Ti⁺³, V⁺³, And V⁺⁴Paramagnetic ions, positron-emitting metals using various positron-emitting tomography methods, and compounds containing non-radioactive paramagnetic metal ions.

Thus, in one embodiment, the anti-soltilin antibody or soltilin-binding fragment thereof of the invention can be labeled with a fluorescent label, chemiluminescent label, paramagnetic label, radioisotope label or enzyme label. Fragment-labeled antibodies can be used to detect or measure the presence or amount of sortilin in a subject's brain. The method may include detection or measurement of in vivo imaging of the anti-sortilin antibody or sortilin binding fragment bound to the sortilin, including exvivo imaging of the anti-sortilin antibody or sortilin binding fragment bound to such sortilin. obtain.

In a further aspect, the invention relates to an expression vector encoding one or more polypeptide chains of an antibody of the invention or an antigen-binding fragment thereof. Such expression vectors can be used for recombinant production of the antibodies and antigen binding fragments of the invention.

The expression vector in the context of the present invention can be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (nucleic acid sequences containing a suitable set of expression control elements). Examples of such vectors include SV40 derivatives, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the anti-sortilin antibody-encoding nucleic acid is, for example, a linear expression element (eg, as described in Sykes and Johnston, Nat Biotech 12,355-59 (1997)), compacted. Nucleic acid vector (eg, as described in US Pat. No. 6,077,835 and / or WO 00/70087), pBR322, pUC 19/18, or pUC. Naked DNA or RNA vectors containing plasmid vectors such as 118/119, "midge" minimum size nucleic acid vector (eg, as described in Schakowski et al., MoI Ther 3,793-800 (2001)). At or at CaPO₄Precipitated nucleic acid vector constructs such as precipitate constructs (eg, WO 00/46147, Benvenisty and Reshef, PNAS USA 83,9551-55 (1986), Wigler et al., Cell 14,725 (1978), and Coraro and Pearson. , Somatic Cell Genetics 2,603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, eg, US Pat. No. 5,589,466 and US Pat. No. 5,973,972).

In one embodiment, the vector is suitable for expression of an anti-sortilin antibody or antigen-binding fragment thereof in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vector (Van Heeke &amp; Schuster, J Biol Chem 264,5503-5509 (1989), pET vector (Novagen, Madison, WI), etc.). Can be mentioned.

The expression vector can further or instead be a suitable vector for expression in a yeast system. Any vector suitable for expression in the yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as α factor, alcohol oxidase and PGH (F. Ausubel et al., Ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience). New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al.Methods Mol.Biol.824,329-358 (2012), Celik, E. et al.Biotechnol.Adv .30 (5), 1108-1118 (2012), Li, P.et al.Appl.Biochem.Biotechnol.142 (2), 105-124 (2007), Boeer, E.et al.Appl.Microbiol.Biotechnol.77 (3), 513-523 (2007), van der Vaart, JM Methods (Outlined in Mol.Biol.178,359-366 (2002), and Holliger, P.Methods Mol.Biol.178,349-357 (2002)).

In the expression vector of the invention, the anti-sortilin antibody-encoding nucleic acid may contain or be bound to any suitable promoter, enhancer, and other expression-promoting element. Examples of such elements are potent expression promoters (eg, human CMV IE promoters / enhancers and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly (A) termination sequences, Escherichia coli (eg, human CMV IE promoters / enhancers and HIV LTR promoters). The origin of replication of the plasmid product in E. coli), the antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (eg, a polylinker). Nucleic acids can also include inducible promoters as opposed to constitutive promoters such as CMV IE (those skilled in the art will appreciate that such term is, in fact, a descriptive term for the degree of gene expression under certain conditions. Will recognize).

In yet another embodiment, the invention is a transfectoma producing an antibody of the invention as defined herein, or an antigen-binding fragment thereof or a bispecific molecule of the invention as defined herein. For recombinant eukaryotic or prokaryotic host cells, such as (transfectoma). Examples of host cells include yeast, bacteria, and mammalian cells (such as CHO or HEK cells). For example, in one embodiment, the invention provides a cell comprising a nucleic acid stably integrated into a cellular genome comprising a sequence code for expression of the anti-saltylin antibody of the invention or an antigen-binding fragment thereof. In another embodiment, the invention comprises an unfused nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising a sequence code for expression of the anti-saltylin antibody or antigen-binding fragment thereof of the invention. Provides cells containing.

In a further embodiment, the invention is a method for producing the anti-saltylin antibody of the invention, a) the step of culturing the hybridoma or host cell of the invention as described above herein, and b). The present invention relates to a method comprising a step of purifying an antibody of the present invention from a medium.

In one embodiment, the invention is a preparation that, when such terms are used herein, comprises an anti-sortilin antibody as defined herein and is not capable of binding to sortilin. Or relating to a preparation that is substantially free of naturally occurring antibodies that does not substantially alter the anti-sortilin functionality of the preparation. Thus, such preparations do not include naturally occurring serum, or purified derivatives of such serum, and are a mixture of an anti-sortilin antibody and another antibody that does not alter the functionality of the anti-sortilin antibody in the preparation. Including, where such functionality is (i) binding affinity for sortilin (K)._D); (ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis in the brain; (v) Ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice; And / or (vi) the ability to provide treatment for frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when administered chronically.

The present invention particularly relates to such an anti-saltylin antibody having a structural change in its amino acid sequence (either in its CDR, variable region, framework residue and / or constant region) as compared to the structure of the native anti-saltylin antibody. In the preparation of, the structural changes significantly altered the functionality of the anti-saltylin antibody monoclonal antibody compared to the functionality exhibited by the native anti-saltylin antibody (ie, more than 20% difference in functionality, 40). Over% difference, over 60% difference, over 80% difference, over 100% difference, over 150% difference, over 2x difference, over 4x difference, over 5x difference, or over 10x (Super difference); where such functionality is: (i) binding affinity for soltilin (K)_D); (ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis in humans; (vi) Ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice; / Or the ability to increase concentrations and / or (vi) of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and / or Alzheimer's disease (AD) when administered chronically The ability to provide treatment, especially with respect to preparations in which such altered functionality is the result of structural changes and is therefore inseparable from it.

The term "substantially free" of naturally occurring antibodies is used in such preparations in such preparations, in such preparations, or in such preparations, which does not substantially alter the sortilin binding properties of such preparations. Refers to the complete absence of inclusion of such naturally occurring antibodies at certain concentrations. An antibody is said to be "isolated" if it has no naturally occurring counterpart or is separated or purified from its naturally occurring components.

The term "spontaneous antibody", when it comes to such preparations, is an antibody that is induced in living humans or other animals as a natural result of the functioning of their immune system (spontaneous autoantibodies). (Including antibodies).

Accordingly, the preparations of the invention do not exclude such preparations containing anti-sortilin antibodies, and additional antibodies that can be intentionally added that can bind to epitopes not retained by sortilin, and in fact clearly it. Including. Such preparations include, in particular, embodiments thereof that show improved efficacy in treating frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS). ..

The antibodies of the antigen-binding fragment of the present invention are various cell lines such as human cell lines, non-human mammalian cell lines, and insect cell lines such as CHO cell line, HEK cell line, BHK-21 cell line, mouse cell. Can be produced in strains (such as myeloma cell lines), fibrosarma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (detailed herein by reference). Dumont et al, 2015, Crit Rev Biotechnol. Sep 18: 1-13.).

In yet another embodiment, the invention is (i) an anti-saltylin antibody or antigen-binding fragment thereof, or preparation thereof as defined herein, as such terms are defined herein. It relates to a preparation comprising such an anti-saltylin antibody or an antigen-binding fragment thereof, and (ii) a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

The pharmaceutical composition is pharmaceutically acceptable according to conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 2013. It can be formulated with possible carriers or diluents as well as any other known adjuncts and excipients.

Pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients should be suitable for the selected compounds and methods of administration of the invention. Compatibility for carriers and other ingredients of a pharmaceutical composition is determined based on the absence of significant adverse effects on the desired biological properties of the selected compound or pharmaceutical composition of the invention (eg, epitopes). Not significant effect on binding (less than 10% relative inhibition, less than 5% relative inhibition, etc.).

The pharmaceutical composition of the present invention comprises diluents, fillers, salts, buffers, detergents (eg, nonionic detergents such as Tween-20 or Tween-80), stabilizers (eg, sugars or proteins). No amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in pharmaceutical compositions may also be included. Diluents are selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solution, dextrose solution, and Hanks solution. In addition, the pharmaceutical composition or formulation may also include other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. The compositions include proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (eg, latex functionalized sepharose, agarose, cellulose, etc.), polymer amino acids, amino acid copolymers, and lipid aggregates (eg, latex functionalized). It can also include large, slowly metabolized polymers (eg, oil droplets or liposomes).

The actual dose level of the active ingredient in the pharmaceutical composition of the invention is varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response to a particular patient, composition, and method of administration. obtain. The dose level selected is combined with the activity of the particular composition of the invention used, or its amide, route of administration, time of administration, rate of excretion of the particular compound used, duration of treatment, specific composition used. Various drugs including other drugs, compounds and / or materials used in the treatment, age, sex, weight, pathology, overall health and past medical history of the patient being treated, and similar factors well known in the medical field. It depends on the dynamic factors.

The pharmaceutical composition may be administered by any suitable route and method, including parenteral, topical, oral or nasal means for prophylactic and / or therapeutic treatment. In one embodiment, the pharmaceutical composition of the invention is administered parenterally. The terms "parenteral administration" and "administered parenterally" as used herein usually refer to methods of administration other than injectable, intestinal and topical administration, epidermal, intravenous. Intramuscular, intraarterial, intramedullary, intracapsular, intraocular, intracardiac, intracutaneous, intraperitoneal, intratenal, transtracheal, subcutaneous, subepithelial, intra-articular, subcapsular, subepidal, intraspinal, intracranial Includes intra-, intrathoracic, epidural and intrathoracic injections and infusions. Further suitable routes for administering the compounds of the invention in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art. In one embodiment, the pharmaceutical composition is administered intravenously or by subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption retarders that are physiologically compatible with the compounds of the invention. Can be mentioned.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, physiological saline, phosphate buffered saline, ethanol, dextrose, polyols (glycerol, propylene glycol, polyethylene glycol, etc.). , And suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacant gum and injectable organic acid esters (such as ethyl oleate), and / or various buffers. Liquid is mentioned. Other carriers are well known in the pharmaceutical field.

Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is envisioned, unless conventional vehicles or agents are incompatible with the active compound.

Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants.

The pharmaceutical composition of the present invention comprises pharmaceutically acceptable antioxidants such as (1) water-soluble antioxidants (ascorbic acid, cysteine hydrochloride, sodium bicarbonate, sodium metabisulfite, sodium sulfite, etc.); (2). ) Oil-soluble antioxidants (ascorbic palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.); and (3) metal chelating agents (citrate, It may also contain ethylenedidiamine tetraacetic acid (EDTA), sorbitol, tartrate acid, phosphoric acid, etc.).

The pharmaceutical composition of the present invention may also contain isotonic agents such as saccharides, polyalcohol (mannitol, sorbitol, glycerol, etc.) or sodium chloride in the composition.

The pharmaceutical compositions of the present invention are suitable for the route of administration of choice, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which can improve the shelf life or effectiveness of the pharmaceutical composition. It may also contain one or more adjuncts. The compounds of the invention can be prepared with carriers that protect the compound from immediate release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. It may be included alone or with wax or other materials known in the art. Methods for preparing such formulations are generally known to those of skill in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Their use in the pharmaceutical compositions of the present invention is envisioned, unless conventional vehicles or agents are incompatible with the active compound. Auxiliary active compounds can also be incorporated into the composition.

Pharmaceutical compositions for injection typically must be sterile and stable under conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol) and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic acid esters (such as ethyl oleate). It can be an aqueous or non-aqueous solvent or dispersion medium. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants. In many cases, it will be preferable to include an isotonic agent, such as a saccharide, polyalcohol (glycerol, mannitol, sorbitol, etc.), or sodium chloride in the composition. Sustained absorption of the injectable composition can be achieved by including in the composition substances that delay the absorption of the antibody, such as monostearate and gelatin. Sterilized injection solutions can be prepared by incorporating the required amount of active compound in a suitable solvent containing, for example, one or a combination of components as listed above, and if necessary, subsequent sterile microfiltration. .. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from, for example, the components listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum-dried and freeze-dried (lyophilized) to obtain powders of the active ingredient and any additional desired ingredients from those solutions that have been sterile filtered. ).

Sterile injectable solutions can be prepared by incorporating the required amount of active compound in a suitable solvent containing one or a combination of components as listed above and, if necessary, subsequent sterile microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from the components listed above. For sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum-dried and freeze-dried (lyophilized) to obtain powders of the active ingredient and any additional desired ingredients from those solutions that have been sterile filtered. ).

The above methods of treatment and the dosing regimen in the uses described herein are tailored to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, some divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the requirements of the treatment situation. May be good. The parenteral composition can be formulated in a unit dosage form for ease of administration and uniformity of administration. Unit dosage forms as used herein refer to physically separate units suitable as a unified dose for the subject being treated; each unit is associated with the required pharmaceutical carrier. It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect. The specification of the unit dosage form of the present invention is that (a) the unique properties of the active compound and the specific therapeutic effect obtained, and (b) the treatment of such active compound in an individual. It is subject to and directly dependent on the limitations specific to the technical field of formulation.

Effective doses and dosing regimens of anti-sortilin antibodies will depend on the disease or condition being treated and may be determined by one of ordinary skill in the art. An exemplary non-limiting range of therapeutically effective amounts of the antibodies of the invention is about 0.1-10 mg / kg / body weight, such as about 0.1-5 mg / kg / body weight, such as about 0.1-2 mg / kg / body weight. For example, about 0.1-1 mg / kg / body weight, for example about 0.15, about 0.2, about 0.5, about 1, about 1.5 or about 2 mg / kg / body weight.

A physician or veterinarian with conventional skills in the art can easily determine and prescribe an effective amount of the required pharmaceutical composition. For example, a physician or veterinarian may start with a dose of anti-sortilin antibody used in a pharmaceutical composition at a lower level than required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Can increase to. In general, a suitable daily dose of the composition of the invention would be the amount of compound that is the lowest effective dose to produce a therapeutic effect. Such effective doses generally depend on the factors mentioned above. Administration can be, for example, intravenously, intramuscularly, intraperitoneally, or subcutaneously, eg, proximal to the site of the target. If desired, the effective daily dose of the pharmaceutical composition is optionally in unit dosage form, administered separately at appropriate intervals throughout the day, 2, 3, 4, 5, 6 times. Or it can be administered as a higher divided dose. The compound of the present invention can be administered alone, but it is preferable to administer the compound as a pharmaceutical composition as described above.

Labeled antibodies of the invention or antigen-binding fragments thereof can be used for diagnostic purposes to detect, diagnose, or monitor a disease or disorder. The present invention provides the detection or diagnosis of neurodegenerative or cognitive disorders or disorders, including, but not limited to, FTD, ALS or TDP43 proteinosis (such as Alzheimer's disease (AD)), and is (a) soltilin specific. The step of measuring the presence of a pyroglutamyl Aβ fragment in a subject's cell or tissue sample using one or more antibodies that bind to; (b) control levels of the antigen, eg, normal tissue sample. An increase in the measured level of the antigen compared to the level in, thereby comprising a control level of the antigen, indicates a disease or disorder or an indication of the severity of the disease or disorder.

The antibodies of the invention or antigen-binding fragments thereof can be used to measure soltilin or antigen-binding fragments of soltilin in biological samples using immunohistochemical methods well known in the art. Other antibody-based methods useful for detecting proteins include enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay assay (RIA) and mesoscale discovery platform-based assay (MSD). Immunoassay methods such as. Suitable antibody labels can be used in such kits and methods, and known labels in the art include enzyme labels (such as alkaline phosphatase and glucose oxidase); radioactive isotope labels (iodine (iodine (iodine)).¹²⁵I,¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (^99mTc); etc.); and luminescent labels (such as luminol and luciferase); and fluorescent labels (such as fluorescein and rhodamine).

The presence of labeled anti-sortilin antibodies or their sortilin-binding fragments can be detected in vivo for diagnostic purposes. In one embodiment, the diagnosis is a) the step of administering to the subject an effective amount of such a labeled molecule; b) after administration, waiting for a predetermined time interval to place the labeled molecule at the site of Aβ deposition (if). (If any) concentrate to allow unbound labeled molecules to be removed until background levels; c) determine background levels; and d) label molecules above background levels. Detection comprises the step of detecting a labeled molecule in a subject such that the subject suffers from a disease or disorder or indicates the severity of the disease or disorder. According to such an embodiment, the molecule is labeled with an imaging moiety suitable for detection using a particular imaging system known to those of skill in the art. Background levels can be determined by a variety of methods known in the art, including comparing the amount of labeled antibody detected to standard values predetermined for a particular imaging system. Methods and systems that can be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), And ultrasonography.

In a further aspect, the invention relates to an antibody of the invention or an antigen-binding fragment thereof for medical use.

In a further aspect, the invention relates to an antibody of the invention, or an antigen-binding fragment thereof, for use in treating a disease associated with decreased PGRN levels in a patient's brain.

In a further aspect, the invention relates to the use of an antibody of the invention, or an antigen-binding fragment thereof, in the manufacture of a drug for treating a disease associated with decreased PGRN levels in a patient's brain.

In a further embodiment, the invention is a method of preventing or treating a disease associated with reduced PGRN levels in a patient's brain, the step of administering an effective dose of the antibody of the invention, or an antigen-binding fragment thereof. Regarding methods including.

In the use and method of those embodiments of the invention, the disease is preferably FTD; ALS; or TDP43 proteinosis (such as AD).

Preferably, in the use and method of those embodiments of the invention, the treatment is long-term, preferably over at least 2 weeks, eg at least 1 month, 6 months, 1 year or more.

In a further aspect, the invention provides a kit comprising the antibody of the invention, or an antigen binding fragment thereof.

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The enumeration or description herein of a document that appears to have been previously published is not necessarily construed as a confirmation that the document is part of prior art or is common general knowledge.

Embodiments As will be apparent from the text and examples, the present invention further relates to the following embodiments.

1. An antibody that specifically binds to sortilin and is capable of inhibiting the binding of PGRN to sortilin, or an antigen-binding fragment thereof.

2. The antibody according to Embodiment 1, or an antigen-binding fragment thereof, wherein the antibody comprises or comprises an intact antibody.

3. The antigen-binding fragment is an Fv fragment (eg, single-chain Fv or disulfide-bonded Fv); a Fab-like fragment (eg, Fab fragment or F (ab')).₂Fragments); and domain antibodies (eg single V)_HVariable area or V_LThe antibody according to embodiment 1 or 2, or an antigen-binding fragment thereof, comprising or consisting of an antigen-binding fragment selected from the group consisting of (variable regions).

4. The antibody according to any preceding embodiment, or an antigen-binding fragment thereof, wherein the antibody is selected from the group consisting of antibodies of subtypes IgG1, IgG2, IgG3 or IgG4.

5. The antibody according to any of the preceding embodiments, or an antigen-binding fragment thereof, wherein the antibody or an antigen-binding fragment thereof specifically binds to the D region of sortilin defined in SEQ ID NO: 170.

6. Any preceding embodiment, wherein the antibody or fragment thereof specifically binds to at least 3 contiguous amino acids in the D region of sortilin defined in SEQ ID NO: 170, eg, 4, 5, 6 or 7 contiguous amino acids. An antibody or an antigen-binding fragment thereof.

7. The antibody or antigen binding fragment has the following properties: (i) Binding affinity for sortilin of 0.5-10nM, eg 1-5nM or 1-2nM (K)_D), (Ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis; (v) One or more of the ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice. The antibody according to the preceding embodiment, or an antigen-binding fragment thereof.

8. Embodiment 7 comprising that the ability of an antibody or fragment thereof to reduce PGRN binding to sortilin reduces PGRN binding to sortilin by 10% or more; for example, by 20% or more; or by 30% or more. The antibody or antigen-binding fragment thereof described in 1.

