CN116964089A - Methods of using anti-sortilin antibodies - Google Patents

Abstract

The present disclosure relates generally to the use of antibodies that specifically bind to one or more epitopes within sortilin (e.g., human sortilin or mammalian sortilin) for treating and/or delaying progression of a disease, disorder, or injury in an individual in need thereof.

Description

Methods of using anti-sortilin antibodies

Cross Reference to Related Applications

The application claims the benefit of U.S. provisional application No. 63/120,670 filed on 12/2/2020 and U.S. provisional application No. 63/271,658 filed on 25/10/2021, which are incorporated herein by reference in their entireties.

Submitting sequence Listing in ASCII TEXT File

The following contents of the submissions regarding ASCII TEXT file are incorporated herein by reference in their entirety: a Computer Readable Form (CRF) of the sequence listing (file name: 735022003640seqlist. Txt, date recorded: 2021, 11, 29, size: 55,902 bytes).

FIELD

The present disclosure relates to therapeutic uses of anti-sortilin antibodies.

Background

Sortilin is a type I transmembrane protein that acts both as a receptor for several ligands and for degradation in sorting of selected cargo from the Trans Golgi Network (TGN) to late endosomes and lysosomes. Sortilin binds to secreted protein granulin Precursors (PGRNs) and targets them for lysosomal degradation, thereby down-regulating extracellular levels of granulin precursors (Hu, F et al, neuron 68, 654-667). Consistent with this, the lack of sortilin significantly increased plasma granule protein precursor levels in both in vivo mouse models and in vitro human cells (Carrasquick, M.M et al, (2010) Am Jhum Genet 87,890-897; lee, W.C et al, (2014) Hum Mol Genet 23, 1467-1478). In addition, sortilin polymorphisms have been shown to correlate closely with human granulin precursor serum levels (Carrasquisllo MM et al, (2010), am J Hum Genet.10;87 (6): 890-7).

The granulin Precursors (PGRNs) encoded by the GRN genes are secreted, growth factor-like, nutritional and anti-inflammatory proteins that also function as fat factors involved in diet-induced obesity and insulin resistance (Nguyen DA et al, (2013), trends in Endocrinology and Metabolism,24, 597-606).

The deficiency of granulin precursors accounts for about 25% of all heritable forms of frontotemporal dementia (FTD), an early-onset neurodegenerative disease. Patients with heterozygous loss of function mutations in the granulin precursors have about 50% reduced extracellular protein levels, and they will always develop FTD, making granulin precursors the causative gene of the disease (Baker, M et al, (2006) Nature 442,916-919; carecchi M et al, (2011) J alzheimer's Dis 27,781-790; cruts, M et al, (2008) Trends gene 24,186-194; galimberti, d et al, (2010) J alzheimer's Dis 19, 171-177).

Granulin precursors are also associated with parkinson's disease. The GRN parkinson's disease risk allele is associated with reduced levels of granulin precursors in plasma, cerebrospinal fluid and brain. Furthermore, loss of homozygosity of the granulin precursor function causes neuronal ceroid lipofuscinosis, while loss of heterozygosity of the granulin precursor function causes frontotemporal dementia (FTD), sometimes accompanied by Parkinson's performance (Smith et al, am J Hum Genet (2012) 90 (6): 1102-1107; boeve et al, brain (2006) 129 (Pt 11): 3103-3114; and Wauters et al, neurobiol agent (2018) 67:84-94). In addition, reduced plasma granulin precursor levels have been associated with increased severity of Parkinson's disease (Yao et al, neuroscienett (2020) 725:134873). The granule protein precursor deficiency also worsens the results of rodent parkinsonism models (Martens et al, J Clin Invest (2012) 122 (11): 3955-3959), whereas the delivery of granule protein precursor genes improved those results (Van Kampen et al, PLoS One (2014) 7;9 (5): e 97032).

Furthermore, granulin precursor mutant alleles have been identified in Alzheimer's disease patients (Seelaar, H et al, (2011), journal of neurology, neurosporey, and psychiatry82, 476-486). Importantly, granulin precursors act protectively in several disease models, with increased granulin precursor levels accelerating recovery of ischemic behavior (Tao, J et al, (2012) Brain Res 1436,130-136; egashira, y et al, (2013) J Neuroinflammation 10,105), attenuating pathology in amyotrophic lateral sclerosis models (Laird, A.S et al, (2010), PLoS One 5, el 3368) and arthritis (Tang, W et al, (2011), science 332, 478-484), and preventing memory defects in alzheimer's disease models (Minami, s.s et al, (2014), nature med20, 1157-1164).

Through its diverse interactions with proteins such as granulin precursors, sortilin and various ligands thereof, has been shown to be involved in a variety of diseases, disorders and conditions such as frontotemporal dementia (FTD), amyotrophic Lateral Sclerosis (ALS), amyotrophic lateral sclerosis-frontotemporal dementia phenotypes, alzheimer's disease, parkinson's disease, depression, neuropsychiatric disorders, vascular dementia, seizures, retinal dystrophy, age-related macular degeneration, glaucoma, traumatic brain injury, aging, wound healing, stroke, arthritis and atherosclerotic vascular disease.

