Agents, uses and methods for therapy

Abstract

The present invention relates to agents, uses and methods for therapy, in particular to monoclonal anti-sortin antibodies which have been found to be useful in correcting defective levels of granulin precursors (PGRNs). In particular, these antibodies may be used in the treatment of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) as well as other neurodegenerative disorders, such as Alzheimer's disease (AD).

Description

[0001] This application is a divisional application of the Chinese patent application with application number 2018800479188, application date July 18, 2018, and invention name “Agents, uses and methods for treatment”. Technical Field

[0002] The present invention relates to monoclonal anti-Sortilin antibodies that are useful in correcting defective levels of progranulin (PGRN). Specifically, these antibodies can be used for the treatment of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In addition, it is expected that these monoclonal antibodies can also be used to treat neurodegenerative disorders such as Alzheimer's disease (AD).

[0003] References to sequence listings

[0004] This application includes one or more sequence listings (pursuant to 37 CFR 1.821 et seq.), which are disclosed in the form of a computer-readable medium (file name: 0993_ST25.txt, created on June 22, 2016, and 144 kB in size), which is incorporated herein by reference in its entirety. Background Art

[0005] Sortilin is a receptor that has been reported to mediate the pro-apoptotic effects of pro-neurotrophins and to mediate the trafficking and sorting of neurotrophin receptors ( 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). A number of sortilin ligands have been identified, including neurotensin, whose high affinity binding site was mapped by X-ray crystallography to the beta propeller channel of 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). Recently, sortilin was shown to function as a high affinity receptor for the growth factor progranulin (PGRN) (PGRN, Hu et al., Neuron. 2010 Nov 18;68(4):654-67).

[0006] PGRN (proepitheliin, progranulin-epitheliin, PC cell-derived growth factor, acrogranin) is a secreted glycosylated protein with anti-inflammatory and neurotrophic effects (for a recent review, Nguyen, Trends Endocrinol Metab. 2013 Dec; 24(12):597-606). PGRN is proteolytically cleaved into acrogranin, but much remains to be learned about the physiological roles of PGRN and acrogranin and the identity of their receptors. PGRN has been implicated in several cellular functions, including cell cycle regulation and cell motility (He, Z. & Bateman, A., J. MoI. Med. 57:600-612 (2003); Monami, G. et al., Cancer Cell Biology ... Res. [Cancer Research] (5 (5: 7103-7110 (2006)), wound repair, inflammation (Zhu, J. et al., Cell [Cell] 777: 867-878 (2002)), induction of growth factors (such as vascular endothelial growth factor (VEGF)) (Tangkeangsiπsin, W. & Serrero, G, Carcinogenesis [Carcinogenesis] 25. 1587-1592 (2004)) and tumorigenesis (He, Z. & Bateman, A., J. MoI. Med. [Journal of Molecular Medicine] 81: 600-612 (2003), Monami, G. et al., Cancer Res [Cancer Research] (5 (5: 7103-7110 (2006); Serrero, G., Biochem Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 505-409-413 (2003), Lu, R & Serrero, G., Proc. Natl Acad Sci U.S.A. [Proceedings of the National Academy of Sciences of the United States of America] 98 142-147 (2001); Liau, L M., et al., Cancer Res [Cancer Research]. 60: 1353-1360 (2000)). PGRN has been reported to bind to TNF receptors (Tang W et al., Science [Science] 2011, 332(6028): 478-84), but this observation has been questioned by others (Chen et al., J Neurosci. [Journal of Neuroscience] 2013, 33(21): 9202-9213).

[0007] Binding of PGRN to sortilin has been mapped to the neurotensin site and reported to be mediated exclusively by the PGRN C-terminal domain in a manner similar to neurotensin (Zheng et al., PLoS One. 2011; 6(6):e21023; Lee et al., Hum Mol Genet. 2013), and consistent with this, neurotensin has been shown to block the interaction of sortilin with PGRN and other ligands. Upon binding, sortilin mediates lysosomal clearance of PGRN and thereby regulates extracellular PGRN levels (Hu et al., 2010). Thus, knockdown or overexpression of sortilin has been shown to modulate extracellular PGRN levels in cell culture (Carrasquillo et al., Am J Hum Genet. 2010 Dec 10; 87(6):890-7), and in mice, sortilin deficiency was reported to increase PGRN levels and restore plasma and brain PGRN levels in PGRN+ / - mice (Hu et al., 2010). Interestingly, single nucleotide polymorphisms (SNPs) near sortilin were associated with reduced plasma PGRN levels 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 key extracellular PGRN regulator.

[0008] PGRN has been linked to frontotemporal dementia (FTD, a progressive dementia characterized by behavioral and semantic changes) as well as frontotemporal lobar degeneration (FTLD) and neuronal inclusions or tau inclusions containing TAR DNA binding protein-43 (TDP-43) (Baker et al., 2006, Nature. 2006 Aug 24;442(7105):916-9; Cruts 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)). A large proportion of sporadic and familial FTD cases show TDP-43 pathology similar to ALS (approximately 50%), and FTD-TDP43 and ALS are considered by some to constitute a spectrum of diseases (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 Aug;8(8):423-434) due to shared pathological and genetic factors and some overlap in symptomatology. There are no disease-modifying treatment options available for FTD. A small group of frontotemporal dementia patients with TDP-43 pathology have loss-of-function mutations in the granulin gene (GRN), resulting in PGRN haploinsufficiency. Over 75 different mutations in the granulin gene, all resulting in reduced PGRN levels and / or function, have been associated with FTD and it is believed that elevating extracellular PGRN in plasma and brain will impede the disease process.

[0009] PGRN mutations have also been linked to Alzheimer's disease (AD) (Sheng et al., 2014, Gene. [Gene] June 1, 2014; 542 (2): 141-5; Brouwers et al., 2008, Neurology. [Neurology] August 26, 2008; 71 (9): 656-64), indicating that PGRN deficiency may play an important role in the pathogenesis of AD. In addition, the neuroprotective effect of PGRN has been observed in mouse AD models (Minami et al., 2014, Nat Med. [Natural Medicine] October 2014; 20 (10): 1157-64), providing support for the view that enhanced PGRN may be beneficial in AD and possibly other neurodegenerative diseases.

[0010] This application describes the generation and characterization of anti-human sortilin antibodies that can modulate PGRN in cell models and mice. Those antibodies unexpectedly bind to a region on sortilin that is far from the previously reported progranulin binding site (the so-called neurotensin-site) and are still able to increase extracellular PGRN.

[0011] The inventors have defined six sortilin binding regions and identified effective antibodies to the binding regions ("E regions"). These antibodies do not block the binding of PGRN to sortilin, but affect / increase PGRN levels, indicating that these antibodies have a novel mode of action. Because PGRN has neuroprotective and anti-inflammatory effects, the inventors' findings indicate that such antibodies targeting sortilin may have beneficial effects in the treatment of a range of neurodegenerative disorders, including FTD / FTLD. A subset of FTD / FTLD patients with TDP-43 pathology carry mutations in the gene encoding PGRN, resulting in PGRN haploinsufficiency. It is expected that sortilin antibodies may hinder this PGRN deficiency and may have similar therapeutic benefits for patients with other TDP-43 proteinopathies, including ALS, in which PGRN levels can affect TDP43 function and pathology, as well as other neurodegenerative diseases, including AD, in which increased PGRN function may be neuroprotective. Summary of the invention

[0012] The inventors of the present invention have generated monoclonal antibodies that are capable of unexpectedly binding to a novel region of sortilin designated as the "E-region" as defined in SEQ ID NO: 146 and are capable of modulating PGRN levels in the brain of a patient.

[0013] The present invention also relates to a method for preventing or treating a disease associated with reduced PGRN levels in the patient's brain, the method comprising administering an effective dose of an antibody or antigen-binding fragment thereof that is bound to the E-region of sortin. In particular, these diseases include FTD, ALS, and protein diseases (such as AD, PD). BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The steps in antibody selection are described. AE refers to the regions assigned to the corresponding sortilin binding antibodies based on the shuffled constructs as described in Example 1 and SEQ ID No 147-155. "Other" refers to antibodies that cannot be assigned to a region and can bind at the interface between A-region and B-region. Tet refers to antibodies that also bind Tetraodon-sortilin.

[0015] Figures 2A-2C Antibody region assignments based on binding to sortilin shuffled constructs are shown.

[0016] Figure 2A Shown is a linear illustration of the shuffled constructs used for antibody region assignment as described in Example 1. Sortilin shuffled constructs were generated based on the human sortilin sequence (SEQ ID NO: 145) (segments depicted in grey) in which amino acid residues were exchanged for the corresponding amino acids from the Tetraodon sortilin sequence (depicted in black) (SEQ ID NO: 149) (Examples 1-3).

[0017] Figure 2B Shown is the predicted structure of the shuffled construct illustrated linearly in A. Dark residues indicate residues that were changed into the corresponding Tetraodon sequence in the shuffled construct.

[0018] Figure 2C The binding mode of the antibody that is assigned to D-district and E district category respectively is illustrated. "+" indicates to be combined with given reorganization construct, and "-" indicates to lack combination. Based on the binding mode with different reorganization constructs, it is antibody distribution region. The gained antibody region category is indicated by AE. For the D and E district antibodies illustrated, both are combined with the human sequence (all gray) as indicated by "+" and are not combined with the Tetraodon sequence (all black) as indicated by "-", and the E district antibody combines the hB45678 reorganization construct, but the D district antibody does not combine, thereby produces the binding location illustrated in Figure A. For the E district antibody, it is observed to be combined with the following reorganization regions: hsort, hB06-10, B12390, hB45678. These antibodies are not combined with hB01-05 and Tetraodon construct.

[0019] These antibodies did not bind to the complete Tetraodon sortilin, with two exceptions. The two antibodies that bound to the Tetraodon sequence are denoted "tet". "Other" refers to antibodies that could not be assigned to a region.

[0020] Figure 3 Binding affinities of human E-region antibodies are shown. Binding affinities (EC50, ng / ml) to sortilin constructs were determined by biofilm interferometry as described in Example 2 using Octet384RED. NB indicates no binding. Values ​​between 0.1 ng / ml-10 ng / ml and >10 ng / ml indicate binding. Region assignments were based on the binding patterns of antibodies to different sortilin shuffled constructs ( Figures 2A-2C ). The lack of binding to the sortilin constructs hB01-05 and Tetra indicates that these antibodies bind to the E region.

[0021] Figure 4 Cross-blocking between E-region antibodies is shown. Each antibody binds to human wild-type sortilin to form antibody-sortilin complex. All other E domain antibodies are tested for binding to preformed antibody-sortilin complex (Example 8). The interference of binding to antibody-sortilin is determined by analyzing the cross-blocking between sortilin antibodies from the same or different domains. The binding of antibodies to sortilin-ECD-His (as described above) is measured by biofilm interferometry using Octet 384RED. The left column indicates one-level (fixed) antibody and the top row indicates secondary antibody (antibody tested for fixed antibody). The binding of one-level and secondary antibody to sortilin-ECD-His will all produce a response value higher than 0.1 and indicate that both antibodies are bound to different regions of the protein. A response value less than 0.1 shows the lack of binding of the secondary antibody and the effective cross-blocking by the fixed (one-level) antibody, which indicates that the two antibodies are bound to the same region of sortilin. 'x' indicates no binding, and therefore the antibodies block each other. 'b' indicates binding of both antibodies to Sortilin and therefore do not cross-block each other.

[0022] Twenty-two of the 26 antibodies from the E domain cross-blocked all antibodies from the panel, and the remaining four antibodies cross-blocked 20 of the 26 antibodies, indicating that most antibodies bind to the same or adjacent regions on sortilin.

[0023] Figure 5 The effect of sortilin antibodies on extracellular PGRN in neuron-differentiated induced pluripotent stem cells (iPSCs) generated from an apparently healthy male (18 years old) is shown. (Example 9)

[0024] Figure 6Plasma PGRN levels in knock-in (KI) mice of human sortilin treated with sortilin human E-region binding antibodies are shown (Example 10). Antibody #30 increases plasma PGRN levels compared to mice treated with isotype control antibody anti-HEL.

[0025] Mice were injected with 10 mg / kg of the test antibody by subcutaneous administration. They were sacrificed 48 hours later and blood samples were collected for analysis. Plasma PGRN was measured by ELISA. Data are presented as mean ± SD. Data were analyzed by t-test. ***p < 0.001

[0026] Fig. 7A and 7B The effects of sortilin antibodies on extracellular PGRN in neuron-differentiated induced pluripotent stem cells (iPSCs) generated from apparently healthy individuals and from a PGRN R493X patient are shown (Example 11).

[0027] Figures 8A-8F Shown are representative peptides covering the conformational epitope of antibody 30. All peptides shown were shown to be protected from exchange greater than 0.5D (Example 12). DETAILED DESCRIPTION

[0028] As used herein, the term "Sortilin" is synonymous with "Sortilin protein" (identified in, e.g., UniProt as Q99523, 1 and 2). The amino acid numbering of Sortilin is given as follows relative to SEQ ID NO: 145, with methionine (M) being amino acid 1:

[0029] MERPWGAADG LSRWPHGLGL LLLLQLLPPS TLSQDRLDAP PPPAAPLPRW

[0030] SGPIGVSWGL RAAAAGGAFP RGGRWRRSAP GEDEECGRVR DFVAKLANNT

[0031] HQHVFDDLRG SVSLSWVGDS TGVILVLTTF HVPLVIMTFG QSKLYRSEDY

[0032] GKNFKDITDL INNTFIRTEF GMAIGPENSG KVVLTAEVSG GSRGGRIFRS

[0033] SDFAKNFVQT DLPFHPLTQM MYSPQNSDYL LALSTENGLW VSKNFGGKWE

[0034] EIHKAVCLAK WGSDNTIFFT TYANGSCKAD LGALELWRTSDLGKSFKTIG

[0035] VKIYSFGLGG RFLFASVMAD KDTTRRIHVS TDQGDTWSMA QLPSVGQEQF

[0036] YSILAANDDM VFMHVDEPGD TGFGTIFTSD DRGIVYSKSL DRHLYTTTGG

[0037] ETDFTNVTSL RGVYITSVLS EDNSIQTMIT FDQGGRWTHL RKPENSECDA

[0038] TAKNKNECSL HIHASYSISQ KLNVPMAPLS EPNAVGIVIA HGSVGDAISV

[0039] MVPDVYISDD GGYSWTKMLE GPHYYTILDS GGIIVAIEHS SRPINVIKFS

[0040] TDEGQCWQTY TFTRDPIYFT GLASEPGARS MNISIWGFTE SFLTSQWVSY

[0041] TIDFKDILER NCEEKDYTIW LAHSTDPEDY EDGCILGYKE QFLRLRKSSV

[0042] CQNGRDYVVT KQPSICLCSL EDFLCDFGYY RPENDSKCVE QPELKGHDLE

[0043] FCLYGREEHL TTNGYRKIPG DKCQGGVNPV REVKDLKKKC TSNFLSPEKQ

[0044] NSKSNSVPII LAIVGLMLVT VVAGVLIVKK YVCGGRFLVH RYSVLQQHAE

[0045] ANGVDGVDAL DTASHTNKSG YHDDSDEDLLE

[0046] As used herein, the term "E region" is intended to refer to the region on Sortilin consisting of the amino acids in SEQ ID NO: 146 as shown below (corresponding to residues 612-753 of SEQ ID NO: 145):

[0047] CEEKDYTIW LAHSTDPEDY EDGCILGYKEQFLRLRKSSVCQNGRDYVVT KQPSICLCSL

[0048] EDFLCDFGYY RPENDSKCVE QPELKGHDLEFCLYGREEHL TTNGYRKIPG

[0049] DKCQGGVNPV REVKDLKKKC TSNFLSPEKQNSKSNS

[0050] For the E region antibodies, binding was observed to the following shuffled regions: hsort, hB06-10, B12390, hB45678. These antibodies did not bind to hB01-05 and tetra.