9. With an IC50 of 22nM or less, eg, 22nM-1nM, or 10nM-1nM, or 5nM-1nM, said antibody or fragment thereof, antibody or fragment thereof, that reduces and / or inhibits PGRN binding to sortilin. , The antibody according to embodiment 7 or 8, or an antigen-binding fragment thereof, comprising reducing and / or inhibiting PGRN binding to sortilin.

10. An antibody according to any preceding embodiment, which is a human or humanized antibody, or an antigen-binding fragment thereof.

11. Light chain variable regions containing one or more of the CDRs 1-3 light chains listed for each of the antibodies defined in Table 5, or 4 or less amino acid differences, or 3 or less amino acid differences, or 2 The antibody according to any preceding embodiment, or an antigen-binding fragment thereof, comprising an amino acid sequence having one or less amino acid differences or one or less amino acid differences.

12. The antibody of Embodiment 11, or an antigen-binding fragment thereof, comprising a light chain variable region comprising CDR1-3 light chains listed for each of the antibodies defined in Table 5.

13. The antibody according to embodiment 11 or 12, or an antigen-binding fragment thereof, comprising or comprising a light chain variable region comprising or consisting of the amino acid sequence VL listed for each of the antibodies defined in Table 5.

14. The antibody according to any of embodiments 11-13, or an antigen-binding fragment thereof, comprising a light chain comprising or consisting of the VL amino acid sequence listed for each of the antibodies defined in Table 5.

15. One or more CDR1 to triple chains listed for each of the antibodies defined in Table 5, or 4 or less amino acid differences, or 3 or less amino acid differences, or 2 or less amino acid differences, Alternatively, an antibody according to any preceding embodiment, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising an amino acid sequence having one or less amino acid differences.

16. The antibody of embodiment 15, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a CDR1 to triple chain listed for each of the antibodies defined in Table 5.

17. An antibody according to embodiment 15 or 16, or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising or consisting of the amino acid sequence of VH listed for each of the antibodies defined in Table 5.

18. An antibody according to any of embodiments 15-17, or an antigen-binding fragment thereof, comprising a heavy chain comprising or consisting of the amino acid sequence VL listed for each of the antibodies defined in Table 5.

19. Contains the light chain variable region containing or consisting of the VL amino acid sequences listed for each of the antibodies defined in Table 5, and the VH amino acid sequences listed for each of the antibodies defined in Table 5. The antibody, or antigen-binding fragment thereof, according to any preceding embodiment, comprising or consisting of a heavy chain variable region.

20. A light chain containing or consisting of a VL amino acid sequence listed for each of the antibodies defined in Table 5 and a VH amino acid sequence listed for each of the antibodies defined in Table 5 or An antibody, or antigen-binding fragment thereof, according to any preceding embodiment, comprising a heavy chain comprising.

21. The antibody according to any of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof competes with the antibody according to embodiment 20 for binding to sortilin, or an antigen-binding fragment thereof. Fragment.

22. The antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody or antigen-binding fragment comprises an Fc region.

23. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment further comprises a portion for increasing the in vivo half-life.

24. The antibody or antigen binding thereof according to embodiment 22, wherein the moiety for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, glycosylation groups, fatty acids and dextran. Fragment.

25. An antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment further comprises a detectable moiety.

26. The antibody of embodiment 25, or an antigen-binding fragment thereof, wherein the detectable moiety is selected from the group consisting of a fluorescent label; a chemiluminescent label; a paramagnetic label; a radioisotope label; or an enzyme label.

27. The antibody or antigen-binding fragment thereof according to embodiment 25 or 26, wherein the detectable moiety comprises or comprises a radioisotope.

28. Radioisotopes^99mTc,¹¹¹In,⁶⁷Ga,⁶⁸Ga,⁷²As,⁸⁹Zr,¹²³I and²⁰¹The antibody or antigen-binding fragment thereof according to embodiment 26 or 27, selected from the group consisting of Tl.

29. The antibody or antigen-binding fragment thereof according to embodiment 25, wherein the detectable moiety comprises or consists of a paramagnetic isotope.

30. Paramagnetic isotopes,¹⁵⁷Gd,⁵⁵Mn,¹⁶²Dy,⁵²Cr and⁵⁶The antibody or antigen-binding fragment thereof according to embodiment 29, selected from the group consisting of Fe.

31. The antibody or antigen-binding fragment thereof according to any of embodiments 25-30, wherein the detectable moiety is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasonic imaging.

32. The antibody or antigen-binding fragment thereof according to any of embodiments 25-31, wherein the detectable moiety is indirectly bound to the antibody or antigen-binding fragment thereof via the binding moiety.

33. The binding moiety is 1,4,7,10-tetraazacyclododecane-1,4,7,10, a derivative of tetraacetic acid (DOTA), deferroxamine (DFO), a derivative of diethylenetriaminepentaacetic acid (DTPA), S. -2- (4-isothiocyanatobenzyl) -1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and 1,4,8,11-tetraazacyclododecane- The antibody or antigen-binding fragment thereof according to embodiment 32, selected from the group consisting of derivatives of 1,4,8,11-tetraacetic acid (TETA).

34. An isolated nucleic acid molecule encoding the antibody according to any of embodiments 1-33, or an antigen-binding fragment thereof.

35. The nucleic acid molecule according to embodiment 34, wherein the molecule is a cDNA molecule.

36. A vector comprising the nucleic acid molecule according to embodiment 34 or 35.

37. Recombinant host cell comprising the nucleic acid molecule according to any of embodiments 34-36.

38. The method for producing the antibody or antigen-binding fragment according to any one of embodiments 1-33, under conditions that allow the expression of the encoded antibody or antigen-binding fragment thereof, embodiment 37. A method comprising the step of culturing a host cell according to.

39. A preparation comprising the antibody according to any one of the preceding embodiments, or an antigen-binding fragment thereof, wherein the preparation is not capable of binding to sortilin or the anti-sortilin function of the preparation. Substantially free of naturally occurring antibodies that do not substantially alter sex, said functionality is (i) binding affinity for sortilin (K)._D); (ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis; (v) Ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice; and / or (vi) chronically administered A preparation selected from the group consisting of the ability to provide treatment for frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when it is done.

40. A preparation comprising the monoclonal antibody according to any one of the preceding embodiments or an antigen-binding fragment thereof, wherein the monoclonal antibody has a structural change in its amino acid sequence as compared with the structure of a natural anti-saltylin antibody. Due to the structural changes, the monoclonal antibody exhibits altered functionality compared to the functionality exhibited by the natural anti-saltylin antibody, which is (i) binding affinity for soltilin (K)._D); (ii) Ability to reduce and / or inhibit PGRN binding to sortilin; (iii) Ability to reduce and / or inhibit PGRN clearance by sortilin-expressing cells; (iv) PGRN by sortilin-expressing cells Ability to reduce and / or inhibit endocytosis; (v) Ability to increase the amount and / or concentration of PGRN in plasma in human-sortilin-expressing knock-in mice; and / or (vi) chronically administered A preparation that is capable of providing treatment for frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when it is done.

41. A pharmaceutical composition comprising the antibody according to any one of embodiments 1-33, or an antigen-binding fragment thereof, or the preparation according to any one of embodiments 39-40, and a pharmaceutically acceptable carrier. ..

42. The antibody according to any one of embodiments 1-33, or an antigen-binding fragment thereof, or the preparation according to any one of embodiments 39-40 for medical use.

43. Antibodies according to any of embodiments 1-33, or antigen-binding fragments thereof, or embodiments thereof, for use in the prevention and / or treatment of diseases associated with reduced PGRN levels in a patient's brain. The preparation according to any one of forms 39-40.

44. The antibody according to any of embodiments 1-33, or an antigen-binding fragment thereof, or performed in the manufacture of a drug for preventing and / or treating a disease associated with decreased PGRN levels in a patient's brain. Use of the preparation according to any one of forms 39-40.

45. The antibody for use according to embodiment 43, or an antigen-binding fragment thereof, or the use according to embodiment 44, wherein the disease is selected from the group consisting of FTD; ALS; TDP43 proteinosis (such as AD). ..

46. A method of preventing or treating a disease associated with decreased PGRN levels in a patient's brain, wherein an effective dose of the antibody or fragment thereof according to any of embodiments 1-33, embodiments 39-. A method comprising the step of administering the preparation according to any one of 40 or the pharmaceutical composition according to embodiment 41.

47. The antibody for use according to embodiment 43, or an antigen-binding fragment thereof, or according to embodiment 44, wherein the disease is selected from the group consisting of FTD; ALS; or TDP43 proteinosis (such as AD). Use, or the method according to embodiment 46.

48. Antibodies for use according to embodiment 46 or 47, or antigen-binding fragments thereof; or uses; or methods, wherein the treatment is long-term.

49. Antibodies for use, or antigen-binding fragments thereof; or methods thereof, according to embodiment 48, wherein the long-term treatment spans at least 2 weeks, eg at least 1 month, 6 months, 1 year or more. ..

50. It is possible to specifically bind to sortilin and inhibit the binding of PGRN to sortilin, but the binding does not inhibit or substantially inhibit the binding of neurotensin or AF38469 to sortilin, performed. The antibody according to any one of embodiments 1 to 33, or an antigen-binding fragment thereof, the preparation according to any one of embodiments 39 to 40, or the pharmaceutical composition according to embodiment 41.

51. A kit comprising the antibody according to any one of embodiments 1-33, or an antigen-binding fragment thereof, the preparation according to any one of embodiments 39-40, or the pharmaceutical composition according to embodiment 41. ..

Accordingly, preferred non-limiting examples of carrying out a particular aspect of the invention are described with reference to the accompanying figures.