Novel therapeutic antibodies targeting sortilin are one solution to treat diseases associated with sortilin or granulin precursor activity. However, administration of monoclonal antibodies can present challenges for such therapeutic uses. For example, therapeutic antibodies typically have limited oral bioavailability, so they are typically administered intravenously, subcutaneously, or intramuscularly (Ovacik, M and Lin, L, (2018) Clin fransl Sci 11, 540-552). Among the available administration options, subcutaneous administration is most convenient, as it can be done at home and often by the patient, while intravenous administration delivers higher systemic exposure. Because of the high systemic doses required for delivery to the cerebrospinal fluid (CSF), intravenous administration is often employed because subcutaneous administration cannot deliver a sufficiently high dose. However, intravenous administration can be challenging for patients with neurodegenerative diseases such as alzheimer's disease, parkinson's disease, or FTD, where treatment can be administered over the course of years, otherwise patient compliance can be difficult.

Thus, there is a need for therapeutic antibodies that specifically bind sortilin to treat one or more diseases, disorders, and conditions associated with sortilin activity. In addition, there is a need to identify methods of treating patients with the correct dose and methods of administering the dose in a manner that reduces patient compliance.

All references, including patents, patent applications, and publications, cited herein are hereby incorporated by reference in their entirety.

Disclosure of Invention

In one aspect, provided herein is a method of treating or delaying progression of a disease, disorder, or injury in an individual comprising administering to the individual an anti-sortilin antibody at a dose of at least about 6mg/kg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder or injury, the method comprising administering to the individual an anti-sortilin antibody at a dose of at least about 6mg/kg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no. In some embodiments, the increase in the level of the granulin precursor comprises an increase in the level of the granulin precursor in cerebrospinal fluid of the subject, plasma of the subject, or both.

In another aspect, provided herein is a method of treating or delaying progression of a disease, disorder, or injury in an individual comprising administering to the individual an anti-sortilin antibody by subcutaneous injection at a dose of about 150mg and about 600mg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder or injury, the method comprising administering an anti-sortilin antibody to the individual by subcutaneous injection at a dose of about 150mg and about 600mg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In some embodiments that may be combined with any of the preceding embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion or by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of at least about 6mg/kg, at least about 15mg/kg, at least about 30mg/kg, or at least about 60 mg/kg. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of between about 6mg/kg and about 30 mg/kg. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 6mg/kg, about 15mg/kg, about 30mg/kg, or about 60 mg/kg.

In some embodiments, the method comprises: (a) Administering an anti-sortilin antibody by intravenous infusion at an initial dose of about 60mg/kg, followed by one or more lower doses of anti-sortilin antibody of between about 6mg/kg and about 59mg/kg, or between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg; (b) Administering an anti-sortilin antibody by intravenous infusion at an initial dose of about 30mg/kg, followed by one or more lower doses of the anti-sortilin antibody of between about 6mg/kg and about 29mg/kg, or between about 6mg/kg and about 15 mg/kg; (c) Administering an anti-sortilin antibody at an initial dose of about 15mg/kg by intravenous infusion followed by one or more lower doses of the anti-sortilin antibody between about 6mg/kg and about 14 mg/kg; (d) Administering an anti-sortilin antibody by intravenous infusion at an initial dose of about 6mg/kg, followed by one or more higher doses of anti-sortilin antibody between about 7mg/kg and about 30mg/kg, between about 15mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg; (e) Administering an anti-sortilin antibody by intravenous infusion at an initial dose of about 15mg/kg, followed by one or more higher doses of the anti-sortilin antibody of between about 16mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg; or (f) administering the anti-sortilin antibody by intravenous infusion at an initial dose of about 30mg/kg, followed by one or more higher doses of the anti-sortilin antibody of between about 31mg/kg and about 60 mg/kg.

In some embodiments, the method comprises administering the anti-sortilin antibody about once every four weeks or less frequently. In some embodiments, the method comprises administering the anti-sortilin antibody about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 15mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 15mg/kg about once every six weeks or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 30mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 30mg/kg about once every six weeks or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 60mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody by intravenous infusion at a dose of about 60mg/kg about once every six weeks or about once every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of at least about 270mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of about 150mg and about 600mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of any one of about 150mg, about 270mg, about 300mg, or about 600mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of about 150mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of about 270mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of about 300mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody at a dose of about 600mg by subcutaneous injection. In some embodiments, the method comprises administering the anti-sortilin antibody by subcutaneous injection at any frequency of about every two weeks, about every four weeks, about every six weeks, or about every eight weeks. In some embodiments, the method comprises administering the anti-sortilin antibody about once every four weeks by subcutaneous injection. In some embodiments that can be combined with any of the preceding embodiments, the anti-sortilin antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the anti-sortilin antibody has a human IgG1 isotype. In some embodiments, the anti-sortilin antibody comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering. In some embodiments, an anti-sortilin antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:25 or 26. In some embodiments, the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32. In some embodiments, the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:32, and a heavy chain of an amino acid sequence of seq id no. In some embodiments, the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO: 31.