[0051] The PGRN gene (proepithelial factor, progranulin-epithelial factor, PC cell-derived growth factor, acrogranin) encodes a 68.5 kDa secreted glycoprotein with 7.5-fold repeats of a smaller granulin motif (ranging from 6 kDa-25 kDa) that can be proteolytically cleaved from the precursor PGRN (He, Z. & Bateman, J. MoI. Med. [Journal of Molecular Medicine] 81: 600-6X2 (2003)). In non-neuronal cells, PGRN has been associated with multiple events, such as cell cycle regulation and cell motility (He, Z. & Bateman, A., J. MoI. Med. [Journal of Molecular Medicine] 57: 600-612 (2003); Monami, G. et al., Cancer Cell Biology, 2003). Res. [Cancer Research] (5 (5: 7103-7110 (2006)), wound repair, inflammation (Zhu, J. et al., Cell [Cell] 777: 867-878 (2002)), induction of growth factors (such as vascular endothelial growth factor (VEGF)) (Tangkeangsiπsin, W. & Serrero, G, Carcinogenesis [Carcinogenesis] 25. 1587-1592 (2004)) and tumorigenesis (He, Z. & Bateman, A., J. MoI. Med. [Journal of Molecular Medicine] 81: 600-612 (2003), Monami, G. et al., Cancer Res [Cancer Research] (5 (5: 7103-7110 (2006); Serrero, G., Biochem Biophys. Res. Commun. [Biochemical and Biophysical Research Communications] 505-409-413 (2003), Lu, R & Serrero, G., Proc. Natl Acad Sa U.SA [Proceedings of the National Academy of Sciences of the United States of America] 98 142-147 (2001); Liau, L M. et al., Cancer Res. [Cancer Research] 60: 1353-1360 (2000)).

[0052] PGRN mutations cause haploinsufficiency (Baker, M. et al., Nature 442:916-919 (2006); Cruts, M. et al., Nature 442:920-924 (2006)) and are known to be present in nearly 50% of familial FTD cases, making PGRN mutations a major genetic contributor to FTD (Cruts, M. & Van Broeckhoven, C, Trends Genet. 24:186-194 (2008); Le Ber, I. et al., Brain 129:3051-3065 (2006)). The loss-of-function heterozygous trait of PGRN mutations means that in healthy individuals, PGRN expression plays a critical role in protecting healthy individuals from developing FTD in a dose-dependent manner.

[0053] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule or, according to some embodiments of the present invention, a fragment of an immunoglobulin molecule that has the ability to bind to an epitope of a molecule ("antigen"). Naturally occurring antibodies typically comprise a tetramer, which is typically composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant domain, which is typically composed of three domains (CH1, CH2, and CH3). The heavy chain can have any isotype, including IgG (IgG1, IgG2, IgG3, and IgG4 subtypes). Each light chain consists of a light chain variable domain (abbreviated herein as VL) and a light chain constant domain (CL). The light chain includes a kappa chain and a lambda chain. The heavy and light chain variable domains are typically responsible for antigen recognition, while the heavy and light chain constant domains can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL domains may be further subdivided into hypervariable regions called "complementarity determining regions" interspersed with more conserved regions called "framework regions" (FRs). Each VH and VL is composed of three CDR domains and four FR domains, arranged from amino terminus to carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The heavy and light chain variable domains contain a binding domain that interacts with an antigen. Of particular relevance are antibodies and antigen-binding fragments thereof that have been "isolated" so as to exist in a different physical environment from that in which it may exist in nature or that have been modified so as to differ in amino acid sequence from a naturally occurring antibody.

[0054] The term "epitope" means an antigenic determinant that is capable of specific binding to an antibody. An epitope is usually composed of surface groups such as amino acids or sugar side chain molecules, and usually has specific three-dimensional structural characteristics and specific charge characteristics. The difference between a conformational epitope and a linear epitope is that binding to the former, but not the latter, is often lost in the presence of a denaturing solvent. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not 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 residue is within the range of influence of the specific antigen binding peptide).

[0055] As used herein, the term "antigen-binding fragment of an antibody" means a fragment, part, region or domain of an antibody that is capable of binding to an epitope (regardless of how it is produced (e.g., via cleavage, recombinant, synthetic, etc.)), and thus the term "antigen binding" is intended to mean the same as "epitope binding", such that, for example, "antigen-binding fragment of an antibody" is intended to be the same as "epitope-binding fragment of an antibody". An antigen-binding fragment may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such an antibody, and despite being able to bind to such an epitope, may exhibit specificity, affinity or selectivity for such an epitope that is different from an epitope of such an antibody. However, preferably, an antigen-binding fragment contains all 6 CDR domains of such an antibody. An antigen-binding fragment of an antibody may be part of or comprise a single polypeptide chain (e.g., scFv), or may be part of or comprise two or more polypeptide chains (each having an amino terminus and a carboxyl terminus) (e.g., diabodies, Fab fragments, Fab2 fragments, etc.). Antibody fragments exhibiting antigen binding ability can be obtained, for example, by protease cleavage of intact antibodies. More preferably, although the two domains VL and VH of the Fv fragment are naturally encoded by a single gene or a polynucleotide (e.g., its encoding cDNA) encoding such a gene sequence, the two domains can be connected by a flexible linker using a recombinant method, and the flexible linker enables the two domains to become a single protein chain in which the VL region and the VH region associate to form a monovalent antigen binding molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science [Science] 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. (USA) [Proceedings of the National Academy of Sciences of the United States] 85: 5879-5883). Alternatively, by using a flexible linker that is too short (e.g., less than about 9 residues) to allow the VL and VH domains of a single polypeptide chain to associate together, bispecific antibodies, diabodies or similar molecules (in which two such polypeptide chains associate together to form a bivalent antigen-binding molecule) can be formed (for a description of diabodies, see, e.g., PNAS USA 90 (14), 6444-8 (1993)).Examples of antigen-binding fragments encompassed by the present invention include (i) Fab' or Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO 2007059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge domain; (iii) Fd fragments consisting essentially of a VH domain and a CH1 domain; (iv) Fv fragments, consisting essentially of a VL and a VH domain, (v) dAb fragments (Ward et al., Nature 341, 544-546 (1989)), consisting essentially of a VH domain, and also referred to as domain antibodies (Holt et al.; Trends Biotechnol. 2003 November; 2i(ll):484-90); (vi) camelid antibodies or nanobodies (Revets et al.; Expert Opin Biol Ther. [Expert view on biological theory] January 2005; 5_(1): 1 11-24) and (vii) isolated complementary determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by isolated genes, they can be connected by synthetic joints using a recombinant method, which enables them to become a single protein chain in which the VL domain and the VH domain are paired to form a monovalent molecule (referred to as a single-chain antibody or single-chain Fv (scFv); see, for example, Bird et al., Science [Science] 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). These and other useful antibody fragments in the context of the present invention are further discussed herein. It should also be understood that, unless otherwise indicated, the term antibody also includes antibody-like polypeptides, such as chimeric antibodies and humanized antibodies provided by any known technology (such as enzymatic cleavage, peptide synthesis and recombinant technology) and antibody fragments (antigen binding fragments) that retain the ability to bind to the antigen. The antibodies thus produced can have any isotype. As used herein, "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3 or IgG4) encoded by the heavy chain constant domain gene. Such antibody fragments are obtained using conventional techniques known to those of ordinary skill in the art; suitable fragments that can bind to the desired epitope can be easily screened for practicality in the same manner as complete antibodies.

[0056] The term "bispecific antibody" refers to an antibody containing two independent antigen binding fragments that each target 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 prepared using compensating amino acid changes in the constant domain of the HC of the parent monospecific bivalent antibody molecule. The resulting heterodimeric antibody contains one Fab contributed by two different parent monospecific antibodies. Amino acid changes in the Fc domain result in increased stability of heterodimeric antibodies with bispecificity that are stable over time. (Ridgway et al., Protein Engineering 9, 617-621 (1996); Gunasekaran et al., JBC 285, 19637-1 (2010); Moore et al., MAB 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., MAB 5:5 646-654 (2013)). Bispecific antibodies may also include molecules produced using ScFv fusions. The two monospecific scFvs are then independently linked to an Fc domain capable of forming a stable heterodimer to generate a single bispecific molecule (Mabry et al., PEDS 23:3 115-127 (2010)). Bispecific molecules have dual binding capabilities.

[0057] "Anti-sortilin antibodies" or "sortilin antibodies" (used interchangeably herein depending on the context in which it is written) are antibodies or antigen-binding fragments thereof that specifically bind to sortilin, and in particular to sortilin E-region SEQ ID NO: 146. Anti-sortilin antibodies that bind to sortilin E-region will typically bind to a conformational or linear epitope of 1, 2, 3, 4, 5, 6 or 7 consecutive amino acids within the E-region with an affinity (IC50) of or below 22 nM, such as between 22 nM and 1 nM, between 10 nM and 1 nM, or between 5 nM and 1 nM, or even higher, such as at about 1 pM or 1 pM to 5 pM.

[0058] As used herein, the term "human antibody" (which may be abbreviated as "humAb" or "HuMab") is intended to include antibodies having variable and constant domains derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or in vivo by somatic mutation).

[0059] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody molecule preparation of a single molecule composition. Conventional monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. In certain embodiments, a monoclonal antibody may be composed of more than one Fab domain, thereby increasing the specificity for more than one target. The term "monoclonal antibody" or "monoclonal antibody composition" is not intended to be limited to any specific production method (e.g., recombinant, transgenic, hybridoma, etc.).

[0060] The term "antibody XX" is intended to mean an antibody or antigen-binding fragment thereof (e.g., antibody "6003-056") comprising or consisting of a light chain, a light chain variable domain, or a light chain variable domain CDR1-3 as defined by its corresponding SEQ ID NO and a heavy chain, a heavy chain variable domain, or a heavy chain variable domain CDR1-3 as defined by its corresponding SEQ ID NO. In certain embodiments, an antibody or antigen-binding fragment thereof is defined by including its entire heavy chain variable domain as defined by its SEQ ID NO and its light chain variable domain as defined by its SEQ ID NO.

[0061] The numbering of amino acid residues can be performed as follows: (the international ImMunoGeneTics information ) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. & Lesk, AM (1987), or Canonical structures For The Hypervariable domains Of Immunoglobulins. J. Mol. Biol. 196, 901-917.

[0062] As used herein, an antibody or antigen-binding fragment thereof is said to "specifically" bind to a region of another molecule (i.e., an epitope) if it reacts or associates with that epitope more frequently, more rapidly, with a longer duration, and / or with greater affinity or avidity than an alternative epitope. In one embodiment, an antibody or antigen-binding fragment thereof of the invention binds to its target (sortilin) ​​at least 10 times more strongly than another molecule; preferably at least 50 times more strongly and more preferably at least 100 times more strongly. Preferably, the antibody or antigen-binding fragment thereof binds under physiological conditions (e.g., in vivo). Thus, by "specifically binding to sortilin", we include the ability of the antibody or antigen-binding fragment thereof to bind to sortilin with such specificity and / or under such conditions. Methods suitable for determining such binding are known to those of ordinary skill in the art, and exemplary methods are described in the accompanying Examples. As used herein, in the context of antibody binding to a predetermined antigen, the term "binding" typically refers to binding with a specificity corresponding to about 10 -7 M or less (e.g. about 10 -8 M or smaller, such as about 10 -9 M or less) when antigen is used as ligand and antibody as analyte. When determined by, for example, surface plasmon resonance (SPR) technology in a 3000 or T200 instrument, and binds to a predetermined antigen with an affinity corresponding to a KD that is at least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower, such as at least 100,000 times lower, than the affinity of the antibody for binding to a non-specific antigen other than the predetermined antigen or a closely related antigen, as determined by, for example, surface plasmon resonance (SPR) technology in a 3000 or T200 instrument, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lower. The amount by which the affinity is made lower depends on the KD of the antibody, so that when the KD of the antibody is very low (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is made lower than the affinity for the non-specific antigen may be at least 10,000 times. In particular, the present invention relates to anti-sortilin antibodies that exhibit a binding affinity corresponding to or below 22 nM (such as between 22 nM and 1 nM, between 10 nM and 1 nM, or between 5 nM and 1 nM) when determined, for example, by biofilm interferometry using Octet384RED (Example 7).

[0063] In certain embodiments of the invention, the invention relates to an antibody or antigen-binding fragment thereof that is capable of competing with humAb antibody 30 or humAb antibody 900 for binding to sortilin. In another embodiment, the invention relates to an antibody or antigen-binding fragment thereof that is capable of competing with antibody 30 for binding to the E region of sortilin as defined in SEQ ID NO: 146. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using an immunoprecipitation assay with human sortilin immobilized on the surface of the cell. The chip is incubated with a reference antibody (e.g., antibody "30" or "900"), with and without the antibody polypeptide to be tested. Alternatively, a paired mapping approach can be used, in which a reference antibody (e.g., antibody "30" or "900") is immobilized to On the surface of the chip, human sortilin antigen is bound to the immobilized antibody, and the ability of the secondary antibody to simultaneously bind human sortilin is then tested (see ' Assay Handbook

[00136] Assay Manual], GE Healthcare Life Sciences, 29-0194-00AA 05 / 2012; the disclosure of which is incorporated herein by reference). Alternatively, Octet 384 Red (Examples 7 & 8) or similar methods are used to demonstrate competitive binding.

[0064] As used herein, the term "kd" (sec-1 or 1 / s) refers to the dissociation rate constant of a specific antibody-antigen interaction. This value is also known as the koff value.

[0065] As used herein, the term "ka" (M-1 x sec-1 or 1 / Msec) refers to the association rate constant for a specific antibody-antigen interaction.

[0066] As used herein, the term "KD" (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction and is obtained by dividing kd by ka.

[0067] As used herein, the term "KA" (M-1 or 1 / M) refers to the association equilibrium constant for a specific antibody-antigen interaction and is obtained by dividing ka by kd.

[0068] In one embodiment, the present invention relates to antibodies or antigen-binding fragments thereof that exhibit one or more of the following properties:

[0069] (i) a binding affinity (K) for Sortilin between 0.5 nM and 10 nM (e.g., 1 nM to 5 nM or 1 nM to 2 nM) D );

[0070] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0071] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0072] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin.

[0073] The term "reducing and / or inhibiting the ability of cells expressing sortilin to clear PGRN" includes the ability to increase the concentration of PGRN in the culture medium by at least 20% (such as between 25% and 500%, between 25% and 400%, or between 25% and 200%), as measured by an ELISA assay as disclosed in Example 9.

[0074] “The ability to increase the amount and / or concentration of PGRN in the plasma of a knock-in mouse expressing human sortilin” includes the ability to increase the concentration of PGRN in plasma by at least 25% but preferably between 50% and 500%, as measured by an ELISA assay as disclosed in Example 10.

[0075] In some antibodies, only a portion of the CDRs (ie, the subset of CDR residues required for binding, termed SDRs) need to be retained for binding in the humanized antibody. The immunogenicity of murine antibodies can be minimized by molecular modeling and / or empirically or as described in Gonzales, NR et al., (2004) "SDR Grafting Of A Murine Antibody Using Multiple Human Germline Templates To Minimize Its Immunogenicity," Mol. Immunol. 41:863-872, based on previous studies (e.g., residues H60-H65 in CDR H2 are generally undesirable) from Kabat CDR regions located outside the Chothia hypervariable loop (see, 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 OfImmunoglobulins [typical structures of hypervariable regions of immunoglobulins]," J. Mol. Biol. [Journal of Molecular Biology] 196: 901-917) identifies CDR residues that do not contact relevant epitopes and are not in SDRs. In such humanized antibodies, at positions where one or more donor CDR residues are absent or the entire donor CDR is omitted, the amino acid occupying that position may be the amino acid occupying the corresponding position (by Kabat numbering) in the acceptor antibody sequence. The number of such substitutions of acceptor to donor amino acids in the included CDRs reflects the balance of competing considerations. Such substitutions are potentially advantageous in reducing the number of mouse amino acids in the humanized antibody and therefore in reducing potential immunogenicity. However, substitutions may also cause changes in affinity, and a significant reduction in affinity is preferably avoided. The substitution positions within the CDRs and the amino acids to be substituted may also be selected empirically.

[0076] The fact that a single amino acid change in a CDR residue can result in loss of functional binding (Rudikoff, S. et al., (1982) "Single Amino Acid Substitution Altering Antigen-binding Specificity," Proc. Natl. Acad. Sci. (USA) 79(6): 1979-1983) provides a means for systematically identifying alternative functional CDR sequences. In a preferred method for obtaining such variant CDRs, a polynucleotide encoding a CDR is mutated (e.g., via random mutagenesis or by a site-directed method (e.g., polymerase chain-mediated amplification using primers encoding the mutation locus)) to generate a CDR having a substituted amino acid residue. By comparing the identity of the relevant residue in the original (functional) CDR sequence with the identity of the substituted (non-functional) variant CDR sequence, the BLOSUM62.iij substitution score for the substitution can be identified.The BLOSUM system provides an amino acid substitution matrix created by analyzing sequence databases for reliable alignment (Eddy, SR (2004) "Where Did The BLOSUM62 Alignment Score Matrix Come From?" Nature Biotech. [Nature Biotech] 22(8): 1035-1036; Henikoff, JG (1992) "Amino acidsubstitution matrices from protein blocks," Proc. Natl. Acad. Sci. (USA) [Proceedings of the National Academy of Sciences of the United States of America] 89: 10915-10919; Karlin, S. et al., (1990) "Methods For Assessing The Statistical Significance OfMolecular Sequence Features By Using General Scoring Schemes [Methods for evaluating the statistical significance of molecular sequence features by using a universal scoring scheme]" Proc. Natl. Acad. Sci. (USA) [Proceedings of the National Academy of Sciences of the United States of America] 87: 2264-2268; Altschul, SF (1991) "Amino Acid Substitution Matrices From An Information Theoretic Perspective [Amino acid substitution matrix from the perspective of information theory]" J.Mol.Biol. [Journal of Molecular Biology] 219, 555-565. Currently, the most advanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table 1 presents the BLOSUM62.iij substitution scores (the higher the score, the more conservative the substitution, and therefore the more likely it is that the substitution will not affect the function). If the antigen-binding fragment comprising the resulting CDR cannot bind to sortilin, for example, the BLOSUM62.iij substitution score is considered to be insufficiently conservative, and new candidate substitutions with higher substitution scores are selected and generated. Thus, for example, if the original residue is glutamic acid (E) and the non-functional replacement residue is histidine (H), the BLOSUM62.iij substitution score will be 0, and more conservative changes (such as to aspartic acid, asparagine, glutamine or lysine) are preferred.