<p>Examples 1-3 illustrate the formation of sortilin constructs. Example 1 discloses shuffle constructs. Example 2 discloses the expression of the sortilin construct. Example 3 discloses purification of the sortilin construct.</p><p>Examples 4-7 illustrate the production of sortilin antibodies Example 4 discloses immunization and hybridomas. Example 5 discloses sequence analysis. Example 6 discloses purification of the antibody. Example 7 discloses the production of mouse antibodies.</p><p>Examples 8-17 illustrate the characterization of sortilin antibodies. Examples 8 disclose binding to sortilin. Example 9 discloses the cross-blocking ability of sortilin antibodies. Example 10 discloses HTRF PGRN-sortilin binding. Example 11 discloses NTS binding. Example 12 discloses cellular PGRN binding and endocytosis. Example 13 discloses extracellular PGRN levels. Example 14 discloses iPSC PGRN levels. Example 15 discloses plasma PGRN levels. Example 16 discloses epitope mapping by HDX. Example 17 discloses microdialysis of PGRN in the brain.</p><p>Example 1 A distantly related species (tetraodone) that designs, constructs, and produces so-called "shuffle constructs" for both hybridoma screening processes and antibody panel diversification, and has significantly lower sequence homology. ), A series of chimeric saltylin molecules containing amino acid sequences derived from both were prepared. The principle that the entire sortilin structure and functionality of these chimeric constructs can be retained, except for the loss of antibody binding to specific chimeric constructs, would indicate improved binding of specific exchanged regions. Soluble extracellular space (ECD, aa) 1-755) The construct was labeled with either a BAP tag (biotin acceptor peptide) and co-expression of a biotin ligase or His tag allowed "in vitro" biotinification of the protein, allowing for easy purification. Expression vectors encoding the following proteins were prepared: SORT-ECDBAP, SORT-ECDBAP-hB01-05, SORT-ECDBAP-hB06-10, SORT-ECDBAP-hB12390, SORT-ECDBAP-hB45678, SORT-ECDBAP-tetra, SORT, SORT-tetra.</p><p> Sortilin sequences are found in SEQ ID NOs: 169-180, and FIG. 2 shows a schematic representation of antibody region allocation based on binding to the sortilin shuffle construct.</p><p>Example 2 For antibody expression, suitable heavy and light chain vectors as described in Examples 4, 5 and 6 were co-expressed in HEK-293F cells.</p><p>Example 3: Purification of His-tagged sortilin SORTECDHis was expressed in HEK-293F cells. His-tags in proteins allow purification by immobilized metal affinity chromatography. In this process, NiNTA Superflow Cartridge (Qiagen), 50mM NAH₂PO₄Equilibrate with 300 mM NaCl and 10 mM imidazole pH 8.0. The column is loaded with His-tagged protein with a residence time of 1 minute. Column, 50 mM NAH₂PO₄Wash with 300 mM NaCl and 20 mM imidazole pH 8.0. Protein, 50 mM NAH₂PO₄Elute with 300 mM NaCl and 250 mM imidazole pH 8.0. The protein is then dialyzed against PBS using Slide-A-Lyzer (Thermo Scientific) with a cutoff of 10.000 mwco. After dialysis, the protein is sterile filtered using a 0.2 micron SFCA filter (Thermo Scientific).</p><p> HEK293 cells were transfected with a human wild-type (WT) sortilin expression vector to generate an S18-HEK cell line. Stable transfected cells were obtained after passage in the presence of the drug of choice. Individual clones were selected by diluted cloning. The clones were characterized for sortilin mRNA expression using QPCR. The highest expressed clones were then analyzed by FACS (Guava, Millipore) using an anti-sortilin polyclonal antibody (polyclonal goat soltilin biotinylated Ab, Cat.No: BAF2934 (R &amp; D Systems)) and the surface of sortilin. The expression level was determined.</p><p>Example 4 Immunization Procedure for A-Transgenic Mice The antibody HuMab Soltilin is a HuMAb mouse strain HCo12, HCo17, HCo20, HCo12-BALB / c, HCo17-BALB / c and HCo20-BALB / c (human monoclonal antibody; Medarex Inc.). , San Jose, CA, USA) from immunization. These mice are double knocked out of the mouse immunoglobulin (Ig) heavy chain and the mouse κ light chain, which substantially inactivates antibody expression in complete mice. Various mouse strains are transgeniced by insertion of human Ig heavy chain and human Igκ light chain loci, which differ in the number of human VH (heavy chain variable region) and VL (light chain variable region) genes. .. HCo12-BALB / c mice were obtained by crossbreeding with KCo5-BALB / c (κ light chain transgenic) mice.</p><p> Forty-eight mice were alternately immunized with 20 μg SORTECDHis (SEQ ID NO: 179) intraperitoneally (IP) and subcutaneously (SC, caudal basal) with the same protein at 14-day intervals. Up to 8 immunizations were performed with 4 IPs and 4 SCs.</p><p> In one protocol, the first immunization was performed with SORTECDHis in a complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA) and the next immunization was performed in an incomplete Freund's adjuvant (IFA). The second protocol used SAS as an adjunct in all immunization steps. When serum antibody titers were found to be sufficient (1/50 or less serum dilutions were found to be positive in the antigen-specific screening assay in at least two consecutive biweekly screening events). , Mice were intravenously (IV) twice more boosted with 10 μg SORTECDHis protein in 100 μL PBS 4 and 3 days prior to fusion.</p><p>B-HuMab hybridoma-generation HuMAb mice with sufficient antigen-specific antibody titer expression as defined above were killed and the spleen and lymph nodes adjacent to the abdominal aorta and vena cava were harvested. Fusion of splenocytes and lymph node cells with mouse myeloma cell lines is performed by electrofusion using the CEEF 50 Electrofusion System (Cyto Pulse Sciences, Glen Burnie, MD, USA), basically according to the manufacturer's instructions. rice field. Fused cells in 10% Fetal Clone I in HyQ mADCF-Mab (Perbio) Bovine serum (Perbio), 1 mM sodium pyruvate (Cambrex), 0.5 U / mL penicillin, 0.5 U / mL streptomycin (Cambrex), 50 μM 2-mercaptoethanol (Invitrogen), 600 ng / mL interleukin 6 ( It was seeded in a fusion medium containing IL-6) (Strathmann), 1 × HAT (Sigma) and 0.5 mg / mL Invitrogen. After 10 days, the supernatant was collected and the cells were subjected to 10% Fetal Clone I in HyQ mADCF-Mab. Washed with collection medium containing Bovine serum, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, 600 ng / mL IL-6 and 1 × proHT (Cambrex). The supernatant of the hybridoma culture was screened by a primary screening assay and streptavidin beads bound to SORTECDBAPhB06-10 (SEQ ID NO: 176), SORTECDBAPhB12390 (SEQ ID NO: 177) to produce hybridoma-producing humans (SEQ ID NO: 177). Or chimeric) anti-saultylin antibody was detected. Hybridoma cells from the best primary wells were seeded in semi-solid medium prepared from 40% Clone Media (Genetix, Hampshire, UK) and 60% HyQ 2 x complete medium (Hyclone, Waltham, USA). For each primary well, wells in a Genetix black 6-well plate were sown. From each well, ClonePix Twenty-five subclones were collected using system (Genetix). Subclones were collected in the collection medium. After 7 days, the supernatant of the subclone was screened again for sortilin-specific human IgG binding and human IgG concentration was measured using Octet 384 red (Fortebio, Menlo Park, USA). From each primary well, select the best subclone in expansion medium containing only 600 ng / mL IL-6, 0.5 U / mL penicillin, 0.5 U / mL streptomycin and 1 × proHT. Deployed in. This subclone was expanded from one 96-well plate well to one 24-well plate well, to four 24-well plate wells, and to six 6-well plate wells. The clones obtained by this process were referred to as primary clones (PCs).</p><p> The anti-sortilin HuMab antibody of the present invention was identified and sequenced.</p><p>Example 5: Sequence Analysis of Soltilin-Specific HuMab Variable Regions and Cloning in Expression Vectors Using the SMART RACE cDNA Amplification Kit (Clontech), use the SMART RACE cDNA Amplification Kit (Clontech) to generate 0.2-5 × 106 hybrid doma cells. 5'-RACE-Complementary DNA (cDNA) was prepared from 100 ng of total RNA. VH and VL coding regions are amplified by PCR and ligation-independent cloning (Aslanidis, C. and PJde Jong, Nucleic Acids Res) In 1990; 18 (20): 6069-74), it was cloned directly in the frame in p33G1f and p33κ expression vectors, including the human IgG1 / κ constant region coding sequence. For each antibody, 16 VL clones and 16 VH clones were sequenced. Clone with a suitable open reading frame (ORF) was selected for further research and expression. Vectors for all combinations of heavy and light chains were transiently co-expressed in FreestyleTM 293-F cells using 293fectin.</p><p> The resulting sequences are shown herein in a sequence listing (SEQ ID NOs: 1-168). The CDR sequence was defined according to published guidelines.</p><p>Example 6: Purification of Antibodies The supernatant of the culture is filtered over a 0.2 μm dead-end filter and loaded onto a 5 mL protein A column (rProtein A FF, Amersham Bioscience) with 0.1 M citrate-NaOH, Eluent at pH 3. The eluate was immediately neutralized with 2M Tris-HCl, pH 9 and 12.6 mM NaH.₂PO₄, 140 mM NaCl, pH 7.4 (B. Braun) on O / N (overnight) dialysis. After dialysis, the sample was sterile filtered on a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE and concentration was measured by turbidimetry and absorbance at 280 nm. The purified antibody was divided and stored at -80 ° C. After thawing, the purified antibody aliquot was kept at 4 ° C. Mass spectrometry was performed to determine the molecular weights of the antibody heavy and light chains expressed by hybridomas.</p><p>Example 7: Generation of Mouse Antibodies (1F2 and 5E1) Immunogen Chimera Immunogen h Soltilin-FC, (Human Soltilin AA from SEQ ID NO: 169 (78-756)) and Human IgG1-FC AA from SEQ ID NO: 169 (104) The synthetic gene encoding -330) was cloned into pcDNA3.1 and used for expression using the Invitrogen freestyle system. Antigen was purified from the cell culture supernatant by protein-A affinity chromatography using the standard procedure for antibody purification described above for human antibodies.</p><p>Hybridoma-producing h sortilin-FC was used as an immunogen to immunize 5 BALB / c mice. Mice with a sufficient immune response were selected for cell fusion and hybridoma production. Hybridoma supernatants were screened by ELISA using h sortilin-ECD as the coating antigen. A total of 18 hybridoma cell lines obtained from 9 parent clones were generated.</p><p>Expression hybridomas were first grown in full growth medium, DMEM containing 10% FBS + antibiotics, and then adapted to CD hybridoma medium (Invitrogen) for expression experiments.</p><p>Purified mouse monoclonal antibodies were purified from hybridoma cell culture supernatants with protein-G sepharose according to standard procedures recommended by the supplier (GE healthcare).</p><p>Example 8: Affinity of Sortilin-Specific HuMab and Mouse Antibodies to the Recombined Extracellular Region of Sortilin The rate of binding of anti-sortilin HuMab antibody to sortilin was determined using Octet 384 RED (Fortebio, Menlo Park, USA). A 2 μg / ml HuMab solution was prepared by dilution in a sample diluent (ForteBio, art. No. 18-5028). Prot A sensor (ForteBio, art.no.18-0004) with kinetics buffer for at least 600 seconds. Pre-moistened with buffer) (1:10 sample diluent in PBS). Subsequently, the sensor was fixed with HuMab solution for 600 seconds. The baseline reaction was obtained by immersion in Kinetics buffer for 120 seconds. Meetings of SORTECD constructs took place during the 1000 second incubation. This was followed by dissociation in kinetic buffer for 100 seconds. After dissociation, the sensor was regenerated (10 mM glycine pH 1.0) and neutralized 3 times over 5 seconds (kinetic buffer). All HuMab were analyzed using 4 concentrations of SORTECD construct (10, 5, 2.5 and 1.25 μg / ml). A molecular weight of 76.8 kDA was used for SORTECDHis. The data were fitted with ForteBio Analysis 6.4 software using global full fit. The results are shown in Figures 3 and 4.</p><p>Example 9: Anti-Soltilin HuMab antibody cross-block An antibody cross-block test was performed using Octet 384 RED (Fortebio, Menlo Park, USA). A 2 μg / ml HuMab antibody solution was prepared by dilution in a sample diluent (ForteBio, art. No. 18-5028). An amine reactivity sensor (ForteBio, art.no.18-0008) was used to immobilize HuMab. HuMab was diluted in MES pH 6.0 buffer (18-5027) prior to coupling to the amine reactivity sensor. Coupling was performed at 30 ° C and 1000 rpm as follows: The amine reactivity sensor was pre-moistened in PBS followed by EDC / NHS (ForteBio) for 300 seconds (as directed by the manufacturer). .Art.no.18-1033 / 18-1034) Activated with an activation solution. The activation sensor was immobilized with HuMab for 600 seconds. Immobilization sensors were quenched for the remaining amine reactivity with ethanolamine (ForteBio, cat). no.18-1039). After quenching, the sensor was placed in PBS until it was used. Cross-block analysis begins by setting the baseline reaction at 30 ° C and 1000 rpm. The baseline reaction was obtained by immersing in the sample diluent for 120 seconds. The SORTECDHis meeting was held for 300 seconds, followed immediately by the HuMab meeting for 300 seconds. After the HuMab meeting, the sensor was regenerated (10 mM glycine pH 1.0) and neutralized 3 times over 5 seconds (sample diluent). The data was processed using ForteBio Analysis 6.4 software.