In some embodiments, in some embodiments of their combination, the method further comprises measuring the level of granulin precursor in a blood or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, the method further comprises measuring the level of sortilin in leukocytes in a blood sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, the method further comprises measuring the level of neurofilament light chain (NF-L), tau, one or more biomarkers of neuroinflammation, one or more biomarkers of complement function, and/or one or more biomarkers of microglial activity in a blood or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, (a) the one or more biomarkers of neuroinflammation is selected from IL-6, SPP1, IFI2712A, CHIT1, YKL-40, GFAP, YWHAE, CSF1, AIF1, LY86, CD86, and TOP2A; (b) The one or more inflammatory biomarkers are selected from osteopontin (SPP 1), YWHAE (14-3-3 protein epsilon), allograft inflammatory factor 1 (AIF 1), colony stimulating factor 1 (CSF 1), chitinase 1 (CHIT 1), lymphocyte antigen 86 (LY 86), and CD86; (c) One or more biomarkers of complement function selected from the group consisting of C1qb and C1qc; and/or (d) the biomarker of microglial activity(s) is (are) selected from the group consisting of YKL-40, GFAP and interleukin-6.

In some embodiments that may be combined with any of the preceding embodiments, administering to the subject a dose of 6mg/kg or 15mg/kg of the anti-sortilin antibody by intravenous infusion results in an increase in the level of the granulin precursor in the plasma of the subject of at least about 1.4-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, or at least about 3-fold as compared to the level of the granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the plasma of the subject occurs within about 1 day after administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 6mg/kg, and an increase in the level of progranulin in the plasma of the subject occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 17 days, about 24 days, about 29 days, or about 42 days after administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 15mg/kg, and an increase in the level of progranulin in the plasma of the subject is present about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 17 days, about 24 days, about 29 days, about 42 days, or about 56 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of 6mg/kg, 15mg/kg, 30mg/kg, or 60mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of the granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% as compared to the level of the granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 6mg/kg, and an increase in the level of granulin precursor in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, or about 28 days after administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 15mg/kg, and an increase in the level of progranulin in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, or about 42 days after administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 30mg/kg, and an increase in the level of progranulin in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody. In some embodiments, the dose of the anti-sortilin antibody is 60mg/kg, and an increase in the level of progranulin in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 150mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the subject's plasma by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of the granulin precursor in the subject's plasma prior to administration of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 300mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the subject's plasma by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of the granulin precursor in the subject's plasma prior to administration of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 600mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of the granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, or about 28 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of 6mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of the granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% as compared to the level of the granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 29 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of 15mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 29 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of 30mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 29 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of 60mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the cerebrospinal fluid of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 150mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 300mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, administering a dose of about 600mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of the granulin precursor in the cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of the granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor in the cerebrospinal fluid of the subject occurs about 1 day, about 7 days, about 14 days, or about 21 days after administration of the anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, the disease, disorder, or injury is selected from frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic Lateral Sclerosis (ALS), traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, or osteoarthritis. In some embodiments, the individual is at risk of a disease, disorder, or injury. In some embodiments, the disease, disorder, or injury is parkinson's disease. In some embodiments, the individual is at risk for parkinson's disease. In some embodiments, the disease, disorder, or injury is sporadic parkinson's disease. In some embodiments, the individual has at least one pathogenic mutation in the GBA1 gene. In some embodiments, the individual is homozygous or heterozygous for at least one pathogenic mutation in the GBA1 gene. In some embodiments, the at least one pathogenic mutation in the GBA1 gene is selected from the group consisting of c.1226a > G, c.1448t > C, IVS2+1g > a, recNcil, 84insGG, and any combination thereof; or wherein the at least one pathogenic mutation in the GBA1 gene is a mutation in the GBA1 gene that results in an amino acid substitution in the GBA1 gene product selected from the group consisting of N370S, L444P, R W, H255Q, D409H, E326K, T369M, R496H and any combination thereof. In some embodiments, parkinson's disease is classified as between stage I and stage III based on Hoehn and Yahr criteria. In some embodiments, the individual is receiving one or more treatments for parkinson's disease prior to administration of the anti-sortilin antibody. In some embodiments, the individual continues to receive one or more treatments for parkinson's disease after starting treatment with the anti-sortilin antibody. In some embodiments, the methods comprise administering an anti-sortilin antibody in combination with one or more treatments for parkinson's disease. In some embodiments, the one or more parkinson's disease treatments are selected from glutamate antagonists, anticholinergic agents, dopamine agonists, levodopa (L-DOPA and decarboxylase [ DDC ] inhibitors), monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, beta blockers, selective serotonin uptake inhibitors (SSRI), tricyclic antidepressants (TCA) or indomethacin. In some embodiments, the method further comprises measuring the level of granulin precursors, GCase and/or a-synuclein in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, the method further comprises measuring the level of the biomarker of one or more lysosomal functions in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, the one or more biomarkers of lysosomal function are selected from the group consisting of GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD). In some embodiments, the method further comprises assessing cognitive function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, the cognitive function is assessed using montreal cognitive assessment (MoCA). In some embodiments, the method further comprises assessing the motor function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, motor function is assessed using a motion disorder institute (MDS) sponsored revision Unified Parkinson's Disease Rating Scale (UPDRS) third part (MDS-UPDRS third part) or UPDRS total table. In some embodiments, the method comprises administering an anti-sortilin antibody to an individual for at least about 6 months or at least about 24 weeks.