[0077]

[0078] The present invention therefore contemplates the use of random mutagenesis for identifying improved CDRs. In the context of the present invention, conservative substitutions can be defined by substitutions within the amino acid classes reflected in one or more of the following three tables:

[0079] Conservatively substituted amino acid residue categories:

[0080]

[0081]

[0082] Alternative conservative amino acid residue substitution categories:

[0083]

[0084] Physical and functional classification of amino acid residues for their substitutability:

[0085]

[0086] More conservative substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0087] Additional sets of amino acids may also be formulated using the principles described in, for example, Creighton (1984) Proteins: Structure and Molecular Properties (2nd ed. 1993), WH Freeman and Company.

[0088] Phage display technology can be used alternatively to increase (or decrease) CDR affinity. This technique, known as affinity maturation, employs mutagenesis or "CDR walking" and reselection using a target antigen or its antigenic antigen-binding fragment to identify antibodies with CDRs that bind to the antigen with higher (or lower) affinity when compared to the initial antibody or parent antibody (see, e.g., Glaser et al., (1992) J. Immunology [Journal of Immunology] 149: 3903). Mutagenesis of entire codons rather than single nucleotides produces a semi-random spectrum of amino acid mutations. A library consisting of a group of variant clones can be constructed, each clone differing by a single amino acid change in a single CDR, and these clones contain variants representing every possible amino acid substitution of each CDR residue. Mutants with increased (or decreased) binding affinity to the antigen can be screened by contacting the immobilized mutants with a labeled antigen. Any screening method known in the art (e.g., ELISA) can be used to identify mutant antibodies with increased or decreased affinity for the antigen (see Wu et al., 1998, Proc. Natl. Acad. Sci. (USA) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDR walking that randomizes the light chain may potentially be used (see, e.g., Schier et al., 1996, J. Mol. Bio. 263:551).

[0089] Methods for accomplishing such affinity maturation are described, for example, in: 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, BJ et al. (2010) "Stability And CDR Composition Biases Enrich Binder Functionality Landscapes [Stability and CDR Composition Deviation Enrich Binder Functionality Landscapes]", J. Mol. Biol. [Journal of Molecular Biology] 401(1):84-96; Montgomery, DL et al. (2009) "Affinity Maturation And Characterization Of A Human Monoclonal Antibody Against HIV-1gp41 [Affinity Maturation and Characterization of Human Monoclonal Antibodies Against HIV-1gp41]", MAntibodies [M Antibodies] 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 Human Antibody Library And Directed Against The Internal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fantibodies With Improved HIV-1 Neutralization Potency And Breadth [Affinity maturation of a set of Fabs with improved HIV-1 neutralization potency and breadth was generated by targeted diversification of the CDR-H2 loop of a monoclonal F antibody derived from a synthetic naive human antibody library and directed against the internal trimeric coiled-coil of Gp41], Virology 393(1):112-119; Finlay, WJ et 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 [Affinity maturation of humanized rat antibodies for anti-RAGE therapy: comprehensive mutagenesis reveals a high level of mutational plasticity both within and outside the complementarity determining regions]", J. Mol. Biol. [Journal of Molecular Biology] 388(3):541-558; Bostrom, J. et al. (2009) "Improving Antibody Binding Affinity And Specificity For Therapeutic Development", Methods Mol. Biol. [Molecular Biology Methods], 525:353-376; Steidl, S. et al. (2008) "In Vitro Affinity Maturation Of Human GM-CSF Antibodies By Targeted CDR-Diversification", Mol. Immunol. [Molecular Immunology] 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.

[0090] Thus, the sequence of the CDR variants of the included antibodies or their antigen-binding fragments can be different from the sequence of the CDR of the parent antibody by substitution; for example, substituted 4 amino acid residues, 3 amino acid residues, 2 amino acid residues or 1 of the amino acid residues. According to embodiments of the present invention, it is further envisioned that the amino acids in the CDR regions can be substituted with conservative substitutions as defined in Table 3 above.

[0091] The term "transgenic non-human animal" refers to a non-human animal having a genome comprising one or more human heavy and / or light chain transgenes or transchromosomes (integrated or not integrated into the natural genomic DNA of the animal) and capable of expressing fully human antibodies. For example, a transgenic mouse can have a human light chain transgene and a human heavy chain transgene or a human heavy chain transchromosome, so that when immunized with a sortin antigen and / or a cell expressing sortin, the mouse produces human anti-sortin antibodies. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as is the case with transgenic mice, such as HuMAb mice, such as HCo7 or HCo12 mice, or the human heavy chain transgene can be maintained outside the chromosome, such as the transchromosome KM mice described in WO 02 / 43478. Such transgenic and transchromosome mice (collectively referred to herein as "transgenic mice") can produce a variety of human monoclonal antibody isotypes (such as IgG, IgA, IgM, IgD and / or IgE) against a given antigen by undergoing VDJ recombination and isotype conversion.

[0092] Transgenic non-human animals can also be used to produce antibodies against specific antigens by introducing genes encoding such specific antibodies (e.g., by operably linking the antibody genes to genes encoding proteins expressed in the milk of the animal).

[0093] As used herein, the term "treatment" or "treating" means to improve, slow, attenuate or reverse the progression or severity of a disease or disorder, or to improve, slow, attenuate or reverse one or more symptoms or side effects of such a disease or disorder. For purposes of the present invention, "treatment" or "treating" additionally means a method for obtaining a beneficial or desired clinical result, wherein a "beneficial or desired clinical result" includes, but is not limited to, relief of symptoms, reduction in the extent of the disorder or disease, a stabilized (i.e., non-exacerbated) disease or disorder state, a delay or slowing of the progression of a disease or disorder state, an improvement or alleviation of a disease or disorder state, and relief of a disease or disorder, whether partially or completely, detectable or undetectable.

[0094] When applied to the antibodies or their antigen-binding fragments of the present invention, "effective amount" refers to an amount sufficient to achieve the desired biological effect or desired therapeutic outcome (including but not limited to clinical outcome) at the desired dosage and for the desired time period. When applied to the antibodies or their antigen-binding fragments of the present invention, the phrase "therapeutically effective amount" is intended to represent an amount of an antibody or its antigen-binding fragment sufficient to improve, alleviate, stabilize, reverse, slow down, weaken or delay the progress of an obstacle or disease state, or the progress of the symptoms of the obstacle or disease. In an embodiment, the method of the present invention provides for the administration of an antibody or its antigen-binding fragment in combination with other compounds. In such cases, an "effective amount" is an amount of the combination sufficient to cause the expected biological effect.

[0095] A therapeutically effective amount of an anti-sortilin antibody or antigen-binding fragment thereof of the invention can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-sortilin antibody or antigen-binding fragment thereof to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the beneficial therapeutic effects.

[0096] The antibodies of the present invention are preferably human or humanized antibodies.

[0097] The numbering of the amino acid residues in this region can be performed using, for example: (the international ImMunoGeneTics information ) or Kabat, EA, Wu, TT, Perry, HM, Gottesmann, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, U.S. Department of Health and Human Services; Chothia, C. & Lesk, AM (1987), or Canonical structures For The Hypervariabledomains Of Immunoglobulins. J. Mol. Biol. 196, 901-917.

[0098] Antibody 6003-028:

[0099] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0100] (a) a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 1;

[0101] (b) a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 2;

[0102] (c) a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 3;

[0103] (d) a light chain CDR1 having the amino acid sequence of SEQ ID NO: 4;

[0104] (e) a light chain CDR2 having the amino acid sequence of SEQ ID NO: 5; and

[0105] (f) a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6;

[0106] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:109 and the light chain variable domain of SEQ ID NO:110.

[0107] Antibody 6003-056:

[0108] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0109] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:7;

[0110] b. A heavy chain variable domain CDR 2 comprising SEQ ID NO: 8;

[0111] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 9;

[0112] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 10;

[0113] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 11; and

[0114] f. Light chain variable domain CDR 3 comprising SEQ ID NO:12.

[0115] Preferably, the monoclonal antibody may comprise or consist of a heavy chain variable domain of SEQ ID NO:111 and a light chain variable domain of SEQ ID NO:112.

[0116] Antibody 6003-1286:

[0117] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0118] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 13;

[0119] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 14;

[0120] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 15;

[0121] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 16;

[0122] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 17; and

[0123] f. Light chain variable domain CDR 3 comprising SEQ ID NO:18.

[0124] Preferably, the monoclonal antibody may comprise or consist of a heavy chain variable domain of SEQ ID NO:113 and a light chain variable domain of SEQ ID NO:114.

[0125] Antibody 6003-030:

[0126] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0127] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 19;

[0128] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 20;

[0129] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 21;

[0130] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 22;

[0131] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 23; and

[0132] f. Light chain variable domain CDR 3 comprising SEQ ID NO:24.

[0133] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:115 and the light chain variable domain of SEQ ID NO:116.

[0134] Antibody 6003-1277:

[0135] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0136] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 25;

[0137] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 26;

[0138] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 27;

[0139] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 28;

[0140] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 29; and

[0141] f. Light chain variable domain CDR 3 comprising SEQ ID NO:30.

[0142] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:117 and the light chain variable domain of SEQ ID NO:118.

[0143] Antibody 6003-381:

[0144] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0145] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:31;

[0146] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 32;

[0147] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 33;

[0148] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 34;

[0149] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 35; and

[0150] f. Light chain variable domain CDR 3 comprising SEQ ID NO:36.

[0151] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:119 and the light chain variable domain of SEQ ID NO:120.

[0152] Antibody 6003-083:

[0153] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0154] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 37;

[0155] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 38;

[0156] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 39;

[0157] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 40;

[0158] e. a light chain variable domain CDR 2 comprising SEQ ID NO:41; and

[0159] f. Light chain variable domain CDR 3 comprising SEQ ID NO:42.

[0160] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:121 and the light chain variable domain of SEQ ID NO:122.

[0161] Antibody 6003-799:

[0162] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0163] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:43;

[0164] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 44;

[0165] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 45;

[0166] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 46;

[0167] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 47; and

[0168] f. Light chain variable domain CDR 3 comprising SEQ ID NO:48.

[0169] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:123 and the light chain variable domain of SEQ ID NO:124.

[0170] Antibody 6003-910:

[0171] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0172] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:49;

[0173] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 50;

[0174] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 51;

[0175] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 52;

[0176] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 53; and

[0177] f. Light chain variable domain CDR 3 comprising SEQ ID NO:54.

[0178] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:125 and the light chain variable domain of SEQ ID NO:126.

[0179] Antibody 6003-423:

[0180] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0181] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:55;

[0182] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 56;

[0183] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 57;

[0184] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 58;

[0185] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 59; and

[0186] f. Light chain variable domain CDR 3 comprising SEQ ID NO:60.

[0187] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:127 and the light chain variable domain of SEQ ID NO:128.

[0188] Antibody 6003-822:

[0189] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0190] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:61;

[0191] b. A heavy chain variable domain CDR 2 comprising SEQ ID NO: 62;

[0192] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 63;

[0193] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 64;

[0194] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 65; and

[0195] f. Light chain variable domain CDR 3 comprising SEQ ID NO:66.

[0196] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:129 and the light chain variable domain of SEQ ID NO:130.

[0197] Antibody 6003-886:

[0198] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0199] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 67;

[0200] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 68;

[0201] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 69;

[0202] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 70;

[0203] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 71; and

[0204] f. Light chain variable domain CDR 3 comprising SEQ ID NO:72.

[0205] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:131 and the light chain variable domain of SEQ ID NO:132.

[0206] Antibody 6003-072:

[0207] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0208] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:73;

[0209] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 74;

[0210] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 75;

[0211] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 76;

[0212] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 77; and

[0213] f. Light chain variable domain CDR 3 comprising SEQ ID NO:78.

[0214] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:133 and the light chain variable domain of SEQ ID NO:134.

[0215] Antibody 6003-900:

[0216] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0217] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:79;

[0218] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 80;

[0219] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 81;

[0220] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 82;

[0221] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 83; and

[0222] f. Light chain variable domain CDR 3 comprising SEQ ID NO:84.

[0223] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:135 and the light chain variable domain of SEQ ID NO:136.

[0224] Antibody 6003-936:

[0225] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0226] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 85;

[0227] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 86;

[0228] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 87;

[0229] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 88;

[0230] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 89; and

[0231] f. Light chain variable domain CDR 3 comprising SEQ ID NO:90.

[0232] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:137 and the light chain variable domain of SEQ ID NO:138.

[0233] Antibody 6003-408:

[0234] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0235] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:91;

[0236] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 92;

[0237] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 93;

[0238] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 94;

[0239] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 95; and

[0240] f. Light chain variable domain CDR 3 comprising SEQ ID NO:96.

[0241] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:139 and the light chain variable domain of SEQ ID NO:140.

[0242] Antibody 6003-471:

[0243] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0244] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:97;

[0245] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 98;

[0246] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 99;

[0247] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 100;

[0248] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 101; and

[0249] f. Light chain variable domain CDR 3 comprising SEQ ID NO:102.

[0250] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:141 and the light chain variable domain of SEQ ID NO:142.

[0251] Antibody 6003-972:

[0252] Therefore, the present invention relates to an antibody or antigen-binding fragment thereof comprising or consisting of:

[0253] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 103;

[0254] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 104;

[0255] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 105;

[0256] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 106;

[0257] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 107; and

[0258] f. Light chain variable domain CDR 3 comprising SEQ ID NO:108.

[0259] Preferably, the monoclonal antibody may comprise or consist of the heavy chain variable domain of SEQ ID NO:143 and the light chain variable domain of SEQ ID NO:144.

[0260] According to one embodiment, the above-mentioned antibody may further include variants having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference with the CDR1, CDR2 and / or CDR3 (HC and / or VC) sequences.

[0261] In addition, the antibody can be in a composition together with a pharmaceutically acceptable carrier. The antibodies of the present invention can be used in therapy. Specifically, the antibodies of the present invention can be used to treat FTD or ALS or protein diseases (such as Alzheimer's disease (AD)).

[0262] The treatment envisaged by the present invention may be long-term and the patient may be treated for at least 2 weeks (eg, for at least 1 month, 6 months, 1 year or longer).

[0263] The antibodies of the present invention may be, for example, monoclonal antibodies produced by the hybridoma method, which was first described by Kohler et al., Nature 256, 495 (1975), or may be monoclonal antibodies produced by recombinant DNA or other methods. Monoclonal antibodies may also be isolated from phage antibody display libraries using, for example, techniques described in Clackson et al. (Nature 352, 624-628 (1991)) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may be obtained from any suitable source. Thus, for example, monoclonal antibodies may be obtained from hybridomas prepared from murine splenic B lymphocytes obtained from mice immunized with an antigen of interest or a nucleic acid encoding an antigen of interest, for example, in the form of cells expressing the antigen on the surface. Monoclonal antibodies can also be obtained from hybridomas derived from immunized humans or from antibody-expressing cells of non-human mammals (eg, rats, rabbits, dogs, sheep, goats, primates, etc.).

[0264] In one embodiment, the antibodies of the invention are human antibodies. Human monoclonal antibodies to Sortilin can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. Such transgenic and transchromosomal mice include mice referred to herein as HuMAb mice and KM mice, respectively.

[0265] The HuMAb mouse contains a human immunoglobulin gene minilocus that encodes unrearranged human heavy chain variable and constant (μ and γ) and light chain variable and constant (κ) immunoglobulin sequences together with targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Thus, these mice display reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113, 49-101 (1994); Lonberg, N. and Huszar, D., Intern. Rev. Immunol. 13, 65-93 (1995), and Harding, F. and Lonberg, N., Ann. NY Acad. Sci. 764, 536-546 (1995)). The preparation of HuMAb mice is described in detail in 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. Immunol. 152, 2912-2920 (1994), Taylor, L. et al., International Immunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845-851 (1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187.