</p><p> Antibodies were classified based on their binding profile for different saltylin shuffle constructs (FIG. 2, FIG. 3 and FIG. 4). Octet384, their ability to block each other's binding to wild-type human sortilin ECD to ensure that all antibodies from region D (and region F) bind to the same region in human wild-type sortilin ECD. The characteristics were evaluated in a cross-blocking test using red. For example, when testing antibodies from the same region, the primary antibody blocked the binding of the secondary antibody and vice versa. On the other hand, when antibodies from different regions were tested, there was no cross-blocking as only one region was blocked by the primary antibody and the remaining region was utilized for binding of the secondary antibody. FIG. 7 shows that all D regions and D + antibodies cross block each other, which supports the classification of antibodies against regions D and D + based on the shuffle construct. In addition, these data also support that the chimeric sortilin construct retains similarities to the native human wild-type sortilin ECD.</p><p>Example 10: Evaluation of characteristics of sortilin-PGRN ligand binding in the presence of anti-sortilin antibody The IC50 value of the antibody is measured for the transfer of PGRN binding to sortilin using a uniform time-resolved fluorescence (HTRF, CisBio) assay. (See Figures 5 and 6).</p><p> Experiments were performed on Greiner 384-well, white small volume microtiter plates (784075, Greiner) in 20 μl total volume assay buffer (50 mM phosphate, pH 7.0, 0.1% BSA).</p><p> Antibodies were preincubated with 50 nM HIS-tagged sortilin ECD and 4 nM PGRN (SULU20110924) for 15 minutes at room temperature, then conjugated buffer (50 mM phosphate, pH 7.0, 0.8 mM KF, 0.1%). 7 nM anti-6HIS-d2 and 0.7 nM anti-PGRN-Eu crypto (Cisbio) diluted in BSA) were added. 20 μM neurotensin was used as a positive control and DMSO in buffer was used as a negative control.</p><p> Assay plates were incubated overnight at room temperature for 60 minutes and at 4 ° C, and then the plates were read on an EnVision reader (Perkin Elmer).</p><p> Unlabeled neurotensin and DMSO blanks were used as positive and negative controls for the assay facility, respectively. Dose-response evaluation of the antibody was performed using a concentration of 10 from 1 μM to 50 pM on the 3-fold dilution curve.</p><p> Half-inhibition concentration (IC50) was calculated by non-linear regression using an S-shaped concentration reaction (variable slope) in XLfit 4 (IDBS, UK) (Figs. 5 and 6).</p><p>Example 11: characterization of sortilin-neurotensin binding in the presence of anti-sortilin antibody Sortilin-specific compound AF38469 (Schroeder et al, Bioorg Med Chem Lett. 2014 Jan 1; 24 (1): 177-80, 2014). IC50 to sortilin using scintillation proximity assay (SPA)³It was determined by measuring the transfer of H-neurotensin binding.</p><p> Experiments were performed on a 384-well milky white Optiplate (6007299, Perkin Elmer) with a total volume of 40 μl of assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM CaCl).₂, 0.1% BSA, 0.1% Tween-20).</p><p> Preincubate 150 nM HIS-tagged sortilin with or without 1 μM sortilin-specific antibody (IgG1-6003-045 or IgG1-6003-068) or with a human IgG1 isotype control for 15 minutes at room temperature. Then, the protein solution was added to the well containing AF38469 at a concentration series of 50 μM to 2.5 nM. Incubate the mixture in a shaker at room temperature for 15 minutes, then 5 nM.³H-Neurotensin and Ni-chelate imaging beads (RPNQ0266, Perkin Elmer) were added. The assay was incubated under the same conditions for an additional 60 minutes.</p><p> After 6 hours, the plate was read in ViewLux (exposure time of 360 seconds). Unlabeled neurotensin and DMSO blanks were used as positive and negative controls, respectively.</p><p> Dose-response evaluation for AF38469 was performed at 10 concentrations of 50 μM to 2.5 nM on the 3-fold dilution curve. Half-inhibition concentration (IC50) was calculated by non-linear regression using S-shaped concentration reaction (variable slope) in XLfit 4 (IDBS, UK). The results can be seen in Figure 8.</p><p>Example 12: Characteristic evaluation of sortilin-PGRN ligand binding to the surface of cells in the presence of anti-sortilin antibody Transfected cells transiently transfected with sortilin and stable cell line S18-HEK cells (human saltylin overexpressing HEK cells) Both were used in this assay. The cells were trypsinized and plated in 96-well plates at a density of 42,000 cells per well. For transiently transfected cells, the cells were plate cultured 24 hours after transfection in 96-well plates. The next day, the medium was completely replaced, the test compound diluted in the medium was added to the cells for 30 minutes, and then PGRN was added for 4 hours. At the end of the test (4.5 hours later), cells were fixed and stained for PGRN. All stained plates were analyzed by Cellomics Array Scan (Thermo Fischer) and the average staining intensity of PGRN / cells / wells was used for the analysis.</p><p> The PGRN used in the assay was harvested from the medium after transient transfection of the PGRN expression plasmid in HEK 293 cells. PGRN levels were measured using the PGRN ELISA kit (R &amp; D).</p><p> The PGRN added to the cells is readily bound and incorporated, which results in an increased fluorescence signal in the sortilin-transfected wells. The addition of neurotensin prevented sortilin binding to PGRN, and the PGRN fluorescence intensity was similar to the control level, indicating that PGRN was not bound or incorporated in the presence of neurotensin. ..</p><p> Sortilin HumAb (45 and 68) both blocked the uptake of PGRN with the same efficacy as neurotensin. The isotype control antibody, B12, had no effect on PGRN endocytosis or binding. The results can be seen in Figure 9.</p><p>Example 13: Effect of antibody on extracellular PGRN levels Both HEK293 and S18-HEK cells were found to continuously secrete PGRN into the medium without stimulation.</p><p> Antibodies and control agents were added to S18-HEK cells and their effect on PGRN was evaluated. Addition of neurotensin, known peptide soltilin ligands, or human antibodies 45, 68 and 811 to S18-HEK cells increased PGRN in cell culture medium. Two of the Soltilin human antibodies (45 and 68) increased PGRN levels to 202% and 201%, respectively, with effects similar to neurotensin. Antibody 811 increased PGRN to 146% in medium compared to control B12 and the isotype control antibody was used as a negative control in all tests, which showed no effect on PGRN levels. These observations indicate that the sortilin antibody tested inhibits the sortilin-mediated internalization of PGRN, thereby increasing extracellular PGRN.</p><p> On day 1, S18-HEK cells were seeded in 96-well plates. After 24 hours, the medium was completely replaced with medium alone (control) or medium supplemented with test compound. Unless otherwise specified, all compounds were tested at 10uM and antibodies at 100nM. Medium was collected on day 3 and analyzed using PGRN ELISA (R &amp; D). Cell viability was evaluated by Cell TiterGlo (Pro Mega) to evaluate the cytotoxic effect of the compound. PGRN levels in medium were analyzed by ELISA and the values were normalized to control wells. The results can be seen in Figure 10.</p><p>Example 14: ELISA assay for extracellular PGRN in iPSC Induced pluripotent stem cells (iPSCs) are described elsewhere (Rasmussen et al., Stem Cell Reports. 2014 Sep 9; 3 (3). ): 404-13.) Generated by non-integrative reprogramming of human fibroblasts (normal human skin fibroblasts in an 18-year-old man; Lonza). The NHDF K1_shp53 strain was used for these tests. iPSCs were first generated in mTESR medium and then cultured in a monolayer in Pluripro (Cell Guidance System). The cells were re-cultured on a poly-L-ornithine / laminin coated dish and they were placed in N3 medium (RA, 0.5 mM GlutaMAX, 0.5 &amp;) with 500 ng / mL brain (noggin) and 10 μM SB431542. By culturing in 50% DMEM / F12 + 50% Neurobasal medium supplemented with 0.5% N2, 1% B27 with NEA, 50 μM 2-mercaptoethanol and 2.5 mg / mL insulin). Nerve differentiation was started on day 0. The medium was refreshed daily. Eleven days after brain / SB431542 induction, cells were divided by dispase and re-cultured in poly-L-ornithine / laminin in N3 medium. From that point on, N3 medium was freshened every 2-3 days and cells were split approximately every 10-14 days using accutase.</p><p> Neurodifferentiated iPSC cells were plate-cultured into 96-well plates. After 1 week, the antibody was added to the cells. Medium from cells was collected at 48 or 96 hours, analyzed by human PGRN ELISA (Enzo Life sciences), and samples were analyzed according to the manufacturer's instructions.</p><p> Soltilin human antibodies (45 and 68) tested increased PGRN levels at variable levels in the medium at 48 and 96 hours. B12 and anti-Hel are control isotype antibodies (negative controls). The data are shown as mean ± SD. The data were analyzed by one-way Anova followed by Danette's analysis.^*p &lt;0.05;^**p &lt;0.01. The results can be seen in Figure 12.</p><p>Example 15 To analyze the effect of the antibody on plasma PGRN levels, humanized sortilin KI mice were given single or multiple infusions (10 mg / kg) of sortilin antibody or isotype control by subcutaneous infusion. Animals were anesthetized and slaughtered at various points after dosing and plasma PGRN levels were determined by ELISA.</p><p> A. Time-lapse study: Mice were treated with antibody (sortilin humab or control ab) and slaughtered at various time points. Mice treated with the control antibody (anti-Hel) showed no changes in plasma PGRN, whereas mice treated with sortilin humab45 had increased PGRN levels, which peaked between 24-48 hours. Then, it gradually decreased from around 4 days. The PGRN level was still high on the 7th day.</p><p> B. Subchronic study: Based on data from time-course studies, sortilin KI mice were treated with either Sortilin human antibody 45 or isotype-controlled antibody to maintain stable antibody levels during the subchronic study (4 weeks). Was subcutaneously (sc) administered at 10 mg / kg twice a week. Blood samples were collected at the start of the test and once a week to track changes in plasma PGRN. Plasma PGRN levels at the start of the study were similar in both groups of animals. Higher levels of plasma PGRN were seen from week 1 in mice treated with sortilin antibody 45 and remained high throughout the study. Mice treated with control ab showed no increase in plasma PGRN and were maintained at baseline levels (week 0).</p><p> C. Dose-response study: Different doses (4 doses: 10, 2, 0.4 and 0.1 mg / kg) of sortilin (45) and control antibody (anti-Hel) were injected and the mice were slaughtered on day 2. Plasma PGRN was high at 10 mg / kg and 2 mg / kg in mice treated with sortilin humab, lower doses had no effect on plasma PGRN, which had a dose-dependent effect of sortilin antibody on plasma PGRN levels. Clearly show. Mice treated with the control antibody showed no change in PGRN levels.</p><p> Mice were anesthetized with 0.4 ml Avertin IP, cardiac blood was collected and transferred to a 500 ul kEDTA vial. The sample was held on ice for 15 minutes at 4C until centrifugation at 3600G. Plasma was pipetted into micronic vials and frozen at -20C. PGRN in the sample was measured using the PGRN ELISA kit (Adipogen) according to the manufacturer's instructions. The results can be seen in Figure 13.</p><p>Example 16: Hydrogen / deuterium exchange, followed by epitope mapping of antibodies targeting progranulin-soltilin interactions by mass spectrometry Hydrogen / deuterium exchange, followed by mass spectrometry (HDX-MS), proteins. The exchange rate of skeletal amide hydrogen in is measured. This makes it possible to investigate the conformational dynamics of all protein skeletons except proline residues. The rate of the exchange reaction is determined by the hydrogen-bonded state of the skeletal amide and, to a lower extent, solvent accessibility. For example, slight changes in these two parameters caused by the presence of a ligand can be observed as changes in deuterium uptake.