In some embodiments, the disease, disorder, or injury is frontotemporal dementia (FTD). In some embodiments, the individual: (a) Heterozygous for one or more mutations in the GRN gene, optionally wherein the one or more mutations are loss-of-function mutations; (b) heterozygous for a C9orf72 hexanucleotide repeat expansion; (c) FTD-symptomatic, FTD-symptomatic or pre-symptomatic FTD; and/or (d) has FTD-GRN, or FTD caused by one or more mutations in the GRN gene. In some embodiments, (a) the individual has a pre-symptomatic FTD and: (i) Elevated levels of one or more biomarkers selected from Nfl, SPP1, ywae, AIF1, CSF1, CHIT1, and LY86, and/or (ii) reduced levels of one or more biomarkers selected from NAGK and CTSB; or (b) the individual is free of FTD symptoms and: (i) Heterozygous for one or more mutations in the GRN gene, and/or (ii) has reduced PGRN levels or functions

In some embodiments, the disease, disorder, or injury is alzheimer's disease.

In some embodiments, the disease, disorder, or injury is ALS.

In some embodiments that may be combined with any of the preceding embodiments, the method further comprises measuring the level of granulin precursors, GCase and/or a-synuclein in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, the method further comprises measuring the level of the biomarker of one or more lysosomal functions in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments, the one or more biomarkers of lysosomal function are selected from the group consisting of GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD). In some embodiments that may be combined with any of the preceding embodiments, the method further comprises assessing cognitive function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody. In some embodiments that may be combined with any of the preceding embodiments, the individual is a human.

In another aspect, provided herein is a method of monitoring the treatment of an individual administered an anti-sortilin antibody, comprising measuring the level of one or more biomarkers, wherein the one or more biomarkers are selected from the group consisting of a granule protein precursor, GCase, neurofilament light chain (NF-L), tau, a marker of one or more neuroinflammatory disorders, one or more inflammatory biomarkers, a biomarker of one or more complement functions, one or more biomarkers of microglial activity, or alpha-synuclein, wherein the level of the one or more biomarkers is measured in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of an anti-sortilin antibody.

In another aspect, provided herein is a method of monitoring treatment of an individual administered an anti-sortilin antibody, comprising measuring the level of a biomarker of one or more lysosomal functions in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of an anti-sortilin antibody.

In some embodiments that may be combined with any of the preceding embodiments, the biomarker of lysosomal function(s) is (are) selected from the group consisting of GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD). In some embodiments, the method further comprises assessing the activity of the anti-sortilin antibody in the individual based on the level of the one or more biomarkers in the sample.

In some embodiments that may be combined with any of the preceding embodiments, (a) the one or more markers of neuroinflammation is selected from IL-6, SPP1, IFI2712A, CHIT1, YKL-40, GFAP, YWHAE, CSF1, AIF1, LY86, CD86, and TOP2A; (b) The one or more inflammatory biomarkers are selected from osteopontin (SPP 1), YWHAE (14-3-3 protein epsilon), allograft inflammatory factor 1 (AIF 1), colony stimulating factor 1 (CSF 1), chitinase 1 (CHIT 1), lymphocyte antigen 86 (LY 86), and CD86; (c) One or more biomarkers of complement function selected from the group consisting of C1qb and C1qc; and/or (d) one or more biomarkers of microglial activity selected from the group consisting of YKL-40, GFAP and interleukin-6.

In another aspect, provided herein is an antibody that binds sortilin, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, and wherein the antibody has a human IgG1 isotype and comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

In another aspect, provided herein is an antibody that binds sortilin, wherein the antibody comprises an amino acid sequence comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

In another aspect, provided herein is an anti-sortilin antibody for use in a method of treating or delaying progression of a disease, disorder, or injury in an individual at a dose of at least about 6mg/kg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is an anti-sortilin antibody for use in a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder, or injury, at a dose of at least about 6mg/kg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is an antibody that binds sortilin, for use in a method of treating or delaying progression of a disease, disorder, or injury in an individual, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, and wherein the antibody has a human IgG1 isotype and comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

In another aspect, provided herein is an antibody that binds sortilin for use in a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder, or injury, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, and wherein the antibody has a human IgG1 isotype and comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

In another aspect, provided herein is an antibody that binds sortilin for use in a method of treating or delaying progression of a disease, disorder, or injury in an individual, wherein the antibody comprises an amino acid sequence comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

In another aspect, provided herein is an antibody that binds sortilin for use in a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder, or injury, wherein the antibody comprises an amino acid sequence comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

In another aspect, provided herein is a use of an anti-sortilin antibody at a dose of at least about 6mg/kg in the manufacture of a medicament for treating or delaying progression of a disease, disorder, or injury in an individual, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is a use of an anti-sortilin antibody at a dose of at least about 6mg/kg in the manufacture of a medicament for increasing the level of a granulin precursor in an individual suffering from a disease, disorder, or injury, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In another aspect, provided herein is a method of treating a disease, disorder, or injury or delaying progression in an individual comprising administering to the individual an antibody that binds sortilin, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, and wherein the antibody has a human IgG1 isotype and comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

In another aspect, provided herein is a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder or injury, comprising administering to the individual an antibody that binds sortilin, wherein the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:21, and wherein the antibody has a human IgG1 isotype and comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

In another aspect, provided herein is a method of treating a disease, disorder, or injury or delaying progression in an individual comprising administering to the individual an antibody that binds sortilin, wherein the antibody comprises an amino acid sequence comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