[0266] HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in their endogenous light chain (κ) gene (as described in Chen et al., EMBO J. [Journal of the European Molecular Biology Association] 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), and a KCo5 human κ light chain transgene (as described in Fishwild et al., Nature Biotechnology [Nature Biotechnology] 14, 845-851 (1996)). In addition, HCo7 mice have the HCo7 human heavy chain transgene (as described in US 5,770,429), HCo12 mice have the HCo12 human heavy chain transgene (as described in Example 2 of WO 01 / 14424), HCo17 mice have the HCo17 human heavy chain transgene (as described in Example 2 of WO 01 / 09187), and HCo20 mice have the HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and kappa light chain transgenes in a background homozygous for the disruption of the endogenous mouse heavy chain and kappa light chain loci.

[0267] In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. EMBO J. [Journal of the European Molecular Biology Association] 12, 811-820 (1993), and the endogenous mouse heavy chain gene has been homozygously disrupted 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 [Nature Biotechnology] 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome consisting of chromosome 14 fragment hCF (SC20), as described in WO 02 / 43478. HCo12-Balb / c, HCo17-Balb / c and HCo20-Balb / c mice can be generated by crossing HCo12, HCo17 and HCo20 with KCo5[J / K](Balb) as described in WO 09 / 097006.

[0268] In the KM mouse strain, the endogenous mouse kappa light chain gene has been homozygously disrupted as described in Chen et al. EMBO J. [Journal of the European Molecular Biology Association] 12, 811-820 (1993), and the endogenous mouse heavy chain gene has been homozygously disrupted 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 [Nature Biotechnology] 14, 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome consisting of the chromosome 14 antigen binding fragment hCF (SC20), as described in WO 02 / 43478.

[0269] Splenocytes from these transgenic mice can be used to produce hybridomas secreting human monoclonal antibodies according to well-known technology. Human monoclonal antibodies of the present invention or polyclonal antibodies or antibodies of the present invention derived from other species can also be produced by producing another non-human mammal or plant transgenic for the immunoglobulin heavy chain and light chain sequence of interest and producing antibodies in recyclable form therefrom and transgenicly producing. Combined with transgenic production in mammals, antibodies can be produced and recovered from the milk of goats, cows or other mammals. See, for example, US 5,827,690, US5,756,687, US 5,750,172 and US 5,741,957.

[0270] The antibodies of the present invention may have any isotype. The selection of isotypes will typically be guided by desired effector functions (such as ADCC induction). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either of the human light chain constant domains κ or λ can be used. If desired, the class of the anti-sortin antibody of the present invention can be converted by known methods. For example, an antibody of the present invention that was originally IgM can be converted to an IgG antibody of the present invention. In addition, class switching techniques can be used to convert one IgG subclass into another subclass, such as from IgG1 to IgG2. Therefore, the effector function of the antibody of the present invention can be changed to, for example, IgG1, IgG2, IgG3, IgG4 antibodies by isotype switching for various therapeutic purposes. In one embodiment, the antibody of the present invention is an IgG1 antibody, such as IgG1, κ. An antibody is said to belong to a specific isotype if its amino acid sequence is mostly homologous to that isotype relative to other isotypes.

[0271] In one embodiment, the antibody of the present invention is a full-length antibody, preferably an IgG antibody, in particular an IgG1, κ antibody. In another embodiment, the antibody of the present invention is an antibody antigen-binding fragment or a single-chain antibody.

[0272] Antibodies and antigen-binding fragments thereof can be obtained, for example, by antigen-binding fragmentation using conventional techniques, and the antigen-binding fragments are screened for practicality in the same manner as described herein for intact antibodies. For example, F(ab')2 antigen-binding fragments can be produced by treating antibodies with pepsin. The resulting F(ab')2 antigen-binding fragments can be treated to reduce disulfide bonds, thereby producing Fab' antigen-binding fragments. Fab antigen-binding fragments can be obtained by treating IgG antibodies with papain; Fab' antigen-binding fragments can be obtained by digesting IgG antibodies with pepsin. F(ab') antigen-binding fragments can also be produced by combining Fab' described below via thioether bonds or disulfide bonds. Fab' antigen-binding fragments are antibody antigen-binding fragments obtained by cutting the disulfide bonds of the hinge domain of F(ab')2. Fab'-antigen-binding fragments can be obtained by treating F(ab')2 antigen-binding fragments with a reducing agent (such as dithiothreitol). Antibody antigen-binding fragments can also be produced by expressing nucleic acids encoding such antigen-binding fragments in recombinant cells (see, e.g., Evans et al., J. Immunol. Meth. [Journal of Immunology Methods] 184, 123-38 (1995)). For example, a chimeric gene encoding a portion of a F(ab')2 antigen-binding fragment may include a DNA sequence encoding the CH1 domain and hinge domain of the H chain, followed by a translation stop codon to produce such a truncated antibody antigen-binding fragment molecule.

[0273] In one embodiment, the anti-sortilin antibody is a monovalent antibody, preferably a monovalent antibody with a hinge region deletion as described in WO 2007059782 (which is incorporated herein by reference in its entirety). Thus, in one embodiment, the antibody is a monovalent antibody, wherein the anti-sortilin antibody is constructed by the following method, the method comprising: i) providing a nucleic acid construct encoding the light chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VL region of the selected antigen-specific anti-sortilin antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of Ig are operably linked together, and wherein, in the case of the IgG1 subtype, the nucleotide sequence encoding the CL region has been modified such that in the presence of polyclonal human IgG or when administered to an animal or human, the CL region does not contain any amino acids that can form disulfide bonds or covalent bonds with other peptides comprising the consensus amino acid sequence of the CL region; ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising The invention relates to a method for preparing a monovalent antibody comprising a nucleotide sequence encoding the VH region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of a human Ig, wherein the nucleotide sequence encoding the CH region has been modified so that, in the presence of polyclonal human IgG or when administered to an animal human, the region corresponding to the hinge region and other regions of the CH region (such as the CH3 region) (as required by the Ig subtype) does not contain any amino acid residues that participate in forming a disulfide bond or a covalent or stable non-covalent heavy chain bond with other peptides containing the consistent amino acid sequence of the CH region of a human Ig, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked together; iii) providing a cell expression system for producing the monovalent antibody; iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

[0274] Similarly, in one embodiment, the anti-sortilin antibody is a monovalent antibody comprising:

[0275] (i) a variable domain of an antibody of the invention as described herein, or an antigen-binding portion of such a domain, and

[0276] (ii) a CH domain or a domain thereof comprising a CH2 and a CH3 domain of an immunoglobulin, wherein the CH domain or its domain has been modified such that the domain corresponding to the hinge domain and (if the immunoglobulin is not of the IgG4 subtype) the other domains of the CH domain (such as the CH3 domain) does not contain any amino acid residues that are capable of forming a disulfide bond with the same CH domain or forming other covalent or stable non-covalent inter-heavy chain bonds with the same CH domain in the presence of polyclonal human IgG.

[0277] In a further embodiment, the heavy chain of the monovalent antibody has been modified such that the entire hinge region has been deleted.

[0278] In another further embodiment, the sequence of the monovalent antibody has been modified such that it does not contain any acceptor sites for N-linked glycosylation.

[0279] The present invention also includes "bispecific antibodies" in which the anti-sortilin binding region (e.g., the sortilin binding region of an anti-sortilin monoclonal antibody) is part of a bivalent or multivalent bispecific scaffold that targets more than one epitope (e.g., the second epitope may comprise an epitope of an active transport receptor, such that the bispecific antibody will exhibit improved transcytosis across biological barriers (e.g., the blood-brain barrier)). Thus, in another additional embodiment, a monovalent Fab of an anti-sortilin antibody may be linked to an additional Fab or scFv targeting a different protein to generate a bispecific antibody. The bispecific antibody may have dual functions, such as a therapeutic function conferred by the anti-sortilin binding domain and a transport function that may bind to a receptor molecule to enhance transfer across a biological barrier (e.g., the blood-brain barrier).

[0280] The antibodies and antigen-binding fragments thereof of the present invention also include single-chain antibodies. Single-chain antibodies are peptides in which the heavy chain and light chain Fv domains are connected. In one embodiment, the present invention provides single-chain Fv (scFv), wherein the heavy chain and light chain in the Fv of the anti-sortilin antibody of the present invention are connected into a single peptide chain by a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues). Methods for producing such antibodies are described, for example, in US 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 348, 552-554 (1990). Single-chain antibodies can be monovalent, if only a single VH and VL are used; can be bivalent, if two VH and VL are used; or can be multivalent, if more than two VH and VL are used.

[0281] The antibodies and antigen-binding fragments thereof described herein may be modified by including any suitable number of modified amino acids and / or by associating with such coupling substituents. Suitability in this context is generally determined by the ability to at least substantially retain the sortilin selectivity and / or sortilin specificity associated with the non-derivatized parent anti-sortilin antibody. The inclusion of one or more modified amino acids may be advantageous, for example, in increasing the serum half-life of the polypeptide, reducing the antigenicity of the polypeptide, or increasing the storage stability of the polypeptide. One or more amino acids are modified, for example, simultaneously with translation or after translation during recombinant production (e.g., N-linked glycosylation on NXS / T motifs during expression in mammalian cells), or by synthetic means. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, isoprenylated (e.g., farnesylated, geranyl-geranylated) amino acids, acetylated amino acids, acylated amino acids, pegylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, and the like. References sufficient to guide one of ordinary skill in the art in the modification of amino acids are fairly abundant in the literature. Exemplary protocols are found in Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, NJ. The modified amino acid may, for example, be selected from a glycosylated amino acid, a pegylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid coupled to a lipid moiety, or an amino acid coupled to an organic derivatizing agent.

[0282] The antibodies and antigen-binding fragments thereof of the present invention can also be chemically modified by covalent coupling to a polymer, for example, to increase its circulation half-life. Exemplary polymers and methods of attaching them to peptides are shown in, for example, US 4,766,106; US 4,179,337; US 4,495,285 and US 4,609,546. Additional illustrative polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of between about 1,000 g / mol and about 40,000 g / mol, for example between about 2,000 g / mol and about 20,000 g / mol, for example about 3,000 g / mol-12,000 g / mol).

[0283] The antibodies and antigen-binding fragments thereof of the invention can further be used in diagnostic methods or as diagnostic imaging ligands.

[0284] In one embodiment, antibodies and antigen-binding fragments thereof of the invention comprising one or more radiolabeled amino acids are provided. Radiolabeled anti-sortilin antibodies can be used for both diagnostic and therapeutic purposes (coupling to a radiolabeled molecule is another possible feature). Non-limiting examples of such labels include, but are not limited to, bismuth ( 213 Bi), carbon ( 11 C. 13 C. 14 C), chromium ( 51 Cr), cobalt ( 57 Co. 60 Co、Co、Copper 64 Cu), Dysprosium ( 165 Dy), Erbium ( 169 Er), fluorine ( 18 F), Gadolinium ( 153 Gd, 159 Gd), Gallium ( 68 Ga, 67 Ga), germanium ( 68 Ge), gold ( 198 Au), holmium ( 166 Ho), hydrogen ( 3 H), Indium ( 111 In, 112 In, 113 In, 115 In、Iodine 121 I. 123 I. 125 I. 131 I), iridium ( 192 Ir、Iron 59 Fe), Krypton ( 81m Kr), lanthanum ( 140 La), Lutetium ( 177 Lu), manganese ( 54 Mn), molybdenum ( 99 Mo), nitrogen ( 13 N. 15 N), oxygen ( 15 O), palladium ( 103 Pd), phosphorus ( 32 P), potassium ( 42 K), praseodymium ( 142 Pr), promethium ( 149 Pm), rhenium ( 186 Re, 188 Re), rhodium ( 105 Rh), Rubidium ( 81 Rb, 82 Rb), ruthenium ( 82 Such as 97 Ru), Samarium ( 153 Sm), Scandium (47 Sc), Selenium ( 75 Se), sodium ( 24 Na), Strontium ( 85 Sr. 89 Sr. 92 Sr、Sulphur 35 S), technetium ( 99 Tc), thallium ( 201 Tl), tin ( 113 Sn, 117 Sn), Xenon ( 133 Xe), Ytterbium ( 169 Yb, 175 Yb, 177 Yb), yttrium ( 90 Y) and zinc ( 65Zn). Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902). For example, radioisotopes can be coupled using 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 [Site-specific conjugation of radioiodinated phenylethylamine derivatives to monoclonal antibodies produces increased radioactivity in tumors]", J. Med. Chem. [Journal of Medicinal Chemistry] 36(9): 1255-1261; Rea, DW et al. (1990) "Site-specifically radioiodinated antibody for targeting tumors", Cancer Res. [Cancer Research] 50(3 Suppl): 857s-861s).

[0285] The present invention also provides anti-sortilin antibodies and antigen-binding fragments thereof that are detectably labeled using a fluorescent label (such as a rare earth chelate (e.g., a europium chelate)), a fluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate, 5-carboxyfluorescein, 6-carboxyfluorescein, dichlorotriazinylamine fluorescein), a rhodamine-type label (e.g., 568 (Invitrogen), or dansyl chloride), VIVOTAG680XL FLUOROCHROME TM (Perkin Elmer), phycoerythrin; umbelliferone, lissamine; cyanine; phycoerythrin, Texas Red, (Invitrogen) or its analogs, all of which are suitable for optical detection. Chemiluminescent labels (e.g., luminol, luciferase, luciferin, and aequorin) can be used. Such diagnosis and detection can also be accomplished by coupling the diagnostic molecules of the invention to detectable substances (including but not limited to various enzymes, including but not limited to horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase) or to prosthetic group complexes (such as but not limited to streptavidin / biotin and avidin / biotin).

[0286] Chemiluminescent labels (e.g., luminol, luciferase, luciferin, and aequorin) may be used. Such diagnosis and detection may also be accomplished by coupling the diagnostic molecules of the invention to detectable substances (including, but not limited to, various enzymes, including, but not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase) or to prosthetic group complexes (such as, but not limited to, streptavidin / biotin and avidin / biotin). Paramagnetic labels may also be used, and detection is preferably performed using positron emission tomography (PET) or single photon emission computed tomography (SPECT). Such paramagnetic labels include, but are not limited to, those containing aluminum (Al), barium (Ba), calcium (Ca), cerium (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), rhodium (Rh), ruthenium (Ru), samarium (Sm), sodium (Na), strontium (Sr), terbium (Tb), thulium (Tm), tin (Sn), titanium (Ti), tungsten (W) and zirconium (Zi), and in particular Co. +2 , CR +2 Cr +3 , Cu +2 , Fe +2 , Fe +3 , Ga +3 , Mn +3 、Ni+2 、Ti +3 、V +3 and V +4 Compounds of paramagnetic ions, positron emitting metals using various positron emission tomography techniques as well as non-radioactive paramagnetic metal ions.

[0287] Thus, in one embodiment, the anti-sortilin antibody or sortilin binding fragment thereof of the invention may be labeled with a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label. The labeled antibody fragment may be used to detect or measure the presence or amount of the sortilin in the brain of a subject. Such methods may include in vivo imaging detection or measurement of the anti-sortilin antibody or sortilin binding fragment bound to the sortilin and may include ex vivo imaging of the anti-sortilin antibody or sortilin binding fragment bound to such sortilin.

[0288] In another aspect, the present invention relates to expression vectors encoding one or more polypeptide chains of an antibody of the present invention or an antigen-binding domain thereof. Such expression vectors can be used to recombinantly produce the antibodies and antigen-binding fragments of the present invention.

[0289] In the context of the present invention, the expression vector may be any suitable DNA or RNA vector, including chromosomal vectors, non-chromosomal vectors, and synthetic nucleic acid vectors (comprising a nucleic acid sequence of a set of suitable expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-sortilin antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (as described, for example, in Sykes and Johnston, Nat Biotech [Nature Biotechnology] 12, 355-59 (1997)), a compact nucleic acid vector (as described, for example, in US 6,077,835 and / or WO 00 / 70087), a plasmid vector (such as pBR322, pUC19 / 18 or pUC 118 / 119), a "midge" minimum size nucleic acid vector (as described, for example, in Schakowski et al., Mol Ther [Molecular Therapeutics] 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4 precipitated construct (as described, for example, in 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 use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0290] In one embodiment, the vector is suitable for expressing an anti-sortilin antibody or antigen-binding fragment thereof in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (VanHeeke & Schuster, J Biol Chem [Journal of Biological Chemistry] 264, 5503-5509 (1989)), pET vectors (Novagen, Madison, Wisconsin), and the like.