</p><p> To sub-localize changes in deuterium uptake, the protein is treated with an acid-stabilizing protease (eg, pepsin), which typically produces local regions of 10-15 amino acids. do. Regions that exhibit perturbations in the presence of ligands are directly involved in the binding interface or are allosterically affected by binding events.</p><p>Antibody epitope mapping Deuterium uptake in the extracellular region of sortilin (SEQ ID NO: 188) was measured in the absence and presence of the antibody indicated as mAb30, which does not bind to the mAb45, mAb68, mAb811 and D regions. To ensure that the measurements were taken under steady state conditions, the complex was equilibrated at 25 ° C for 15 minutes before the exchange reaction was initiated. The exchange reaction was initiated by a 1: 9 (v / v) dilution of the protein sample into deuterated buffer (99% D2O, 20 mM Tris, 150 mM NaCl, pDread = 7.6). After various time points (15 seconds, 1 minute, 10 minutes, 1 hour and 8 hours), the exchange reaction was performed with ice-cold quench buffer (2 M glycine, 0.8 M Tris- (2-carboxyethyl) phosphine (TCEP). ), Quenched by 1: 1 (v / v) dilution with pH = 2.3), thereby reducing the pH to 2.46. Quenched samples were immediately placed in a -80 ° freezer and stored until analysis. A fully deuterated control sample is placed in 1: 9 (6M guanididium chloride, 99% D2O, 20mM Tris, 150mM NaCl, pDread = 7.6) in a deuterated modified buffer. Prepared by dilution of v / v), followed by incubation at 25 ° C for 16 hours, then quenching and treating as described above.</p><p> A cooled (0 ° C) reverse phase UPLC with a thawed and home-packed pepsin column (60 μL internal volume, pepsin beads obtained from Thermo Scientific Inc.). -Injected into HDX-System (Waters Inc., USA). Here, the deuterated protein sample was subjected to online pepsin digestion at 20 ° C. and the resulting digestible peptide was separated by reverse phase UPLC. Peptides were ionized by electrospray ionization into a mass spectrometer (Synapt G2 mass spectrometer, Waters Inc, UK), where the peptides were further separated by ion mobility prior to final mass determination.</p><p> Peptides were identified in fully reduced non-deuterated samples by tandem mass spectrometry using a combination of data-independent (MSe) and data-dependent collection.</p><p>Data Analysis Peptide Identification The collected mass spectra were lock mass corrected for GFP and analyzed in PLGS 3.0 to adapt the precursor and fragment ions to the local protein database. The identification of all peptides was carefully evaluated manually.</p><p> Deuterium uptake determination: The collected mass spectrum is GFP-corrected rock mass of sortilin in the absence or presence of antibody using software DynamX 3.0 (Waters Inc., USA). Deuterium uptake was determined for all peptides.</p><p> A peptide was considered to be part of a binding epitope if protection from exchanges greater than 0.5D was observed in the presence of the antibody.</p><p><tables num="10"><img file="JP6979397B2_D0013.tif" /></tables></p><p>Example 17: Microdialysis for assessing progranulin levels in the brains of awake and free-behavior animals A brain ISF progranulin (PRGN) derived from awake and free-behavior mice was evaluated using a push-pull microdialysis method. .. Mice were housed in a single hut at controlled temperature (22 ± 1.5 ° C) and humidity conditions (55-65%) and turned on at a 12:12 hour light / dark cycle (06: 00h). It was bred in). Food and water were freely available. This test was performed in the hippocampus of human soltilin knock-in (hSORT1) mice (22 weeks old). To enable microdialysis in the hippocampus, mice were anesthetized with isoflurane and an intracerebral guide cannula (CMA) was stereotactically implanted in the brain, Paxinos and Franklin. The microdialysis probe was positioned in the hippocampus according to the 2001 illustration (probe tip placement: 3.1 mm posterior and 2.8 mm lateral to the bregma, and 1.3 mm relative to the dura mater). Acrylic cement was used to secure the guide cannula. After implantation of the cannula, mice were recovered from surgery for 7 days prior to dialysis. During the first 5 days, including the day of surgery, the animals had pain and were given antibiotic treatment (Rimadyl and Noromox). Prolongatum). Twenty-four hours prior to the start of the microdialysis experiment, a peristaltic pump was further connected to the outlet tube to prevent loss of peristaltic fluid from the probe by withdrawing peristaltic fluid from the tube. As a perfusion buffer, 25% bovine albumin fraction V (Sigma) was diluted to 0.2% with artificial CSF (aCSF; mM unit: 147 NaCl, 2.7KCl, 1.2CaCl2, 0.85 MgCl2) on the day of use, and 0.1 μm. It was filtered through a membrane of. The actual flow rate of the pump was measured without the probe connected. Sample tubes were weighed before and after sampling over a given period and flow rates were calculated. The pump was then set to have a constant flow rate of 1 μL / min. A 120-minute sampling scheme was used throughout the experimental period to collect 12 samples (12-hour collection) (see Figure 17 for procedure). At the end of the experiment, blood was drawn from the animal, the animal was perfused, and the brain was collected. The dialysate, plasma and brain were stored at -80 ° C until the PRGN was determined by ELISA.</p><p> Measurements of PRGN levels every two hours for 24 hours are shown in Figure 17. For all periods except 24 hours after the start of didialysis fluid collection, PRGN levels were significantly increased in animals treated with increased mab # 45 when compared to those from animals treated with PBS. (Fig. 18a). PRGN levels are stable over time, from 4 hours to 16 hours after probe insertion into the hippocampus. In the first dialysate, PRGN is probably higher due to probe insertion into the hippocampus. It is speculated that PRGN levels were reduced more than 18 and 20 hours after probe insertion, probably due to probe membrane clogging, which occurred in both groups (previously observed in other push-pull tests). rice field).</p><p> Mean ± SEM of 12 dialysis samples 24 hours after antibody or vehicle treatment for each animal and all subsequently pooled animals was taken as a baseline (Figure 18b). Differences between animals treated with mab # 45 and PBS were analyzed using unpaired t-test. Basis levels of PRGN in animals treated with mab # 45 were significantly increased when compared to those from animals treated with PBS (p &lt;0.001, F10.0, DFn, 9 Dfd 7; 3.3). ± 0.3 ng / ml, n = 10 vs 1.1 ± 0.1 ng / ml, n = 8) (Fig. 18b).<u style="single">The present invention may also include the following aspects.</u><u style="single">[1]</u><u style="single"> It specifically binds to sortilin and inhibits or reduces the binding of PGRN to sortilin.</u><u style="single">Antibodies that can be made, or antigen-binding fragments thereof.</u><u style="single">[2]</u><u style="single"> The antigen-binding fragment is an Fv fragment (eg, single-chain Fv or disulfi).</u><u style="single">Do-join Fv); Fab-like fragment (eg Fab fragment, Fab'flagmen</u><u style="single">Or F (ab) 2 fragment); and domain antibody (eg single VH variable region)</u><u style="single">The antibody according to claim 1, or an antibody thereof, selected from the group consisting of (or VL variable region).</u><u style="single">Originally bound fragment.</u><u style="single">[3]</u><u style="single"> The antibody according to claim 1 or 2, wherein the antibody comprises an intact antibody.</u><u style="single">[4]</u><u style="single"> The antibody comprises an antibody of subtype IgG1, IgG2, IgG3 or IgG4.</u><u style="single">The antibody according to any one of claims 1 to 3, which is selected from the group, or an antigen-binding hula thereof.</u><u style="single">Gument.</u><u style="single">[5]</u><u style="single"> The sol in which the antibody or antigen-binding fragment thereof is defined by SEQ ID NO: 170.</u><u style="single">The antibody according to any one of claims 1 to 4, or an antigen thereof, which binds to the D region of chillin.</u><u style="single">Combined fragment.</u><u style="single">[6]</u><u style="single"> The antibody or antigen-binding fragment thereof is of SEQ ID NO: 185, 186 or 187.</u><u style="single">Any one of claims 1-5 that binds to the D region of sortilin defined by any one</u><u style="single">The antibody according to one item, or an antigen-binding fragment thereof.</u><u style="single">[7]</u><u style="single"> The sol in which the antibody or antigen-binding fragment thereof is defined by SEQ ID NO: 180.</u><u style="single">The antibody according to claim 5 or 6, or its antigen binding, which further binds to the A region of thyrin.</u><u style="single">Fragment.</u><u style="single">[8]</u><u style="single"> The antibody or antigen-binding fragment thereof is SEQ ID NO: 181, 182, 183 or</u><u style="single">Claim that further binds to the A region of sortilin as defined by any one of 184.</u><u style="single">The antibody according to 5 or 6, or an antigen-binding fragment thereof.</u><u style="single">[9]</u><u style="single"> The sol in which the antibody or antigen-binding fragment thereof is defined by SEQ ID NO: 170.</u><u style="single">At least 3 consecutive amino acids in the D region of chillin, eg 4, 5, 6 or 7 consecutive amino acids</u><u style="single">The antibody according to any one of claims 1 to 8, which binds to an acid, or an antigen-binding flag thereof thereof.</u><u style="single">Nto.</u><u style="single">[10]</u><u style="single"> The antibody or antigen binding fragment has the following characteristics:</u><u style="single"> Connection of 0.5 ~ 10nM, for example 1 ~ 5nM or 1-2nM, to sortilin</u><u style="single">Affinity (KD);</u><u style="single"> b. Ability to reduce and / or inhibit PGRN binding to sortilin;</u><u style="single"> c. Sortilin-expressing cells reduce PGRN clearance and / or</u><u style="single">Ability to inhibit;</u><u style="single"> d. Sortilin-expressing cells reduce PGRN endocytosis and /</u><u style="single">Or the ability to inhibit;</u><u style="single"> e. Ability to increase the amount and / or concentration of PGRN in the brain, and / or</u><u style="single"> f. Plasma PGRN levels and / or plasma in human-sortilin-expressing knock-in mice.</u><u style="single">Or the ability to increase concentration</u><u style="single">The antibody according to any one of claims 1 to 9, which indicates one or more of the above, or an antigenic binding thereof.</u><u style="single">Combined fragment.</u><u style="single">[11]</u><u style="single"> The antibody or its thereof that reduces and / or inhibits PGRN binding to sortilin.</u><u style="single">The ability of the antigen-binding fragment is 50 using the time-resolved fluorescence assay (HTFR).</u><u style="single">Less than nM, but preferably in IC50s from 10nM to 0.2nM.</u><u style="single">The antibody according to any one of claims 1 to 9, or an antigen-binding fragment thereof.</u><u style="single">[12]</u><u style="single"> The antibody or its thereof that reduces and / or inhibits PGRN binding to sortilin.</u><u style="single">The ability of the antigen-binding fragment is 22 nM or less, for example 22 nM to 1 nM, or 1</u><u style="single">IC50s of 0nM ~ 1nM or 5nM ~ 1nM reduce PGRN binding to sortilin</u><u style="single">The antibody according to any one of claims 1 to 9, which comprises reducing and / or inhibiting.</u><u style="single">, Or its antigen-binding fragment.</u><u style="single">[13]</u><u style="single"> The antibody or antigen-binding fragment thereof is human, humanized, recombinant or chimeric anti-chimeric.</u><u style="single">The antibody according to any one of claims 1 to 12, which is a body, or an antigen-binding flagmen thereof.</u><u style="single">door.</u><u style="single">[14]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 1;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 2;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 3;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 4;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 5;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 6</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[15]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 8.</u><u style="single">The antibody according to claim 14, or an antigen-binding fragment thereof.</u><u style="single">[16]</u><u style="single"> The antibody or antigen binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 7.</u><u style="single">The antibody according to claim 14, or an antigen-binding fragment thereof.</u><u style="single">[17]</u><u style="single"> The heavy chain variable according to claims 15 and 16, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 14, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[18]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 9;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 10;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 11;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 12;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 13;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 14</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[19]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 16.