In another aspect, provided herein is a method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder or injury, wherein the method comprises administering to the individual an antibody that binds sortilin, wherein the antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

Drawings

FIG. 1 is a design of the study described in example 1. CSF = cerebrospinal fluid; d = study day; IV = intravenous; sc=subcutaneous. Study days for acquisition of CSF samples during the study period

Figures 2A-2C show the plasma levels of granulin precursors in subjects in cohort 1 of the study described in examples 1-2. FIG. 2A provides the granulin precursor levels in plasma at the indicated times (initial dose, EOI, 4HR, 8HR, 12 HR) on day 1 of the study for each of subjects 1-8 in cohort 1 with anti-sortilin antibody ALX administered at a dose of 6 mg/kg. The y-axis is marked as 100, 150 and 200ng/ml. Figure 2B provides the granulin precursor levels in plasma at the indicated times (initial dose, EOI, 4HR, 8HR, 12 HR) on study day 1 for each of subjects 1-3 who had a placebo administered in cohort 1. The y-axis is marked as 100, 150 and 200ng/ml. Figure 2C shows the average level of granulin precursor in plasma of cohort 1 subjects administered placebo or antibody ALX on the day of study.

Figures 3A-3C show the plasma levels of granulin precursors in subjects in cohort 2 of the study described in examples 1-2. FIG. 3A provides plasma granulin precursor levels for each of subjects 1-6 in cohort 2 administered with an anti-sortilin antibody ALX at a 15mg/kg dose at the indicated time point on day 1 of the study. Fig. 3B provides plasma granulin precursor levels for each of subjects 1-2 in cohort 2 administered placebo at the indicated time point on day 1 of the study. Figure 3C shows the average level of granulin precursor in plasma of cohort 2 subjects administered placebo or antibody ALX on the day of study.

Figure 4 shows the average level of granulin precursor in plasma for the indicated days of the study for subjects administered anti-sortilin antibody ALX or placebo protein antibody ALX or placebo.

Figures 5A-5D provide plasma levels of granulin precursors in subjects in cohorts 1 and 2 of the study described in examples 1-2. Figure 5A shows the plasma granulin precursor levels of each subject administered placebo in study cohorts 1 and 2 at the indicated days after placebo administration. Each line represents an object. Fig. 5B shows the granulin precursor levels in plasma for each subject administered antibody ALX (6 mg/kg) in cohort 1 for the indicated days after administration of the antibody. Each line represents an object. Fig. 5C shows the granulin precursor levels in plasma for each subject administered antibody ALX (15 mg/kg) in cohort 2 for the indicated days after administration of the antibody. Each line represents an object. Figure 5D shows the average levels of granulin precursors in plasma for subjects administered antibody ALX or placebo in cohorts 1 and 2 for the indicated days after administration of antibody or placebo. All subjects in cohorts 1 and 2 administered placebo were pooled ("pooled placebo"). SD = single dose; IV = intravenous.

Figure 6 shows the change in the levels of granulin precursor in CSF of subjects in cohorts 1 and 2 of the study described in example 1-2. The granulin precursor levels in CSF are shown as the average percent change on the day of investigation as compared to the baseline CSF granulin precursor levels. The figure shows the number of subjects included in the analysis of granulin precursor CSF levels per cohort per study day.

FIGS. 7A-7B provide average concentrations (ng/ml) of anti-sortilin antibody ALX in cynomolgus monkey serum, as described in example 4, administered intravenously once a week for four weeks at doses of 20mg/kg, 60mg/kg and 200mg/kg. Figure 7A shows the average concentration of anti-sortilin antibody ALX in serum at the indicated times (hours) following administration of each dose of antibody on study day 1, day 8 and day 15. Figure 7B shows the average concentration of anti-sortilin antibody ALX in serum at the indicated time (hours) following administration of each dose of antibody on study day 22 and day 29.

Figures 8A-8C show the levels of sortilin expression in intravenous White Blood Cells (WBCs), serum granulin precursors, and CSF granulin precursors in cynomolgus monkeys, as described in example 4, with antibody ALX administered intravenously at doses of 0mg/kg (control), 20mg/kg, 60mg/kg, and 200mg/kg once a week for four weeks. Fig. 8A shows sortilin (SORT 1) expression levels in leukocytes as a percentage of baseline levels at designated times after administration of the first dose of antibody ALX. Fig. 8B shows the percent of granulin precursor levels in serum as baseline levels at the indicated times after administration of the first dose of antibody ALX. Fig. 8C shows the granulin precursor level in CSF as a percentage of baseline level at a specified time after administration of the first dose of antibody ALX. In fig. 8A-8C, the arrows indicate the administration time of the antibody ALX dose.

Figure 9 is a participant treatment graph of the group entry population at various time points during the study. IV = intravenous; SD = single dose; sc=subcutaneous. ^a Subject withdrawal (n=2); ^b the subject exits; ^c a physician decision; ^d physician decision (n=1) and subject withdrawal (n=3); ^e adverse events.

FIGS. 10A-10B show the average concentration (ng/ml) of anti-sortilin antibody ALX in serum and CSF of subjects in a cohort with single intravenous administration of antibody ALX at a dose of 6mg/kg (cohort 1), 15mg/kg (cohort 2), 30mg/kg (cohort 3), 60mg/kg (cohort 4), or single subcutaneous administration of ALX antibody at a dose of 600mg (SDSC cohort), as described in example 5. Fig. 10A shows serum concentrations of ALX at the indicated times (days) following administration of single intravenous or subcutaneous doses of ALX. Fig. 10B shows the average CSF concentration of ALX at a specified time (day) after administration of a single intravenous or subcutaneous dose of ALX.