[0291] The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in: Current Protocols in Molecular Biology, edited by F. Ausubel et al., 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. [Molecular Biology Methods] 824, 329-358 (2012); Celik, E. et al., Biotechnol. Adv. [Biotechnology Progress] 30(5), 1108-1118 (2012); Li, P. et al., Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 142(2), 105-124 (2007); E. et al., Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007); van der Vaart, JM, Methods Mol. Biol. 178, 359-366 (2002) and Holliger, P., Methods Mol. Biol. 178, 349-357 (2002)).

[0292] In the expression vector of the present invention, the nucleic acid encoding the anti-sortin antibody may include or be associated with any suitable promoter, enhancer and other elements that contribute to expression. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer together with RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly (A) termination sequences, replication origins for producing plasmids in Escherichia coli, antibiotic resistance genes as selective markers and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also include an inducible promoter relative to a constitutive promoter (such as CMV IE) (those skilled in the art will recognize that such terms are actually descriptors of the degree of gene expression under certain conditions).

[0293] In even further aspects, the invention relates to recombinant eukaryotic or prokaryotic host cells (such as transfectomas) that produce an antibody of the invention as defined herein or an antigen-binding fragment thereof or a bispecific molecule of the invention as defined herein. 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 the genome of the cell, the genome comprising a sequence encoding the expression of an anti-sortilin antibody of the invention or an antigen-binding fragment thereof. In another embodiment, the invention provides a cell comprising a non-integrating nucleic acid (such as a plasmid, cosmid, phagemid, or linear expression element) comprising a sequence encoding the expression of an anti-sortilin antibody of the invention or an antigen-binding fragment thereof.

[0294] In a further aspect, the invention relates to a method for producing an anti-sortilin antibody of the invention, the method comprising the steps of: a) culturing the hybridoma or host cell of the invention as described above, and b) purifying the antibody of the invention from the culture medium.

[0295] In one embodiment, the invention relates to a preparation, as that term is used herein, comprising an anti-sortilin antibody as defined herein, and being substantially free of naturally occurring antibodies that are unable to bind to sortilin or that do not substantially alter the anti-sortilin functionality of the preparation. Thus, such a preparation does not include naturally occurring serum or a purified derivative of such serum, the preparation comprising a mixture of anti-sortilin antibodies and another antibody that does not alter the functionality of the anti-sortilin antibodies of the preparation, wherein such functionality is:

[0296] (i) Binding affinity for sortilin (K D );

[0297] (ii) a;

[0298] (iii) reducing and / or inhibiting the ability of cells expressing sortilin to clear PGRN;

[0299] (iv) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0300] (v) the ability to increase the amount and / or concentration of PGRN in the plasma of a knock-in mouse expressing human sortilin; the ability to increase the amount and / or concentration of PGRN in the brain and / or

[0301] (vi) The ability to provide treatment of frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when administered chronically.

[0302] The present invention is specifically directed to preparations of an anti-sortilin antibody having a structural alteration in its amino acid sequence (in any of its CDRs, variable domains, framework residues and / or constant domains) relative to the structure of a naturally occurring anti-sortilin antibody, wherein said structural alteration causes the anti-sortilin antibody monoclonal antibody to exhibit significantly altered functionality (i.e., greater than 20% difference, greater than 40% difference, greater than 60% difference, greater than 80% difference, greater than 100% difference, greater than 150% difference, greater than 2-fold difference, greater than 4-fold difference, greater than 5-fold difference, or greater than 10-fold difference in functionality) relative to the functionality exhibited by said naturally occurring anti-sortilin antibody; wherein such functionality is:

[0303] (i) Binding affinity for sortilin (K D );

[0304] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0305] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0306] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin;

[0307] (v) the ability to increase the amount and / or concentration of PGRN in the brain and / or

[0308] (vi) The ability to provide treatment of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and / or Alzheimer's disease (AD) when administered chronically.

[0309] This is especially true where the functionality thus modified is a result of the structural modification and is therefore inseparable from it.

[0310] The term "substantially free" of naturally occurring antibodies refers to the complete absence of such naturally occurring antibodies in such preparations, or the inclusion of such naturally occurring antibodies in such preparations at concentrations that do not materially affect the Sortilin binding properties of such preparations. An antibody is considered "isolated" if it does not have a naturally occurring counterpart or has been separated or purified from components that naturally accompany the antibody.

[0311] The term "naturally produced antibodies" as it relates to such preparations refers to antibodies (including naturally produced autoantibodies) elicited in a living human or other animal as a natural result of the functioning of the immune system of that living human or other animal.

[0312] Thus, the formulations of the present invention do not exclude and do specifically include such formulations containing anti-sortilin antibodies and intentionally added additional antibodies that are capable of binding to epitopes not possessed by sortilin. Such formulations specifically include embodiments thereof in which the formulation exhibits enhanced efficacy in treating frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS).

[0313] Antibodies of the present invention and their antigen-binding fragments can be produced in different cell lines, such as human cell lines, non-human mammalian cell lines and insect cell lines, for example, CHO cell lines, HEK cell lines, BHK-21 cell lines, murine cell lines (such as myeloma cell lines), fibrosarcoma cell lines, PER.C6 cell lines, HKB-11 cell lines, CAP cell lines and HuH-7 human cell lines (Dumont et al., 2015, Crit Rev Biotechnol. [Critical Review of Biotechnology] September 18: 1-13., the contents of which are included herein by reference).

[0314] In an even further aspect, the invention relates to a pharmaceutical composition comprising:

[0315] (i) an anti-sortilin antibody or antigen-binding fragment thereof, both as defined herein, or a preparation comprising such an anti-sortilin antibody or antigen-binding fragment thereof, as that term is defined herein; and

[0316] (ii) a pharmaceutically acceptable carrier.

[0317] The pharmaceutical composition can be formulated with a pharmaceutically acceptable carrier or diluent together with any other known adjuvants and excipients according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 22nd edition, Gennaro ed., Mack Publishing Co., Easton, Pennsylvania, 2013.

[0318] The pharmaceutically acceptable carrier or diluent, together with any other known adjuvants and excipients, should be suitable for the selected compound of the present invention and the selected mode of administration. The suitability of the carrier and other components of the pharmaceutical composition is determined based on the lack of a significant adverse effect on the desired biological properties of the selected compound of the present invention or pharmaceutical composition with respect to epitope binding (e.g., less than a substantial effect, 10% or less relative inhibition, 5% or less relative inhibition, etc.).

[0319] The pharmaceutical compositions of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in the pharmaceutical composition. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc. The composition may also include large, slowly metabolized macromolecules such as proteins, polysaccharides (like chitosan), polylactic acid, polyglycolic acid, and copolymers (e.g., latex-functionalized cross-linked agarose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes).

[0320] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present invention or its amide employed, the route of administration, the time of administration, the excretion rate of the specific compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0321] The pharmaceutical composition can be given by any suitable route and mode, including: parenteral, topical, oral or intranasal means for preventive and / or therapeutic treatment. In one embodiment, the pharmaceutical composition of the present invention is given parenterally. As used herein, the phrases "parenteral administration" and "administered parenterally" mean the mode of administration except enteral and topical administration, usually by injection, and include epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural and intrasternal injection and infusion. Other suitable in vivo and in vitro routes for giving the compounds of the present invention are well known in the art and can be selected by those of ordinary skill in the art. In one embodiment, the pharmaceutical composition is given by intravenous or subcutaneous injection or infusion.

[0322] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents and the like that are physiologically compatible with the compounds of the invention.

[0323] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (such as 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), carboxymethylcellulose colloid solutions, tragacanth gum and injectable organic esters (such as ethyl oleate) and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0324] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injections or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except in the case where any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.

[0325] The proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0326] The pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable antioxidant, for example (1) a water-soluble antioxidant, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) an oil-soluble antioxidant, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) a metal chelator, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0327] The pharmaceutical compositions of the present invention may also contain isotonic agents, such as sugars, polyalcohols (eg, mannitol, sorbitol, glycerol), or sodium chloride in the composition.

[0328] The pharmaceutical composition of the present invention may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which can enhance the shelf life or effectiveness of the pharmaceutical composition. The compound of the present invention can be prepared with a carrier, which can protect the compound from being quickly released, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers (such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid) (alone or with wax) or other materials well known in the art. The method for preparing such a preparation is generally known to those of ordinary skill in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems [slow-release and controlled-release delivery systems], edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0329] In one embodiment, the compounds of the present 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 extemporaneous preparation of sterile injections or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art. Except in the case where any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0330] Pharmaceutical compositions for injection must typically be sterile and stable under production and storage conditions. The composition can be formulated as a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size (in the case of a dispersion) and by using a surfactant. In many cases, it is preferred to include an isotonic agent in the composition, such as a sugar, a polyol (such as glycerol, mannitol, sorbitol) or sodium chloride. Prolonged release of the injectable composition absorb By including a delay antibody in the composition absorbThe invention relates to a method for preparing a sterile injectable solution. The sterile injectable solution can be prepared by mixing the required amount of the active compound in an appropriate solvent with one or a combination of the ingredients listed above, for example, and then sterilizing by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other ingredients required, for example, from those listed above. In the case where sterile powders are used to prepare sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which obtain powders of the active ingredient plus any additional desired ingredients from their previously sterile filtered solutions.

[0331] Sterile injectable solutions can be prepared by mixing the required amount of active compound with one of the above-listed ingredients or a combination of ingredients in an appropriate solvent, followed by sterilization microfiltration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a basic dispersion medium and other required ingredients from those listed above. In the case where sterile powders are used to prepare sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which obtain powders of the active ingredient plus any additional desired ingredients from their previously sterile filtered solutions.

[0332] The dosage regimen in the above treatment methods and the use described herein is adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus may be given, several divided doses may be given over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. For ease of administration and uniformity of dosage, parenteral compositions may be formulated in dosage unit form. As used herein, dosage unit form refers to a physically discrete unit suitable as a single dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect associated with the required pharmaceutical carrier. The description of the dosage unit form of the present invention is subject to indications and is directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the field of compounding such active compounds to obtain sensitive treatment in individuals.

[0333] The effective dosage and dosage regimen of the anti-sortilin antibody depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art. An exemplary, non-limiting range of a therapeutically effective amount of an antibody of the invention is about 0.1 mg / kg-10 mg / kg body weight, such as about 0.1 mg / kg-5 mg / kg body weight, for example about 0.1 mg / kg-2 mg / kg body weight, such as about 0.1-1 mg / kg body weight, for example about 0.15 mg / kg, about 0.2 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg or about 2 mg / kg body weight.

[0334] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start administering the anti-sortin antibody used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, a suitable daily dose of the composition of the present invention will be the amount of the compound at the lowest dose that effectively produces a therapeutic effect. Such an effective dose will generally depend on the above factors. Administration can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, or, for example, administered near the target site. If desired, the effective daily dose of the pharmaceutical composition can be divided into two, three, four, five, six or more sub-doses at appropriate time intervals throughout the day, optionally administered in unit dosage form. Although the compound of the present invention can be administered alone, it is preferred to administer the compound as a pharmaceutical composition as described above.

[0335] The labeled antibodies or antigen-binding fragments thereof of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases or disorders. The present invention provides for the detection or diagnosis of neurodegenerative or cognitive diseases or disorders, including but not limited to FTD, ALS or protein diseases (such as Alzheimer's disease (AD)), the detection or diagnosis comprising: (a) using one or more antibodies that specifically bind to sortin, determining the presence of pyroglutamated Aβ fragments in a subject's cells or tissue sample; and (b) comparing the level of the antigen to a control level (e.g., the level in a normal tissue sample), whereby an increase in the measured level of the antigen compared to the control level of the antigen indicates the disease or disorder, or indicates the severity of the disease or disorder.

[0336] The antibodies or antigen-binding fragments thereof of the invention can be used to assay for sortilin or sortilin antigen-binding fragments in biological samples using immunohistochemical methods well known in the art. Other antibody-based methods useful for detecting proteins include immunoassays such as enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) and mesoscale discovery platform-based assays (MSD). Suitable antibody labels can be used in such kits and methods, and labels known in the art include enzyme labels such as alkaline phosphatase and glucose oxidase; radioisotope labels such as iodine ( 125 I. 131 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99mTc); and luminescent labels, such as luminol and luciferase; and fluorescent markers, such as fluorescein and rhodamine.

[0337] The presence of labeled anti-sortilin antibodies or sortilin binding fragments thereof can be detected in vivo for diagnostic purposes. In one embodiment, the diagnosis comprises: a) administering an effective amount of such labeled molecules to a subject; b) waiting a certain time interval after administration to allow the labeled molecules to aggregate at Aβ deposition sites (if any) and to allow unbound labeled molecules to be cleared to background levels; c) determining the background level; and d) detecting the labeled molecules in the subject, such that detection of labeled molecules above the background level indicates that the subject has the 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 for detection using a specific imaging system known to those of ordinary skill in the art. The background level can be determined by various methods known in the art, including comparing the amount of labeled antibody detected to a previously determined standard value for a specific imaging system. Methods and systems that can be used in the diagnostic methods of the present 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.

[0338] In a further aspect, the invention relates to antibodies or antigen-binding fragments thereof for use in medicine.

[0339] In a further aspect, the invention relates to an antibody or antigen-binding fragment thereof of the invention for use in treating a disease associated with decreased PGRN levels in the brain of a patient.

[0340] In a further aspect, the invention relates to the use of an antibody or antigen-binding fragment thereof of the invention in the manufacture of a medicament for treating a disease associated with reduced PGRN levels in the brain of a patient.

[0341] In a further aspect, the invention relates to a method for preventing or treating a disease associated with decreased PGRN or decreased functional PGRN levels in the brain of a patient, the method comprising administering an effective dose of an antibody or antigen-binding fragment thereof of the invention.

[0342] Preferred are uses and methods according to those aspects of the invention wherein the disease is: FTD, ALS or a proteinopathy such as AD.

[0343] Preferably, in the uses and methods of those aspects of the invention, the treatment is long-term and preferably continues for at least 2 weeks, such as for at least 1 month, 6 months, 1 year or longer.

[0344] In a further aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention.

[0345] Table 5 - Sequence

[0346]

[0347]

[0348]

[0349]

[0350]

[0351] The listing or discussion in the specification of an apparently prior-published document does not necessarily constitute an admission that the document is part of the state of the art or is common general knowledge.

[0352] Implementation

[0353] As should be apparent from the text and the examples, the present invention further relates to the following embodiments:

[0354] 1. An antibody or an antigen-binding fragment thereof, which is capable of specifically binding to sortilin and inhibiting the binding of PGRN to sortilin.

[0355] 2. The antibody or antigen-binding fragment thereof according to embodiment 1, wherein the antibody comprises or consists of a complete antibody.

[0356] 3. The antibody or antigen-binding fragment thereof according to embodiment 1 or 2, wherein the antigen-binding fragment comprises or consists of an antigen-binding fragment selected from the group consisting of: an Fv fragment (e.g., a single-chain Fv or a disulfide-bonded Fv); a Fab-like fragment (e.g., a Fab fragment or a F(ab')2 fragment); and a domain antibody (e.g., a single V H Variable domain or V L variable fields).

[0357] 4. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody is selected from the group consisting of antibodies of subtype IgG1, IgG2, IgG3 or IgG4.

[0358] 5. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment thereof specifically binds to the E region of sortilin as defined in SEQ ID NO:146.

[0359] 6. An antibody or antigen-binding fragment thereof according to any of the preceding embodiments, wherein the antibody or fragment thereof specifically binds to at least 3 consecutive amino acids, such as 4, 5, 6 or 7 consecutive amino acids, in the E region of sortilin as defined in SEQ ID NO: 146.

[0360] 7. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment exhibits one or more of the following properties:

[0361] (i) a binding affinity (K) for Sortilin between 0.5 nM-10 nM (such as 1 nM-5 nM or 1 nM-2 nM), or even higher, such as between 0.5 pM and 500 pM D )

[0362] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0363] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0364] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin.

[0365] 8. The antibody or antigen-binding fragment thereof according to embodiment 5, which is human or humanized.

[0366] 9. The antibody or antigen-binding fragment thereof according to embodiment 6, which is human or humanized.

[0367] 10. The antibody or antigen-binding fragment thereof according to any of the above embodiments, wherein the antibody or antigen-binding fragment thereof is human or humanized.

[0368] 11. An antibody or antigen-binding fragment thereof according to any preceding embodiment, comprising a light chain variable domain comprising one or more of the CDR 1-3 light chains as listed for each of the antibodies defined in Table 5, or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference.