</u><u style="single">, The antibody according to claim 18, or an antigen-binding fragment thereof.</u><u style="single">[20]</u><u style="single"> The antibody or antigen binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 15.</u><u style="single">, The antibody according to claim 18, or an antigen-binding fragment thereof.</u><u style="single">[21]</u><u style="single"> The heavy chain variable according to claims 19 and 20, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 18, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[22]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 17;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 18;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 19;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 20;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 21;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 22</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[23]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 24.</u><u style="single">, The antibody according to claim 22, or an antigen-binding fragment thereof.</u><u style="single">[24]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 23.</u><u style="single">, The antibody according to claim 22, or an antigen-binding fragment thereof.</u><u style="single">[25]</u><u style="single"> The heavy chain variable according to claims 23 and 24, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 22, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[26]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 25;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 26;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 27;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 28;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 29;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 30</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[27]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 32.</u><u style="single">, The antibody according to claim 26, or an antigen-binding fragment thereof.</u><u style="single">[28]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 31.</u><u style="single">, The antibody according to claim 26, or an antigen-binding fragment thereof.</u><u style="single">[29]</u><u style="single"> The heavy chain variable according to claims 27 and 28, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 26, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[30]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 33;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 34;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 35;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 36;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 37;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 38</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[31]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 40.</u><u style="single">, The antibody according to claim 30, or an antigen-binding fragment thereof.</u><u style="single">[32]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 39.</u><u style="single">, The antibody according to claim 30, or an antigen-binding fragment thereof.</u><u style="single">[33]</u><u style="single"> The heavy chain variable according to claims 31 and 32, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">30. The antibody or antigen-binding hula thereof according to claim 30, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[34]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 41;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 42;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 43;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 44;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 45;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 46</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[35]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 48.</u><u style="single">The antibody according to claim 34, or an antigen-binding fragment thereof.</u><u style="single">[36]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 47.</u><u style="single">The antibody according to claim 34, or an antigen-binding fragment thereof.</u><u style="single">[37]</u><u style="single"> The heavy chain variable according to claims 35 and 36, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 34, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[38]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 49;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 50;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 51;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 52;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 53;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 54</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[39]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 56.</u><u style="single">The antibody according to claim 38, or an antigen-binding fragment thereof.</u><u style="single">[40]</u><u style="single"> The antibody or antigen binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 55.</u><u style="single">The antibody according to claim 38, or an antigen-binding fragment thereof.</u><u style="single">[41]</u><u style="single"> The heavy chain variable according to claims 39 and 40, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 38, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[42]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 57;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 58;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 59;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 60;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 61;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 62</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[43]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 64.</u><u style="single">42. The antibody according to claim 42, or an antigen-binding fragment thereof.</u><u style="single">[44]</u><u style="single"> The antibody or antigen binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 63.</u><u style="single">42. The antibody according to claim 42, or an antigen-binding fragment thereof.</u><u style="single">[45]</u><u style="single"> The heavy chain variable according to claims 43 and 44, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 42, which comprises both a region and a light chain variable region, or an antigen-binding antibody thereof.</u><u style="single">Rugment.</u><u style="single">[46]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 65;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 66;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 67;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 68;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 69;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 70</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[47]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 72.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 46.</u><u style="single">[48]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 71.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 46.</u><u style="single">[49]</u><u style="single"> The heavy chain variable according to claims 47 and 48, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">46. The antibody or antigen-binding hula thereof according to claim 46, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[50]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 73;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 74;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 75;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 76;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 77;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 78</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[51]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 80.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 50.</u><u style="single">[52]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 79.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 50.</u><u style="single">[53]</u><u style="single"> The heavy chain variable according to claims 51 and 52, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 50 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[54]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 81;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 82;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 83;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 84;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 85;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 86</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[55]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 88.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 54.</u><u style="single">[56]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 87.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 54.</u><u style="single">[57]</u><u style="single"> The heavy chain variable according to claims 55 and 56, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 54 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[58]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 89;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 90;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 91;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 92;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 93;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 94</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[59]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 96.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 58.</u><u style="single">[60]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 95.</u><u style="single">, The antibody or antigen-binding fragment thereof according to claim 58.</u><u style="single">[61]</u><u style="single"> The heavy chain variable according to claims 59 and 60, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">58. The antibody or antigen-binding hula thereof according to claim 58, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[62]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 97;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 98;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 99;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 100;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 101;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 102</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[63]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 104.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 62.</u><u style="single">[64]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 103.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 62.</u><u style="single">[65]</u><u style="single"> The heavy chain variable according to claims 63 and 64, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">62. The antibody or antigen-binding hula thereof according to claim 62, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[66]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 105;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 106;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 107;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 108;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 109;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 110</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[67]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 112.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 66.</u><u style="single">[68]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 111.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 66.</u><u style="single">[69]</u><u style="single"> The heavy chain variable according to claims 67 and 68, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 66 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[70]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 113;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 114;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 115;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 116;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 117;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 118</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[71]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 120.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 70.</u><u style="single">[72]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 119.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 70.