FIGS. 11A-11B show the mean percent change in plasma and CSF concentrations of granulin precursors of single intravenous administration of antibody ALX at a dose of 6mg/kg (cohort 1), 15mg/kg (cohort 2), 30mg/kg (cohort 3), 60mg/kg (cohort 4), or single subcutaneous administration of ALX antibody at a dose of 600mg (SDSC cohort) as described in example 5. Figure 11A shows the mean percent change from baseline in plasma concentration of granulin precursor at the indicated time (day) after administration of a single dose of antibody ALX. Figure 11B shows the mean percent change in CSF concentration of granulin precursor at a given time (day) after administration of a single dose of antibody ALX relative to baseline.

Figure 12 shows a design of a single dose subcutaneous cohort. A single subcutaneous dose of 150mg antibody ALX was administered to the subject. As indicated by the arrow, a single dose was administered on day 1. Study days for obtaining CSF samples are represented as squares. LP = lumbar puncture; BL = baseline; sc=subcutaneous; EOS = end of study.

Figure 13 shows a design of a multi-dose subcutaneous array. Seven doses of 300mg antibody ALX were administered to subjects (n=10) on days 1, 15, 29, 43, 57, 71 and 85, as indicated by the arrows. Study days for obtaining CSF samples are represented as squares. LP = lumbar puncture; BL = baseline; sc=subcutaneous; HV = healthy volunteer; EOS = end of study.

Fig. 14 shows a design of a multi-dose venous line. 4 doses of 30mg/kg antibody ALX or placebo (antibody ALX: placebo was administered to subjects (n=10) on days 1, 29, 57 and 85 as indicated by the arrow.) were administered at a ratio of 8:2. Study days to obtain CSF samples were represented as squares.lp = lumbar puncture, BL = baseline, IV = intravenous, HV = healthy volunteer, EOS = end of study.

Fig. 15 shows a design of the study described in example 1. CSF = cerebrospinal fluid; DLAE = dose limiting adverse event; IV = intravenous; PD = pharmacodynamics; PK = pharmacokinetics; sc=subcutaneous; SD = single dose. ^a CSF sampling after administration at 2 time points on day 25, 43 or 57; ^b CSF sampling after administration at 2 time points on days 25, 43, 57 or 85; ^c CSF sampling after dosing at 2 time points on day 43, 57 or 85; ^d CSF sampling 1 day after administration at 2 time points on day 25, 43, 57 or 85; ^e follow-up for 12 weeks in 6-mg/kg, 15-mg/kg and 30-mg/kg dose groups; ^f subjects in the 60-mg/kg dose group were followed for 16 weeks; ^g final study visit of 6-mg/kg, 15-mg/kg and 30-mg/kg dose group subjects; ^h final study visit of subjects in the 60-mg/kg dose group; ⁱ post-dose CSF sampling was performed at 2 time points on day 8, day 13, day 18, day 25, or day 43.

Detailed Description

Definition of the definition

As used herein, the term "preventing" includes providing prophylaxis with respect to the occurrence or recurrence of a particular disease, disorder, or condition in an individual. An individual may be susceptible to or susceptible to, or at risk of developing, a particular disease, disorder, or condition, but has not yet been diagnosed with such a disease, disorder, or condition.

As used herein, an individual "at risk of developing a particular disease, disorder, or condition" may or may not have a detectable disease or disease symptom, and may or may not exhibit a detectable disease or disease symptom prior to the methods of treatment described herein. "at risk" means that an individual has one or more risk factors that are measurable parameters associated with the development of a particular disease, disorder, or condition, as known in the art. Individuals with one or more of these risk factors have a higher likelihood of developing a particular disease, disorder, or condition than individuals without one or more of these risk factors.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include reducing the rate of progression, delaying progression, improving or alleviating a pathological state, and alleviating or improving the prognosis of a particular disease, disorder or condition. For example, an individual is successfully "treated" if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated.

An "effective amount" refers to an amount effective to at least achieve the desired therapeutic or prophylactic result over the necessary dosage and period of time. An effective amount may be provided in one or more administrations. The effective amount herein may vary depending on factors such as the disease state, age, sex and weight of the individual, the ability of the treatment to elicit a desired response in the individual, and the like. An effective amount is also an amount of any toxic or detrimental effect of the treatment that is beyond the therapeutic benefit. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening of severity or delaying the onset of a disease, including biochemical, histological and/or behavioral symptoms of the disease, complications thereof, and intermediate pathological phenotypes exhibited during disease progression. For therapeutic use, beneficial or desired results include clinical results, such as reducing one or more symptoms caused by a disease, increasing the quality of life of a patient suffering from a disease, reducing the dosage of other drugs required to treat a disease, e.g., by targeting an effect of enhancing another drug, delaying the progression of a disease, and/or prolonging survival. An effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and if combined with one or more other agents, a single agent may be considered to be administered in an effective amount, if the desired result may be obtained or achieved.