[0369] 12. The antibody or antigen-binding fragment thereof according to embodiment 11, comprising a light chain variable domain comprising CDR 1-3 light chain as listed for each of the antibodies defined in Table 5.

[0370] 13. The antibody or antigen-binding fragment thereof according to embodiment 11 or 12, comprising a light chain variable domain comprising or consisting of the amino acid sequence VL as listed for each of the antibodies defined in Table 5.

[0371] 14. The antibody or antigen-binding fragment thereof according to any one of embodiments 11 to 13, comprising a light chain comprising or consisting of the amino acid sequence of VL as listed for each of the antibodies defined in Table 5.

[0372] 15. An antibody or antigen-binding fragment thereof according to any preceding embodiment, comprising a heavy chain variable domain comprising one or more CDR 1-3 heavy chains as listed for each of the antibodies defined in Table 5, or an amino acid sequence having no more than 4 amino acid differences, or no more than 3 amino acid differences, or no more than 2 amino acid differences, or no more than 1 amino acid difference.

[0373] 16. The antibody or antigen-binding fragment thereof according to embodiment 15, comprising a heavy chain variable domain comprising CDRs 1-3 heavy chain as listed for each of the antibodies defined in Table 5.

[0374] 17. The antibody or antigen-binding fragment thereof according to embodiment 15 or 16, comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of VH as listed for each of the antibodies defined in Table 5.

[0375] 18. The antibody or antigen-binding fragment thereof according to any one of embodiments 15 to 17, comprising a heavy chain comprising or consisting of the amino acid sequence VL as listed for each of the antibodies defined in Table 5.

[0376] 19. An antibody or antigen-binding fragment thereof according to any preceding embodiment, comprising a light chain variable domain comprising or consisting of the amino acid sequence of VL as listed for each of the antibodies defined in Table 5, and a heavy chain variable domain comprising or consisting of the amino acid sequence of VH as listed for each of the antibodies defined in Table 5.

[0377] 20. An antibody or antigen-binding fragment thereof according to any preceding embodiment, comprising a light chain comprising or consisting of the amino acid sequence of VL as listed for each of the antibodies defined in Table 5, and a heavy chain comprising or consisting of the amino acid sequence of VH as listed for each of the antibodies defined in Table 5.

[0378] 21. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment thereof competes with the antibody or antigen-binding fragment thereof defined in embodiment 20 for binding to sortilin.

[0379] 22. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment comprises an Fc region.

[0380] 23. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment further comprises a moiety for increasing half-life in vivo.

[0381] 24. The antibody or antigen-binding fragment thereof according to embodiment 22, wherein the portion for increasing the in vivo half-life is selected from the group consisting of polyethylene glycol (PEG), human serum albumin, a glycosylation group, a fatty acid and a dextran.

[0382] 25. The antibody or antigen-binding fragment thereof according to any preceding embodiment, wherein the antibody or antigen-binding fragment further comprises a detectable portion.

[0383] 26. The antibody or antigen-binding fragment thereof according to embodiment 25, wherein the detectable portion is selected from the group consisting of a fluorescent label, a chemiluminescent label, a paramagnetic label, a radioisotope label, or an enzyme label.

[0384] 27. The antibody or antigen-binding fragment thereof according to embodiment 25 or 26, wherein the detectable portion comprises or consists of a radioactive isotope.

[0385] 28. The antibody or antigen-binding fragment thereof according to embodiment 26 or 27, wherein the radioisotope is selected from the group consisting of: 99m Tc, 111 In, 67 Ga, 68 Ga, 72 As, 89 Zr, 123 I and 201 Tl.

[0386] 29. The antibody or antigen-binding fragment thereof of embodiment 25, wherein the detectable portion comprises or consists of a paramagnetic isotope.

[0387] 30. The antibody or antigen-binding fragment thereof according to embodiment 29, wherein the paramagnetic isotope is selected from the group consisting of: 157 Gd, 55 Mn, 162 Dy,52 Cr and 56 Fe.

[0388] 31. The antibody or antigen-binding fragment thereof according to any one of embodiments 25 to 30, wherein the detectable portion is detectable by imaging techniques such as SPECT, PET, MRI, optical or ultrasound imaging.

[0389] 32. The antibody or antigen-binding fragment thereof according to any one of embodiments 25 to 31, wherein the detectable moiety is indirectly linked to the antibody or antigen-binding fragment thereof via a linking moiety.

[0390] 33. An antibody or antigen-binding fragment thereof according to embodiment 32, wherein the linking portion is selected from the group consisting of derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).

[0391] 34. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33.

[0392] 35. The nucleic acid molecule of embodiment 34, wherein the molecule is a cDNA molecule.

[0393] 36. A vector comprising a nucleic acid molecule as defined in embodiment 34 or 35.

[0394] 37. A recombinant host cell comprising a nucleic acid molecule as defined in any one of embodiments 34-36.

[0395] 38. A method for producing an antibody or antigen-binding fragment as defined in any one of embodiments 1-33, the method comprising culturing a host cell as defined in embodiment 37 under conditions allowing expression of the encoded antibody or antigen-binding fragment thereof.

[0396] 39. A formulation comprising the antibody or antigen-binding fragment thereof according to any one of the above embodiments, wherein the formulation is substantially free of naturally occurring antibodies that are unable to bind to Sortilin or do not substantially alter the anti-Sortilin functionality of the formulation, the functionality being selected from the group consisting of:

[0397] (i) Binding affinity for sortilin (K D);

[0398] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0399] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0400] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin; and / or

[0401] (v) The ability to provide treatment of frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when administered chronically.

[0402] 40. A formulation comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of the above embodiments, wherein the monoclonal antibody has a structural alteration in its amino acid sequence relative to the structure of a naturally occurring anti-sortilin antibody, wherein the structural alteration causes the monoclonal antibody to exhibit altered functionality relative to the functionality exhibited by the naturally occurring anti-sortilin antibody, wherein the functionality is:

[0403] (i) Binding affinity (KD) for sortilin;

[0404] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0405] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0406] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin; and / or

[0407] (v) The ability to provide treatment of frontotemporal dementia (FTD) and / or amyotrophic lateral sclerosis (ALS) when administered chronically.

[0408] 41. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, or a formulation as described in any one of embodiments 39-40, and a pharmaceutically acceptable carrier.

[0409] 42. An antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, or a formulation as described in any one of embodiments 39-40, for use in medicine.

[0410] 43. An antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, or a formulation as described in any one of embodiments 39-40, for use in preventing and / or treating a disease associated with decreased PGRN or decreased functional PGRN levels in the brain of a patient.

[0411] 44. Use of an antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, or a formulation as described in any one of embodiments 39-40, in the manufacture of a medicament for preventing and / or treating a disease associated with reduced PGRN levels in the brain of a patient.

[0412] 45. The antibody or antigen-binding fragment thereof for use according to embodiment 43, or the use according to embodiment 44, wherein the disease is selected from the group consisting of: FTD; ALS; protein diseases such as AD, PD.

[0413] 46. ​​A method for preventing or treating a disease associated with reduced PGRN levels in the brain of a patient, the method comprising administering an effective dose of an antibody or fragment thereof as defined in any one of embodiments 1-33, a formulation as described in any one of embodiments 39-40, or a pharmaceutical composition as described in embodiment 41.

[0414] 47. The antibody or antigen-binding fragment thereof for use according to embodiment 43, or the use according to embodiment 44, or the method according to embodiment 46, wherein the disease is selected from the group consisting of: FTD; ALS; or protein diseases such as AD, PD.

[0415] 48. The antibody or antigen-binding fragment thereof for use; or the use; or the method according to embodiment 46 or 47, wherein the treatment is chronic.

[0416] 49. An antibody or antigen-binding fragment thereof for use; or use; or method according to embodiment 48, wherein the long-term treatment lasts for at least 2 weeks, such as at least 1 month, 6 months, 1 year or longer.

[0417] 50. An antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, a formulation as described in any one of embodiments 39-40, or a pharmaceutical composition as described in embodiment 41, which is capable of specifically binding to Sortilin, but the binding does not inhibit or does not substantially inhibit the binding of neurotensin or AF38469 to Sortilin ( TJ et al., Bioorg Med Chem Lett. 2014 Jan 1;24(1):177-80).

[0418] 51. A kit comprising an antibody or antigen-binding fragment thereof as defined in any one of embodiments 1-33, a formulation as defined in any one of embodiments 39-40, or a pharmaceutical composition as defined in embodiment 41.

[0419] item

[0420] Item 1. An antibody or an antigen-binding fragment thereof that is capable of specifically binding within the E region of sortilin as defined by SEQ ID NO:146.

[0421] Item 2. An antibody or antigen-binding fragment thereof according to Item 1, wherein the antigen-binding fragment is selected from the group consisting of: an Fv fragment (e.g., a single-chain Fv or a disulfide-bonded Fv); a Fab-like fragment (e.g., a Fab fragment, a Fab' fragment or a F(ab)2 fragment); and a domain antibody (e.g., a single VH variable domain or a VL variable domain).

[0422] Item 3. The antibody according to any preceding item, wherein the antibody consists of a whole antibody.

[0423] Item 4. The antibody or antigen-binding fragment thereof according to any preceding item, wherein the antibody is selected from the group consisting of antibodies of subtype IgG1, IgG2, IgG3 or IgG4.

[0424] Item 5. An antibody or antigen-binding fragment thereof according to any of the preceding items, wherein the antibody or antigen-binding fragment thereof binds to the E region of sortilin as defined by SEQ ID NO:146.

[0425] Item 6. An antibody or antigen-binding fragment thereof according to any of the preceding items, wherein the antibody or antigen-binding fragment thereof binds to at least 1 or more consecutive amino acids, such as 1, 2, 3, 4, 5, 6 or 7 consecutive amino acids, within the E region of sortilin as defined by SEQ ID NO:146.

[0426] Item 7. The antibody or antigen-binding fragment thereof according to any preceding item, wherein the antibody or antigen-binding fragment exhibits one or more of the following properties:

[0427] a. A binding affinity (KD) for sortilin between 0.5 nM-10 nM, such as between 1 nM-5 nM or 1 nM-2 nM, or even higher, such as between 0.5 pM and 500 pM

[0428] b. reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0429] c. reducing and / or inhibiting the ability of cells expressing sortilin to internalize PGRN;

[0430] d. Ability to increase the amount and / or concentration of PGRN in the brain, and / or

[0431] e. Ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin.

[0432] Item 8. An antibody or antigen-binding fragment thereof according to any one of the preceding items 1-7, wherein the ability of the antibody or antigen-binding fragment thereof to reduce and / or inhibit the clearance of PGRN includes reducing and / or inhibiting the clearance of PGRN at an IC50 at or below 22nM, such as between 22nM and 1nM, or between 10nM and 1nM, or between 5nM and 1nM.

[0433] Item 9. The antibody or antigen-binding fragment thereof according to any preceding item, wherein the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a recombinant antibody or a chimeric antibody.

[0434] Item 10. The antibody or antigen-binding fragment thereof according to any preceding item, wherein the antibody or antigen-binding fragment thereof comprises:

[0435] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 1;

[0436] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 2;

[0437] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 3;

[0438] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 4;

[0439] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 5; and

[0440] f. Light chain variable domain CDR 3 comprising SEQ ID NO:6.

[0441] Item 11. The antibody or antigen-binding fragment thereof according to Item 10, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:109.

[0442] Item 12. The antibody or antigen-binding fragment thereof according to Item 10, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:110.

[0443] Item 13. An antibody or antigen-binding fragment thereof according to Item 10, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 11 and 12.

[0444] Item 14. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0445] a. A heavy chain variable domain CDR 1 comprising SEQ ID NO: 7;

[0446] b. A heavy chain variable domain CDR 2 comprising SEQ ID NO: 8;

[0447] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 9;

[0448] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 10;

[0449] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 11; and

[0450] f. Light chain variable domain CDR 3 comprising SEQ ID NO:12.

[0451] Item 15. The antibody or antigen-binding fragment thereof according to Item 14, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:111.

[0452] Item 16. An antibody or antigen-binding fragment thereof according to Item 14, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:112.

[0453] Item 17. An antibody or antigen-binding fragment thereof according to Item 14, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 15 and 16.

[0454] Item 18. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0455] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 13;

[0456] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 14;

[0457] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 15;

[0458] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 16;

[0459] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 17; and

[0460] f. Light chain variable domain CDR 3 comprising SEQ ID NO:18.

[0461] Item 19. An antibody or antigen-binding fragment thereof according to Item 18, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which comprises SEQ ID NO:113.

[0462] Item 20. An antibody or antigen-binding fragment thereof according to Item 18, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:114.

[0463] Item 21. The antibody or antigen-binding fragment thereof according to Item 18, wherein the antibody or antigen-binding fragment thereof comprises both the heavy chain variable domain and the light chain variable domain as defined in Items 19 and 20.

[0464] Item 22. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0465] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 19;

[0466] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 20;

[0467] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 21;

[0468] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 22;

[0469] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 23; and

[0470] f. Light chain variable domain CDR 3 comprising SEQ ID NO:24.

[0471] Item 23. An antibody or antigen-binding fragment thereof according to Item 22, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:115.

[0472] Item 24. An antibody or antigen-binding fragment thereof according to Item 22, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:116.

[0473] Item 25. An antibody or antigen-binding fragment thereof according to Item 22, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 23 and 24.

[0474] Item 26. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0475] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 25;

[0476] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 26;

[0477] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 27;

[0478] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 28;

[0479] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 29; and

[0480] f. Light chain variable domain CDR 3 comprising SEQ ID NO:30.

[0481] Item 27. An antibody or antigen-binding fragment thereof according to Item 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:117.

[0482] Item 28. An antibody or antigen-binding fragment thereof according to Item 26, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:118.

[0483] Item 29. An antibody or antigen-binding fragment thereof according to Item 26, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 27 and 28.

[0484] Item 30. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0485] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:31;

[0486] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 32;

[0487] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 33;

[0488] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 34;

[0489] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 35; and

[0490] f. Light chain variable domain CDR 3 comprising SEQ ID NO:36.

[0491] Item 31. An antibody or antigen-binding fragment thereof according to Item 30, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:119.

[0492] Item 32. An antibody or antigen-binding fragment thereof according to Item 30, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:120.

[0493] Item 33. An antibody or antigen-binding fragment thereof according to Item 30, wherein the antibody or antigen-binding fragment thereof comprises both the heavy chain variable domain and the light chain variable domain as defined in Items 31 and 32.

[0494] Item 34. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0495] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 37;

[0496] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 38;

[0497] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 39;

[0498] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 40;

[0499] e. a light chain variable domain CDR 2 comprising SEQ ID NO:41; and

[0500] f. Light chain variable domain CDR 3 comprising SEQ ID NO:42.

[0501] Item 35. An antibody or antigen-binding fragment thereof according to Item 34, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:121.

[0502] Item 36. An antibody or antigen-binding fragment thereof according to Item 34, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:122.

[0503] Item 37. An antibody or antigen-binding fragment thereof according to Item 34, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 35 and 36.

[0504] Item 38. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0505] a. A heavy chain variable domain CDR 1 comprising SEQ ID NO: 43;

[0506] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 44;

[0507] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 45;

[0508] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 46;

[0509] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 47; and

[0510] f. Light chain variable domain CDR 3 comprising SEQ ID NO:48.

[0511] Item 39. An antibody or antigen-binding fragment thereof according to Item 38, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:123.

[0512] Item 40. An antibody or antigen-binding fragment thereof according to Item 38, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:124.

[0513] Item 41. An antibody or antigen-binding fragment thereof according to Item 38, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 39 and 40.

[0514] Item 42. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0515] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:49;

[0516] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 50;

[0517] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 51;

[0518] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 52;

[0519] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 53; and

[0520] f. Light chain variable domain CDR 3 comprising SEQ ID NO:54.

[0521] Item 43. An antibody or antigen-binding fragment thereof according to Item 42, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain comprising SEQ ID NO:125.

[0522] Item 44. An antibody or antigen-binding fragment thereof according to Item 42, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:126.

[0523] Item 45. An antibody or antigen-binding fragment thereof according to Item 42, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 43 and 44.

[0524] Item 46. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0525] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:55;

[0526] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 56;

[0527] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 57;

[0528] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 58;

[0529] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 59; and

[0530] f. Light chain variable domain CDR 3 comprising SEQ ID NO:60.

[0531] Item 47. An antibody or antigen-binding fragment thereof according to Item 46, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:127.

[0532] Item 48. An antibody or antigen-binding fragment thereof according to Item 46, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:128.

[0533] Item 49. An antibody or antigen-binding fragment thereof according to Item 46, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 47 and 48.