</u><u style="single">[73]</u><u style="single"> The heavy chain variable according to claims 71 and 72, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 70 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[74]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 121;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 122;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 123;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 124;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 125;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 126</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[75]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 128.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 74.</u><u style="single">[76]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 127.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 74.</u><u style="single">[77]</u><u style="single"> The heavy chain variable according to claims 75 and 76, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody or antigen-binding hula thereof according to claim 74, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[78]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 129;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 130;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 131;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 132;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 133;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 134</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[79]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 136.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 78.</u><u style="single">[80]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 135.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 78.</u><u style="single">[81]</u><u style="single"> The heavy chain variable according to claims 79 and 80, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 78 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[82]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 137;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 138;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 139;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 140;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 141;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 142</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[83]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 144.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 82.</u><u style="single">[84]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 143.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 82.</u><u style="single">[85]</u><u style="single"> The heavy chain variable according to claims 83 and 84, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 82 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[86]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 145;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 146;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 147;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 148;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 149;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 150</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[87]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 152.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 86.</u><u style="single">[88]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 151.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 86.</u><u style="single">[89]</u><u style="single"> The heavy chain variable according to claims 87 and 88, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 86 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[90]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 153;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 154;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 155;</u><u style="single"> d. Heavy chain variable region H-CDR1 containing SEQ ID NO: 156;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 157;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 158</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[91]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 160.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 90.</u><u style="single">[92]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 159.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 90.</u><u style="single">[93]</u><u style="single"> The heavy chain variable according to claims 91 and 92, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 90 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[94]</u><u style="single"> The antibody or its antigen-binding fragment</u><u style="single"> Light chain variable region L-CDR1 containing SEQ ID NO: 161;</u><u style="single"> b. Light chain variable region L-CDR2 containing SEQ ID NO: 162;</u><u style="single"> c. Light chain variable region L-CDR3 containing SEQ ID NO: 163;</u><u style="single"> d. Heavy chain variable region containing SEQ ID NO: 164 H-CDR1;</u><u style="single"> e. Heavy chain variable region H-CDR2; containing SEQ ID NO: 165;</u><u style="single"> f. Heavy chain variable region H-CDR3 containing SEQ ID NO: 166</u><u style="single">The antibody according to any one of claims 1 to 13, or an antigen-binding fragment thereof.</u><u style="single">。</u><u style="single">[95]</u><u style="single"> The antibody or antigen-binding fragment thereof contains a heavy chain variable region containing SEQ ID NO: 168.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 94.</u><u style="single">[96]</u><u style="single"> The antibody or antigen-binding fragment thereof comprises a light chain variable region comprising SEQ ID NO: 167.</u><u style="single">The antibody or antigen-binding fragment thereof according to claim 94.</u><u style="single">[97]</u><u style="single"> The heavy chain variable according to claims 95 and 96, wherein the antibody or an antigen-binding fragment thereof is used.</u><u style="single">The antibody according to claim 94 or an antigen-binding hula thereof, which comprises both a region and a light chain variable region.</u><u style="single">Gument.</u><u style="single">[98]</u><u style="single"> A preparation containing the antibody according to any one of claims 1 to 97 or an antigen-binding fragment thereof.</u><u style="single">It is a product and the preparation is not capable of binding to sortilin or the preparation.</u><u style="single">Substantially free of naturally occurring antibodies that do not substantially alter the anti-sortilin functionality of the product, said</u><u style="single">Functionality,</u><u style="single"> (i) Binding affinity for sortilin (K)</u>_{<u style="single">D</u>}<u style="single">);</u><u style="single"> (ii) Ability to reduce and / or inhibit PGRN binding to sortilin;</u><u style="single"> (iii) Sortilin-expressing cells reduce PGRN clearance and /</u><u style="single">Or the ability to inhibit;</u><u style="single"> (iv) Sortilin-expressing cells reduce PGRN endocytosis and</u><u style="single">And / or the ability to inhibit;</u><u style="single"> (v) Amount of plasma PGRN and / in human-sortilin-expressing knock-in mice</u><u style="single">Or the ability to increase concentration;</u><u style="single"> (vi) Ability to increase the amount and / or concentration of PGRN in the brain and / or</u><u style="single"> (vii) Frontotemporal dementia (FTD), amyotrophic lateral sclerosis when administered chronically</u><u style="single">Ability to provide treatment for disease (ALS) or Alzheimer's disease (AD)</u><u style="single">A preparation selected from the group consisting of.</u><u style="single">[99]</u><u style="single"> The monoclonal antibody according to any one of claims 1 to 98 or an antigen-binding flag thereof.</u><u style="single">The monoclonal antibody is a preparation containing ment, and the structure of the natural anti-sortilin antibody is</u><u style="single">Compared with, it has a structural change in its amino acid sequence, and the structural change</u><u style="single">By the above-mentioned krona</u><u style="single">Lu-antibody has altered functionality compared to the functionality exhibited by the natural anti-sortilin antibody.</u><u style="single">Shown, the functionality is</u><u style="single"> (i) Binding affinity for sortilin (K)</u>_{<u style="single">D</u>}<u style="single">);</u><u style="single"> (ii) Ability to reduce and / or inhibit PGRN binding to sortilin;</u><u style="single"> (iii) Sortilin-expressing cells reduce PGRN clearance and /</u><u style="single">Or the ability to inhibit;</u><u style="single"> (iv) Sortilin-expressing cells reduce PGRN endocytosis and</u><u style="single">And / or the ability to inhibit;</u><u style="single"> (v) Amount of plasma PGRN and / in human-sortilin-expressing knock-in mice</u><u style="single">Or the ability to increase concentration;</u><u style="single"> (vi) Ability to increase the amount and / or concentration of PGRN in the brain, or</u><u style="single"> (vii) Frontotemporal dementia (FTD), amyotrophic lateral sclerosis when administered chronically</u><u style="single">Ability to provide treatment for disease (ALS) and / or Alzheimer's disease (AD)</u><u style="single">A preparation selected from the group consisting of.</u><u style="single">[100]</u><u style="single"> The antibody according to any one of claims 1 to 97, or an antigen-binding fragment thereof, or</u><u style="single">Includes the preparation according to any one of claims 98-99, and a pharmaceutically acceptable carrier.</u><u style="single">Pharmaceutical composition.</u><u style="single">[101]</u><u style="single"> The antibody according to any one of claims 1 to 97 for medical use, or an antigen thereof.</u><u style="single">The binding fragment, or the preparation according to any one of claims 98 to 99, or a claim.</u><u style="single">Item 100. The pharmaceutical composition according to Item 100.</u><u style="single">[102]</u><u style="single"> For use in treating diseases associated with decreased PGRN levels in a patient's brain,</u><u style="single">The antibody according to any one of claims 1 to 97, or an antigen-binding fragment thereof, or</u><u style="single">The preparation according to any one of claims 98 to 99, or the pharmaceutical composition according to claim 100.</u><u style="single">thing.</u><u style="single">[103]</u><u style="single"> In the manufacture of drugs to treat diseases associated with decreased PGRN levels in the patient's brain</u><u style="single">The antibody according to any one of claims 1 to 97, or an antigen-binding fragment thereof.</u><u style="single">Alternatively, the preparation according to any one of claims 98 to 99, or the doctor according to claim 100.</u><u style="single">Use of drug composition.</u><u style="single">[104]</u><u style="single"> A method of preventing or treating a disease associated with decreased PGRN levels in a patient's brain.</u><u style="single">The antibody according to any one of claims 1 to 97, or an antigen-binding antibody thereof, in an effective dose.</u><u style="single">The rugment, or the preparation according to any one of claims 98 to 99, or claim 10.</u><u style="single">A method comprising the step of administering the pharmaceutical composition according to 0.</u><u style="single">[105]</u><u style="single"> The disease is FTD; ALS; or TDP43 proteinosis (such as AD).</u><u style="single">The antibody for use according to claim 102, or an antigen-binding fragment thereof, or claim.</u><u style="single">The use according to 103, or the method according to claim 104.</u><u style="single">[106]</u><u style="single"> The treatment is long-term, preferably at least 2 weeks, eg at least 1 month.</u><u style="single">Or the use according to claim 102 for at least 6 months, or at least 1 year.</u><u style="single">Antibodies for use, or antigen-binding fragments thereof, or the use according to claim 103.</u><u style="single">Alternatively, the method according to claim 104.</u><u style="single">[107]</u><u style="single"> The antibody according to any one of claims 1 to 97, or an antigen-binding fragment thereof, or</u><u style="single">Is the preparation according to any one of claims 98 to 99, or the pharmaceutical set according to claim 100.</u><u style="single">A kit containing adults.</u><u style="single">[108]</u><u style="single"> Smell of cell lines such as human cell lines, non-human mammalian cell lines, insect, yeast or bacterial cell lines</u><u style="single">The antibody according to any one of claims 1 to 97, or an antigen thereof, produced or produced in the art.</u><u style="single">Combined fragment.</u><u style="single">[109]</u><u style="single"> CHO cell line, HEK cell line, BHK-21 cell line, mouse cell line (myeloma cell line, etc.)</u><u style="single">), Fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and H</u><u style="single">The antibody according to claim 108, or an antigenic conjugate thereof, produced in a uH-7 human cell line.</u><u style="single">Combined fragment.</u></p>