As used herein, administration "in combination" or "in combination" with another compound or composition includes administration simultaneously and/or at different times. The combined or combined administration also includes administration as a co-formulation or as separate compositions, including at different administration frequencies or intervals, and using the same route of administration or different routes of administration.

For the purposes of treatment, prevention or risk reduction, "individual" refers to any animal classified as a mammal, including humans, domestic animals and farm animals, as well as zoo, sports or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual is a human.

Unless otherwise indicated, the terms "sortilin protein" and "sortilin polypeptide" are used interchangeably herein to refer to any native sortilin from any mammalian source, including primates (e.g., humans and cynomolgus monkeys) and rodents (e.g., mice and rats). In some embodiments, the term includes wild-type sequences and naturally occurring variant sequences, such as splice variants or allelic variants. In some embodiments, the term includes "full length," unprocessed sortilin, as well as any form of sortilin produced by processing in a cell. In some embodiments, the sortilin is human sortilin. In some embodiments, the amino acid sequence of an exemplary human sortilin is SEQ ID NO:1.

The terms "anti-sortilin antibody", "sortilin-binding antibody", and "sortilin-specific antibody" refer to an antibody that is capable of binding sortilin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent for targeting sortilin. In one embodiment, the extent of binding of an anti-sortilin antibody to an unrelated, non-sortilin polypeptide is less than about 10% of the binding of the antibody to sortilin, as measured, for example, by a Radioimmunoassay (RIA), a biological layer interferometry assay (e.g., using a ForteBio system), or a Surface Plasmon Resonance (SPR) assay, see, e.g., heart et al, methods Mol Biol (2012) 907:411-42; vishal and Rafique, analytical Biochemistiy (2017) 536, pp.16-31; estep et al, MAbs (2013) 5 (2): 270-8; and Friget et al Analytical Biochemistry (1993) 210 (2): 344-350. In certain embodiments, antibodies that bind sortilin have<1μM，<100nM，<10nM，<1nM，<0.1nM，<0.01nM or<0.001nM (e.g. 10 ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10 ^-9 M to 10 ^-13 M) dissociation constant (KD). In certain embodiments, the anti-sortilin antibody binds to a sortilin epitope that is conserved among sortilins of different species.

The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody". The term "antibody" is used herein in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), including those formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

"Natural antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light ("L") chains and two identical chainsHeavy ("H") chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds in the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain at one end (V _H ) Followed by a plurality of constant domains. Each light chain has a variable domain at one end (V _L ) And has a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable regions.

See, e.g., basic and Clinical Immunology, 8 th edition, daniel p. Sites, abba i.terr and Tristram g.Parslow editions, appleton & Lange, norwalk, CT,1994, page 71, chapter 6 for the structure and properties of different classes of antibodies.

Based on the amino acid sequence of its constant domain, light chains from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda. Immunoglobulins can be assigned to different classes or isotypes based on the amino acid sequence of the heavy chain constant region (CH). There are five classes of immunoglobulins: igA, igD, igE, igG and IgM having heavy chains named α, δ, ε, γ and μ, respectively. The sums are further divided into subclasses (isoforms) based on relatively small differences in CH sequence and function, e.g., humans express the following subclasses: igG1, igG2, igG3, igG4, igA1, and IgA2. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, cellular and Molecular Immunology, 4 th edition (w.b. samundrs co., 2000).

The "variable region" or "variable domain" of an antibody (e.g., an anti-sortilin antibody of the disclosure) refers to the amino terminal domain of the heavy or light chain of the antibody. The variable regions of the heavy and light chains, respectively, may be referred to as "V _H "and" V _L ". These domains are typically the most variable parts of an antibody (relative to other antibodies of the same class) and contain antigen binding sites.

The term "variable" refers to the fact that certain segments of the variable domain differ widely in sequence between antibodies (e.g., anti-sortilin antibodies of the disclosure). The variable region mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the span of the variable domains. Instead, it concentrates in three segments of the light and heavy chain variable regions known as hypervariable regions (HVRs). The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions, principally in a β -sheet configuration, connected by three HVRs, which form loops connecting the β -sheet structure, and in some cases form part of the β -sheet structure. The HVRs in each chain are bound together in close proximity by the FR regions and together with the HVRs from the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth edition, national Institute of Health, bethesda, MD (1991)). The constant regions are not directly involved in binding of antibodies to antigens, but exhibit various effector functions, such as antibodies involved in antibody-dependent cytotoxicity.

An "isolated" antibody, such as an anti-sortilin antibody of the disclosure, is an antibody that has been identified, isolated, and/or recovered from a component (e.g., natural or recombinant) of its production environment. Preferably, the isolated polypeptide does not bind to all other contaminant components from its production environment. Contaminant components from their production environment, such as those produced by recombinant transfected cells, are substances that typically interfere with the research, diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide will be purified: (1) To greater than 95 wt% of antibodies, as determined by, for example, the Lowry method, and in some embodiments, to greater than 99 wt%; (2) A degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotary cup sequencer, or (3) uniformity by SDS-PAGE under non-reducing or reducing conditions using coomassie blue or preferably silver staining. Isolated antibodies include in situ antibodies within recombinant T cells, as at least one component of the natural environment of the antibody will not be present. However, the isolated polypeptide or antibody is typically prepared by at least one purification step.