[0534] Item 50. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0535] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:61;

[0536] b. A heavy chain variable domain CDR 2 comprising SEQ ID NO: 62;

[0537] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 63;

[0538] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 64;

[0539] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 65; and

[0540] f. Light chain variable domain CDR 3 comprising SEQ ID NO:66.

[0541] Item 51. An antibody or antigen-binding fragment thereof according to Item 50, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:129.

[0542] Item 52. An antibody or antigen-binding fragment thereof according to Item 50, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:130.

[0543] Item 53. An antibody or antigen-binding fragment thereof according to Item 50, wherein the antibody or antigen-binding fragment thereof comprises both the heavy chain variable domain and the light chain variable domain as defined in Items 51 and 52.

[0544] Item 54. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0545] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 67;

[0546] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 68;

[0547] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 69;

[0548] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 70;

[0549] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 71; and

[0550] f. Light chain variable domain CDR 3 comprising SEQ ID NO:72.

[0551] Item 55. An antibody or antigen-binding fragment thereof according to Item 54, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which comprises SEQ ID NO:131.

[0552] Item 56. An antibody or antigen-binding fragment thereof according to Item 54, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:132.

[0553] Item 57. An antibody or antigen-binding fragment thereof according to Item 54, wherein the antibody or antigen-binding fragment thereof comprises both the heavy chain variable domain and the light chain variable domain as defined in Items 55 and 56.

[0554] Item 58. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0555] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:73;

[0556] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 74;

[0557] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 75;

[0558] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 76;

[0559] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 77; and

[0560] f. Light chain variable domain CDR 3 comprising SEQ ID NO:78.

[0561] Item 59. An antibody or antigen-binding fragment thereof according to Item 58, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:133.

[0562] Item 60. An antibody or antigen-binding fragment thereof according to Item 58, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:134.

[0563] Item 61. An antibody or antigen-binding fragment thereof according to Item 58, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 59 and 60.

[0564] Item 62. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0565] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:79;

[0566] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 80;

[0567] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 81;

[0568] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 82;

[0569] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 83; and

[0570] f. Light chain variable domain CDR 3 comprising SEQ ID NO:84.

[0571] Item 63. An antibody or antigen-binding fragment thereof according to Item 62, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:135.

[0572] Item 64. An antibody or antigen-binding fragment thereof according to Item 62, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:136.

[0573] Item 65. An antibody or antigen-binding fragment thereof according to Item 62, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 63 and 64.

[0574] Item 66. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0575] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 85;

[0576] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 86;

[0577] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 87;

[0578] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 88;

[0579] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 89; and

[0580] f. Light chain variable domain CDR 3 comprising SEQ ID NO:90.

[0581] Item 67. An antibody or antigen-binding fragment thereof according to Item 66, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:137.

[0582] Item 68. An antibody or antigen-binding fragment thereof according to Item 66, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:138.

[0583] Item 69. An antibody or antigen-binding fragment thereof according to Item 66, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 67 and 68.

[0584] Item 70. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0585] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:91;

[0586] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 92;

[0587] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 93;

[0588] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 94;

[0589] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 95; and

[0590] f. Light chain variable domain CDR 3 comprising SEQ ID NO:96.

[0591] Item 71. An antibody or antigen-binding fragment thereof according to Item 70, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:139.

[0592] Item 72. An antibody or antigen-binding fragment thereof according to Item 70, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:140.

[0593] Item 73. An antibody or antigen-binding fragment thereof according to Item 70, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 71 and 72.

[0594] Item 74. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0595] a. A heavy chain variable domain CDR1 comprising SEQ ID NO:97;

[0596] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 98;

[0597] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 99;

[0598] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 100;

[0599] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 101; and

[0600] f. Light chain variable domain CDR 3 comprising SEQ ID NO:102.

[0601] Item 75. An antibody or antigen-binding fragment thereof according to Item 74, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:141.

[0602] Item 76. An antibody or antigen-binding fragment thereof according to Item 74, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:142.

[0603] Item 77. An antibody or antigen-binding fragment thereof according to Item 74, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 75 and 76.

[0604] Item 78. The antibody or antigen-binding fragment thereof according to any one of Items 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises:

[0605] a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 103;

[0606] b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 104;

[0607] c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 105;

[0608] d. a light chain variable domain CDR 1 comprising SEQ ID NO: 106;

[0609] e. a light chain variable domain CDR 2 comprising SEQ ID NO: 107; and

[0610] f. Light chain variable domain CDR 3 comprising SEQ ID NO:108.

[0611] Item 79. An antibody or antigen-binding fragment thereof according to Item 78, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain, which heavy chain variable domain comprises SEQ ID NO:143.

[0612] Item 80. An antibody or antigen-binding fragment thereof according to Item 78, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable domain, which light chain variable domain comprises SEQ ID NO:144.

[0613] Item 81. An antibody or antigen-binding fragment thereof according to Item 78, wherein the antibody or antigen-binding fragment thereof comprises both a heavy chain variable domain and a light chain variable domain as defined in Items 79 and 80.

[0614] Item 82. A preparation comprising an antibody or antigen-binding fragment thereof according to any one of the preceding items, wherein the preparation is substantially free of naturally occurring antibodies that are unable to bind to Sortilin or do not substantially alter the anti-Sortilin functionality of the preparation, the functionality being selected from the group consisting of:

[0615] (i) Binding affinity for sortilin (K D );

[0616] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0617] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0618] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin; and / or

[0619] (v) The ability to provide treatment of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), or Alzheimer's disease (AD) when administered chronically.

[0620] Item 83. A formulation comprising a monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding items, wherein the monoclonal antibody has a structural alteration in its amino acid sequence relative to the structure of a naturally occurring anti-sortilin antibody, wherein the structural alteration causes the monoclonal antibody to exhibit altered functionality relative to the functionality exhibited by the naturally occurring anti-sortilin antibody, wherein the functionality is selected from the group consisting of:

[0621] (i) Binding affinity (KD) for sortilin;

[0622] (ii) reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN;

[0623] (iii) reducing and / or inhibiting the ability of cells expressing Sortilin to internalize PGRN;

[0624] (iv) the ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin;

[0625] (v) the ability to increase the amount and / or concentration of PGRN in the brain, or

[0626] (vi) The ability to provide treatment of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and / or Alzheimer's disease (AD) when administered chronically.

[0627] Item 84. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as defined in any one of Items 1 to 81, or a formulation as described in any one of Items 82-83, and a pharmaceutically acceptable carrier.

[0628] Item 85. An antibody or antigen-binding fragment thereof as defined in any one of Items 1 to 81, or a preparation as described in any one of Items 82-83, or a pharmaceutical composition as described in Item 84, for use in medicine.

[0629] Item 86. An antibody or antigen-binding fragment thereof as defined in any one of Items 1 to 81, or a preparation as described in any one of Items 82-83, or a pharmaceutical composition as described in Item 84, for use in the treatment of a disease associated with reduced PGRN levels or reduced functional PGRN in the patient's brain.

[0630] Item 87. Use of an antibody or antigen-binding fragment thereof as defined in any one of Items 1 to 81, or a preparation as described in any one of Items 82-83, or a pharmaceutical composition as described in Item 84, in the preparation of a medicament for treating a disease associated with reduced PGRN or reduced functional PGRN levels in the brain of a patient.

[0631] Item 88. A method for preventing or treating a disease associated with reduced PGRN or reduced functional PGRN levels in the brain of a patient, the method comprising administering an effective dose of an antibody or antigen-binding fragment thereof as defined in any of Items 1-81, or a preparation as described in any of Items 82-83, or a pharmaceutical composition as described in Item 84.

[0632] Item 89. An antibody or antigen-binding fragment thereof for use according to Item 86, or the use according to Item 87, or the method according to Item 88, wherein the disease is FTD; ALS; or a protein disease such as AD.

[0633] Item 90. An antibody or antigen-binding fragment thereof for use according to Item 86, or the use according to Item 87, or the method according to Item 88, wherein the treatment is long-term and preferably lasts for at least 2 weeks, such as at least for 1 month, or at least for 6 months, or at least for 1 year.

[0634] Item 91. A kit comprising an antibody or antigen-binding fragment thereof as defined in any one of Items 1-81, or a preparation as described in any one of Items 82-83, or a pharmaceutical composition as described in Item 84.

[0635] Item 92. An antibody or antigen-binding fragment thereof as defined in any of Items 1-81, which has been produced or prepared in a cell line, such as a human cell line, a non-human mammalian cell line, an insect, yeast or bacterial cell line.

[0636] Item 93. The antibody or antigen-binding fragment thereof according to Item 92, which is produced in a CHO cell line, a HEK cell line, a BHK-21 cell line, a murine cell line (such as a myeloma cell line), a fibrosarcoma cell line, a PER.C6 cell line, a HKB-11 cell line, a CAP cell line and a HuH-7 human cell line.

[0637] Preferred, non-limiting embodiments employing certain aspects of the present invention will now be described with reference to the accompanying drawings.

[0638] Example

[0639] Examples 1-3 describe the generation of sortilin constructs

[0640] Example 1 discloses shuffling constructs. Example 2 discloses expression of sortilin constructs. Example 3 discloses purification of sortilin constructs.

[0641] Examples 4-6 describe the generation of antibodies to sortilin

[0642] Example 4 discloses immunization and hybridomas. Example 5 discloses sequence analysis. Example 6 discloses purification of antibodies.

[0643] Examples 7-11 describe the characterization of sortilin antibodies

[0644] Example 7 discloses binding to Sortilin. Example 8 discloses the cross-blocking ability of Sortilin antibodies. Example 9 discloses extracellular PGRN levels in iPSCs. Example 10 discloses plasma PGRN levels.

[0645] Example 1

[0646] So-called "shuffled constructs" were designed, constructed and generated, for both use in hybridoma screening processes and as diversification of antibody panels, to prepare a set of chimeric Sortilin molecules containing amino acid sequences derived from both human Sortilin and a distantly related species (Tetraodon) with significantly reduced sequence homology. The rationale is that the overall Sortilin structure and functionality of these chimeric constructs will be retained, but the loss of antibody binding to certain chimeric constructs will indicate that specific exchange regions are involved in the binding. The soluble extracellular region (ECD, aa 1-755) constructs were labeled with either a BAP tag (biotin acceptor peptide) (enabling "in vitro" biotinylation of the protein by co-expressing a biotin ligase) or a His tag (enabling 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.

[0647] Sortilin sequences can be found in SEQ ID NOs: 147-155 and Figure 2A -C shows a schematic representation of the assignment of antibody regions based on binding to the Sortilin shuffled constructs.

[0648] Example 2

[0649] In the case of antibody expression, appropriate heavy and light chain vectors were co-expressed in HEK-293F cells as described in Examples 4, 5 and 6.

[0650] Example 3: Purification of His-tagged Sortilin

[0651] SORTECDHis is expressed in HEK-293F cells. The His-tag in the protein enables purification by immobilized metal affinity chromatography. In this method, 50mM NAH2PO4, 300mM NaCl and 10mM imidazoles (pH 8.0) are balanced by NiNTA superflow cartridge (Qiagen). The column is loaded with His-tagged protein, wherein the residence time is 1 minute. 50mM NAH2PO4, 300mM NaCl and 20mM imidazoles (pH 8.0) are washed by the column. 50mM NAH2PO4, 300mM NaCl and 250mM imidazoles (pH 8.0) are eluted by protein. Subsequently, the Slide-A-Lyzer (Thermo Scientific) that is retained as 10.000mwco by protein is dialyzed with PBS. After dialysis, the protein was sterile filtered using a 0.2 micron SFCA filter (Thermo Scientific).

[0652] Clones were characterized for Sortilin mRNA expression using qPCR. The highest expressing clones were then analyzed by FACS (Guava, Millipore) using an anti-Sortilin polyclonal antibody (Polyclonal goat Sortilin biotinylated Ab, Catalog No: BAF2934 (R&D Systems)) to determine surface expression levels of Sortilin.

[0653] Example 4

[0654] A-Immunization schedule of transgenic mice

[0655] The antibody HuMab sorting proteins were derived from immunization of the HuMAb mouse strains HCo12, HCo17, HCo20, HCo12-BALB / c, HCo17-BALB / c, and HCo20-BALB / c (human monoclonal antibodies; Medarex Inc., San Jose, CA, USA). These mice were double knocked out for mouse immunoglobulin (Ig) heavy chains and mouse kappa light chains, essentially inactivating antibody expression in a completely murine manner. The different mouse strains were made transgenic by insertion of human Ig heavy chain and human Ig kappa light chain loci and differed in the number of human VH (variable domain of the heavy chain) and VL (variable domain of the light chain) genes. HCo12-BALB / c mice were obtained by crossbreeding with KCo5-BALB / c (kappa light chain transgenic) mice.

[0656] 48 mice were immunized with 20 μg SORTECDHis (SEQ ID NO: 155) intraperitoneally (IP) and with the same protein subcutaneously (SC, at the base of the tail) alternating at 14-day intervals. A maximum of eight immunizations were performed, 4 IP and 4 SC.

[0657] In one protocol, the first immunization was performed with SORTECDHis in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, Michigan, USA), and subsequent immunizations were performed in incomplete Freund's adjuvant (IFA). The second protocol used SAS as an adjuvant in all immunization steps. When serum titers were found to be sufficient (1 / 50 or less serum dilutions were found to be positive in the antigen-specific screening assay for at least two consecutive, biweekly, screening events), mice were additionally boosted twice intravenously (IV) with 10 μg SORTECDHis protein in 100 μL PBS, four and three days prior to fusion.

[0658] B-HuMab hybridoma-generation

[0659] HuMAb mice with sufficient antigen-specific titers as defined above were sacrificed and spleen and lymph nodes from both sides of the abdominal aorta and vena cava were collected. Spleen cells and lymph node cells were fused with mouse myeloma cell lines by electrofusion using a CEEF 50 electrofusion system (Cyto Pulse Sciences, Glen Burnie, Maryland, USA) essentially according to the manufacturer's instructions. The fused cells were plated in fusion medium containing 10% Fetal Clone I 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 (IL-6) (Strathmann), 1x HAT (Sigma), and 0.5 mg / mL kanamycin (Invitrogen) in HyQ mADCF-Mab (Perbio). Ten days later, the supernatant was harvested and the cell culture medium was replaced with harvest medium containing 10% Fetal Clone I bovine serum, 0.5 U / mL penicillin, 0.5 U / mL streptomycin, 600 ng / mL IL-6 and 1 x proHT (Combustion) in HyQ mADCF-Mab. Hybridoma culture supernatants were screened by primary screening assays and streptavidin beads coupled to SORTECDBAP (SEQ ID NO 147), SORTECDBAPhB06-10 (SEQ ID NO 152), SORTECDBAPhB12390 (SEQ ID NO 153) to detect hybridomas producing human (or chimeric) anti-sortilin antibodies. Hybridoma cells from the best primary wells were inoculated in semisolid culture medium, which was made of 40% cloning culture medium (CloneMedia) (Genetix, Hampshire, UK) and 60% HyQ 2x complete culture medium (Hyclone, Waltham, USA). For each primary well, the wells of Genetix black 6-well plates were inoculated. 25 subclones were picked from each well using the ClonePix system (Genetix). Subclones were picked in harvest culture medium. After seven days, subclone supernatants were screened again for sortin-specific human IgG binding and human IgG concentration was measured using Octet 384red (Fortebio, Menlo Park, USA). The best subclones were selected from each primary well and amplified in an amplification culture medium containing only 600ng / mL IL-6, 0.5U / mL penicillin, 0.5U / mL streptomycin and 1x proHT.Subclones were expanded from one 96-well plate well to one 24-well plate well, to four 24-well plate wells, to six 6-well plate wells. The clones obtained by this method were designated as primary clones (PC).

[0660] The anti-sortilin HuMab antibodies of the invention were identified and subjected to sequence analysis.

[0661] Example 5: Sequence Analysis and Cloning of Sortilin-Specific HuMab Variable Domains into Expression Vectors

[0662] From 0.2 to 5x 10 6 Total RNA was prepared from 100 ng of total RNA using the SMARTRACE cDNA amplification kit (Clontech) according to the manufacturer's instructions. VH and VL coding regions were amplified by PCR and cloned directly in p33G1f and p33κ expression vectors (containing human IgG1. / κ constant domain coding sequences) by connecting independent clones (Aslanidis, C. and PJ de Jong, Nucleic Acids Res [Nucleic Acids Research] 1990; 18 (20): 6069-74). For each antibody, 16 VL clones and 16 VH clones were sequenced. Clones with the correct open reading frame (ORF) were selected for further study and expression. 293fectin was used to transiently co-express all combinations of heavy and light chains in FreestyleTM 293-F cells.

[0663] The resulting sequences are shown in the sequence listing herein (SEQ ID NOs: 1-144). The CDR sequences were defined according to published guidelines.

[0664] Example 6: Purification of Antibodies

[0665] The culture supernatant was filtered through a 0.2 μm dead-end filter, loaded on a 5 mL protein A column (rProtein AFF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH (pH 3). The eluate was immediately neutralized with 2 M Tris-HCl (pH 9) and dialyzed O / N (overnight) with 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). After dialysis, the sample was sterile filtered through 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 aliquoted and stored at -80 °C. Once thawed, the purified antibody aliquots were kept at 4 °C. Mass spectrometry was performed to identify the molecular weight of the antibody heavy and light chains expressed by the hybridoma.

[0666] Example 7: Affinity of Sortilin-specific HuMabs for the recombinant extracellular region of Sortilin

[0667] The binding kinetics of the anti-sortilin HuMab antibody to sortilin were determined using Octet 384RED (Fortebio, Menlo Park, USA). A 2 μg / ml HuMab solution was prepared by diluting in sample diluent (Fortebio, item number 18-5028). The Prot A sensor protein (Fortebio, item number 18-0004) was pre-wetted with kinetic buffer (1:10 sample dilution in PBS) for at least 600 seconds. Subsequently, the sensor protein was fixed with the HuMab solution for 600 seconds. A baseline response was obtained by immersion in kinetic buffer for 120 seconds. The association of the SORTECD construct was performed during the 1000 second incubation. This was followed by dissociation in kinetic buffer for 100 seconds. After dissociation, the sensor protein was regenerated (10 mM glycine pH 1.0) and neutralized (kinetic buffer) 3 times, each for 5 seconds. All HuMabs were analyzed using four concentrations of SORTECD constructs (10, 5, 2.5 and 1.25 μg / ml). A molecular weight of 76.8 kDA was used for SORTECD HiS. The data were fitted using a global full fit with ForteBio Analysis 6.4 software. The results are in Figure 3 Shown in.

[0668] Example 8: Antibody Cross-Blocking of Anti-Sortilin Human Antibody (HuMantibody)

[0669] Antibody cross-blocking studies were performed using Octet 384RED (Fortebio, Menlo Park, USA). A 2 μg / ml HuMab antibody solution was prepared by diluting in sample diluent (Fortebio, item number 18-5028). Amine-responsive sensor proteins (Fortebio, item number 18-0008) were used for the immobilization of human antibodies. Human antibodies were diluted in MES pH 6.0 buffer (18-5027) before coupling to the amine-responsive sensor proteins. Coupling was performed at 30°C and 1000 rpm as follows: the amine-responsive sensor proteins were pre-wetted in PBS and then activated with EDC / NHS (Fortebio, item number 18-1033 / 18-1034) activation solution (according to the manufacturer's instructions) for 300 seconds. The activated sensor proteins were fixed with human antibodies during 600 seconds. Due to the reactivity of the remaining amine with ethanolamine (Fortebio, catalog number 18-1039), the fixed sensor protein was quenched. After quenching, the sensor protein was placed in PBS until use. Cross-blocking analysis was started by establishing a baseline response at 30 ° C and 1000 rpm. The baseline response was obtained by immersing in the sample diluent for 120 seconds. The association of SORTECDHis was carried out during 300 seconds, followed by the association of HuMab for 300 seconds. After the association of HuMab, the sensor protein was regenerated (10mM glycine pH 1.0) and neutralized (sample diluent) 3 times, each time for 5 seconds. ForteBio analysis 6.4 software was used to process the data.

[0670] The antibodies were grouped based on their binding profiles on different sortilin shuffled constructs ( Figures 2A-2C and Figure 3 ). To confirm that all antibodies from region E bind to the same region of human wild-type sortilin ECD, their ability to block each other's binding to wild-type human sortilin ECD was characterized in a cross-blocking study using Octet384 red. For example, when antibodies from the same region are tested, the primary antibody will block the binding of the secondary antibody, and vice versa. However, when antibodies from different regions are tested, there will be no cross-blocking, because only one region is blocked by the primary antibody and the remaining region is available for the secondary antibody to bind. Figure 4 It was shown that 22 of the 26 antibodies from the E domain cross-blocked all antibodies from the panel, and the remaining 4 antibodies cross-blocked 20 of the 26 antibodies, confirming that most of these antibodies bind to the same region on Sortilin, and the rest have overlapping binding sites and / or bind around the E region. This confirms the sorting of antibodies to the E region based on the shuffled constructs. Additionally, these data also confirm that the chimeric Sortilin constructs retain similarity to the native human wild-type Sortilin ECD.

[0671] Embodiment 9:

[0672] ELISA assay for extracellular PGRN in iPSCs

[0673] Sortilin E region antibody 900 increased PGRN levels, while control antibody B12 did not affect extracellular progranulin levels. Growth factor-matured iPSC neurons were plated in 96-well plates for assay. Antibodies were added to cell culture medium. Culture medium was collected after 72 hours of exposure and analyzed by human PGRN ELISA (Biovendor) and samples were analyzed according to the manufacturer's instructions. Sortilin human antibody 900 increased PGRN levels in culture medium collected from iPSC neurons, while no effect was observed with control antibody B12 ( Figure 5 ). Culture media without antibody exposed to cells showed no signal in the PGRN ELISA. CellTiter-Glo luminescent cell viability assay (Promega) showed no effect on viability with antibody application. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA followed by Dunnett's analysis ***p<0.001.

[0674] According to the non-integrating method described by Rasmussen et al., iPSC lines were generated by reprogramming human fibroblasts sampled from apparently healthy males (18 years old) (Stem Cell Reports. 2014 September 9; 3(3): 404-13). A clone named NHDF K1_shp53 (deposited as BIONi010-A in the European Bank of induced Pluripotent Stem Cells) was used. iPSCs were cultured as a monolayer on Matrigel (BD Biosciences) in mTeSR1 medium (Stemcell Technologies). Neuronal differentiation was initiated at day 0 by dual SMAD inhibition (100 nM LDN and 10 μM SB431542) in N3 medium (50% DMEM / F12+50% Neurobasal medium supplemented with 0.5% N2, 1% B27 with RA, 0,5 mM GlutaMAX-I, 0,5% NEA, 50 μM 2-mercaptoethanol and 2.5 μg / mL insulin) of iPSCs plated at high density. Cells were split at day 12 and plated on poly-L-ornithine / laminin from now on. LDN and SB431542 were removed from day 13. From this time point on, cells were split approximately every 5 days until neuronal progenitor cells (NPCs) were frozen at day 21 to generate NPC banks. These NPCs were thawed, allowed to proliferate for approximately 4 days, then re-plated at day 25 and subjected to the final maturation medium (with 20 ng / mL BDNF, 10 ng / mL GDNF, 500 μM db-cAMP, 200 μM ascorbic acid N3). At day 32, these cells were subjected to final re-plating into maturation medium in assay plates. FACS studies confirmed surface expression of sortilin receptors on iPSC neurons from other lines generated in a similar manner. Antibodies were administered at day 56 and samples were collected at day 59.

[0675] Example 10 Effect of Antibodies on Plasma PGRN Levels in Mice

[0676] To analyze the effect of antibodies on PGRN levels in plasma, humanized sortilin KI mice were given a single injection of sortilin antibody or isotype control (10 mg / kg) by subcutaneous injection. Animals were anesthetized and sacrificed 48 hours after dosing, and plasma PGRN levels were determined by ELISA.

[0677] Mice are anesthetized intraperitoneally with 0.4ml Avertin, and heart blood is collected and transferred into 500ul kEDTA vials. Samples are kept on ice until centrifuged at 3600G for 15min at 4°C. Plasma is pipetted into micronic vials and frozen at -20°C. PGRN ELISA kit (Adipogen company) is used to measure PGRN in samples according to the manufacturer's instructions.

[0678] Mice treated with sortilin humab #30 showed increased plasma PGRN levels after 48 hours compared to an anti-HEL isotype control antibody. The results can be seen in Figure 6 middle.

[0679] Embodiment 11

[0680] Fig. 7A and 7B Shown are the effects of sortilin antibodies on extracellular PGRN in neuronally differentiated induced pluripotent stem cells (iPSCs) generated from apparently healthy individuals and from a PGRN R493X patient.

[0681] Anti-sortin human antibodies #72, #1277, #83, #900, #799, #886, #28, #471, #1286, #822, #423, #56, #381, #936 increased extracellular PGRN levels, while control antibody B12 did not increase extracellular PGRN levels. iPSC neurons matured by growth factors were plated in 96-well plates for determination. Antibodies were added to cell culture medium. According to the manufacturer's instructions, culture medium from iPSC neurons was collected after 72 hours of exposure and analyzed by human PGRN ELISA (Biovendor). Culture medium without antibodies exposed to cells showed no signal in PGRN ELISA. CellTiter-Glo luminescent cell viability assay (Promega) showed no effect on the viability (quantification of intracellular ATP levels) of antibody applications. Data are expressed as mean ± SEM of three experimental runs, and each run was repeated six times. Data were analyzed by one-way ANOVA followed by Dunnett’s analysis *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0682] The iPSC system will be reprogrammed according to the non-integration method from apparently healthy individuals (18-year-old males) and from human fibroblasts sampled from PGRN R493X patients (65-year-old males). The clones used were named BIONi010-A (apparently healthy individuals) and LUBi001-A (PGRN R493X patients). They are preserved in the European library of induced pluripotent stem cells and distributed by the European Collection of Authenticated Cell Cultures. iPSC is cultured in a monolayer on matrigel (BD Biosciences) in mTeSR1 culture medium (Stemcell Technologies) and Essential8 culture medium (Gibco company). Neuronal differentiation was initiated at day 0 by double SMAD inhibition (100 nM LDN and 10 μM SB431542) in N3 medium (50% DMEM / F12+50% Neurobasal medium supplemented with 0.5% N2, 1% B27 with RA, 0,5 mM GlutaMAX-I, 0,5% NEA, 50 μM 2-mercaptoethanol and 2.5 μg / mL insulin) of iPSCs plated at high density. Cells were split at day 12 and plated on poly-L-ornithine / laminin from now on. From day 13 onwards, LDN and SB431542 were replaced by 20 ng / ml of FGF2. From this time point onwards, cells were split approximately every 5 days until neuronal progenitor cells (NPCs) were frozen at day 21 to generate NPC banks. These NPCs were thawed, allowed to proliferate, then re-plated on day 25 and subjected to maturation medium (N3 with 20ng / mL BDNF, 10ng / mL GDNF, 500μM db-cAMP, 200μM ascorbic acid) from day 26. At day 32, these cells were finally re-plated into maturation medium in assay plates. FACS studies of BIONi010-AiPSC neurons confirmed surface expression of sortilin receptors. Antibodies were administered on day 57, and samples were collected after 72 hours (i.e., day 60).

[0683] Example 12: Epitope mapping of antibodies targeting the progranulin-sortilin interaction by hydrogen / deuterium exchange followed by mass spectrometry

[0684] In hydrogen / deuterium exchange followed by mass spectrometry (HDX-MS), the exchange rate of backbone amide hydrogens in proteins is measured. Thus, it is possible to probe the conformational dynamics of the entire protein backbone, except at proline residues. The rate of the exchange reaction is determined by the hydrogen bonding state of the backbone amides and to a lesser extent by their solvent accessibility. Subtle changes in these two parameters, caused for example by the presence of a ligand, can be observed as a function of deuterium incorporation.

[0685] To localize changes in deuterium incorporation, the protein is treated with an acid-stable protease (e.g., pepsin), which generates localized regions of typically ten to fifteen amino acids. Regions that show perturbations in the presence of ligand are either directly involved in the binding interface or are allosterically affected by the binding event.

[0686] Epitope mapping of antibodies

[0687] Deuterium incorporation of the extracellular region (SEQ ID NO: 156) of sortin was measured in the absence and presence of mAb30 (which binds to the E region). In order to ensure that the measurement is carried out under steady-state conditions, the complex is balanced at 25°C for 15min before the exchange reaction is started. The exchange reaction is started by diluting the protein sample 1:9 (v / v) into a deuterated buffer (99% D2O, 20mM tris, 150mMNaCl, pDread=7.6). After different time points (15s, 1min, 10min, 1h and 8h), the exchange reaction is quenched by diluting with ice-cold quenching buffer (2M glycine, 0.8M tris-(2-carboxyethyl) phosphine (TCEP), pH=2.3) 1:1 (v / v), thereby reducing the pH to 2.46. The quenched sample is immediately placed in a -80°C freezer and stored until analysis. Fully deuterated control samples were prepared by diluting sortilin samples 1:9 (v / v) into deuterated denaturation buffer (6 M guanidine-HCl, 99% D2O, 20 mM tris, 150 mM NaCl, pDread = 7.6) followed by incubation at 25°C for 16 h, after which they were quenched and processed as described above.

[0688] The quenched samples were thawed and injected into a cooled (0°C) reversed-phase UPLC-HDX system (Waters Inc., USA) equipped with a home-size pepsin column (60 μL internal volume, pepsin beads obtained from Thermo Scientific Inc.). Here, deuterated protein samples were subjected to online pepsin digestion at 20°C, and the resulting peptic peptides were separated by reversed-phase UPLC. Prior to final mass determination, the peptides were ionized by electrospray ionization into a mass spectrometer (Synapt G2 mass spectrometer, Waters, UK), where they were further separated by ion mobility.

[0689] Identification of peptides was performed by tandem mass spectrometry on fully reduced and non-deuterated samples using a combination of data-independent (MSe) and data-dependent acquisition.

[0690] Data analysis

[0691] Peptide identification

[0692] Acquired mass spectra were lock mass corrected for GFP and analyzed in PLGS 3.0, which matches precursor and fragment ions to a local protein database.All peptide identifications were carefully assessed manually.

[0693] Determination of deuterium incorporation: The acquired mass spectra were lock mass corrected for GFP and the software DynamX3.0 (Waters, USA) was used to determine deuterium incorporation of all peptides of sortilin in the absence or presence of antibodies.

[0694] A peptide was considered to be part of a binding epitope if protection from exchange greater than 0.5 D was observed in the presence of the antibody.

[0695] Table 1 - Table of conformational epitopes identified by HDX-MS.

[0696]

Claims

1. An antibody or antigen-binding fragment thereof that is capable of specifically binding within the E region of sortilin as defined by SEQ ID NO:

146.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of: Fv fragments (eg, single-chain Fv or disulfide-bonded Fv); Fab-like fragments (eg, Fab fragments, Fab' fragments, or F(ab)2 fragments); and domain antibodies (eg, single VH variable domains or VL variable domains).

3. The antibody according to any preceding claim, wherein the antibody consists of a whole antibody.

4. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody is selected from the group consisting of: Antibodies of the subtype IgG1, IgG2, IgG3 or IgG4.

5. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof binds to the E region of sortilin as defined by SEQ ID NO:

146.

6. An antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof binds to at least 1 or more consecutive amino acids, such as 1, 2, 3, 4, 5, 6 or 7 consecutive amino acids within the E region of sortilin as defined by SEQ ID NO:

146.

7. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment exhibits one or more of the following properties: a. A binding affinity (KD) for sortilin between 0.5 nM-10 nM, such as between 1 nM-5 nM or 1 nM-2 nM, or even higher, such as between 0.5 pM and 500 pM b. reducing and / or inhibiting the ability of cells expressing Sortilin to clear PGRN; c. reducing and / or inhibiting the ability of cells expressing sortilin to internalize PGRN; d. Ability to increase the amount and / or concentration of PGRN in the brain, and / or e. Ability to increase the amount and / or concentration of PGRN in the plasma of knock-in mice expressing human sortilin.

8. An antibody or antigen-binding fragment thereof according to any one of the preceding claims 1-7, wherein the ability of the antibody or antigen-binding fragment thereof to reduce and / or inhibit the clearance of PGRN comprises reducing and / or inhibiting the clearance of PGRN at or below 22nM, such as an IC50 between 22nM and 1nM, or between 10nM and 1nM, or between 5nM and 1nM.

9. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof is a human antibody, a humanized antibody, a recombinant antibody or a chimeric antibody.

10. The antibody or antigen-binding fragment thereof according to any preceding claim, wherein the antibody or antigen-binding fragment thereof comprises: a. A heavy chain variable domain CDR1 comprising SEQ ID NO: 1; b. a heavy chain variable domain CDR 2 comprising SEQ ID NO: 2; c. a heavy chain variable domain CDR 3 comprising SEQ ID NO: 3; d. a light chain variable domain CDR 1 comprising SEQ ID NO: 4; e. a light chain variable domain CDR 2 comprising SEQ ID NO: 5; and f. Light chain variable domain CDR 3 comprising SEQ ID NO:6.

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