The term "monoclonal antibody" as used herein refers to an antibody, such as a monoclonal anti-sortilin antibody of the disclosure, obtained from a substantially homogeneous population of antibodies, i.e., except possibly naturally occurring mutations and/or post-translational modification (e.g., isomerization, amidation, etc.) modifications (e.g., isomerization, amidation, etc.) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized from hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" refers to the characteristics of the antibody as obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by a variety of techniques including, but not limited to, one or more of the following methods, methods of immunizing animals with one or more DNA, virus-like particles, polypeptides and/or cells, including, but not limited to, rats, mice, rabbits, guinea pigs, hamsters and/or chickens, hybridoma methods, B cell cloning methods, recombinant DNA methods, and techniques for producing human or human-like antibodies in animals having a portion or all of a human immunoglobulin locus or a gene encoding a human immunoglobulin sequence.

The terms "full length antibody", "whole antibody" or "whole antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment, e.g., an anti-sortilin antibody of the disclosure. In particular, intact antibodies include those having heavy and light chains including an Fc region. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

"antibody fragments"comprises a portion of an intact antibody, preferably the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, fab ', F (ab') ₂ And Fv fragments; a dimeric antibody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng.8 (10): 1057-1062 (1995)); single chain antibody molecules and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies (e.g., anti-sortilin antibodies of the present disclosure) produces two identical antigen-binding fragments (referred to as "Fab" fragments) and a remaining "Fc" fragment (a name reflecting the ability to crystallize readily). Fab fragments consist of the complete L chain and the variable region domain of the H chain (V _H ) And a first constant domain of a heavy chain (C _H 1) Composition is prepared. Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of antibodies produced single large F (ab') ₂ Fragments, which correspond approximately to two disulfide-linked Fab fragments with different antigen binding activities and are still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments in that at C _H The carboxy terminus of the 1 domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab '-SH is referred to herein as Fab', in which the cysteine residues of the constant domain bear free thiol groups. F (ab') ₂ Antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of two H chains bound together by a disulfide. The effector function of antibodies is determined by sequences in the Fc region, which is also recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. Folding of these two domains produces 6 hypervariable loops (3 loops in each of the H and L chains) that provide amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with less affinity than the entire binding site.

"Single chain Fv" also abbreviated "sFv" or "scFv" is an antibody fragment comprising VH and VL antibody domains linked in a single polypeptide chain. Preferably, the sFv polypeptide further comprises V _H And V _L Polypeptide linkers between domains that enable sFv to form the structures required for antigen binding. For reviews of sFvs, see Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, edited by Rosenburg and Moore, springer-Verlag, new York, pp.269-315.

A "functional fragment" of an antibody (e.g., an anti-sortilin antibody of the disclosure) comprises a portion of an intact antibody, typically including the antigen-binding or variable region of the intact antibody or the F region of an antibody that retains or has modified FcR binding capacity. Examples of antibody fragments include linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term "diabody" refers to a polypeptide which is produced by the construction of a polypeptide in V _H And V _L An sFv fragment (see the preceding paragraph) with a short linker (about 5-10 residues) between domains, thereby enabling interchain pairing of the variable domains instead of intrachain pairing, thereby generating a bivalent fragment, i.e., a small antibody fragment prepared from a fragment having two antigen binding sites. Bispecific dimeric antibodies are heterodimers of two "cross" sFv fragments, wherein V of both antibodies _H And V _L The domains are present on different polypeptide chains.

As used herein, "chimeric antibody" refers to an antibody (immunoglobulin), such as a chimeric anti-sortilin antibody of the present disclosure, in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibody packages of interest hereinScraperAn antibody, wherein the antigen binding region of the antibody is derived from, for example, an antibody produced by immunization of cynomolgus monkeys with an antigen of interest. As used herein, a "humanized antibody" is a subset of a "chimeric antibody".

A "humanized" form of a non-human (e.g., murine) antibody, such as the humanized form of an anti-sortilin antibody of the disclosure, is a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In certain embodiments, the humanized antibody will comprise substantially all of at least one and typically two variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody, e.g., an anti-sortilin antibody of the disclosure, produced by a human and/or prepared using any of the techniques disclosed herein for preparing a human antibody. The definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries and yeast-based platform techniques. Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled, such as immunized xenogeneic mice and by human B cell hybridoma technology.

As used herein, the term "hypervariable region," "HVR," or "HV" refers to a region of an antibody variable domain, such as a region of an antibody variable domain of an anti-sortilin antibody of the disclosure, which is hypervariable in sequence and/or forms a structurally defined loop. Typically, an antibody comprises six HVRs; v (V) _H Three of them (H1, H2, H3), V _L Three of (L1, L2),L3). Of the natural antibodies, H3 and L3 show the greatest diversity of 6 HVRs, particularly H3 is thought to play a unique role in conferring fine specificity to antibodies. Naturally occurring camelid antibodies consisting of heavy chains only are functional and stable in the absence of light chains.

Many HVRs are depicted in use and included herein. In some embodiments, the HVR may be a Kabat Complementarity Determining Region (CDR) based on sequence variability and is most commonly used (Kabat et al, supra). In some embodiments, the HVR may be a Chothia CDR. Chothia refers to the position of the structural loop (Chothia and Lesk J. Mol Biol 196:901-917 (1987)). In some embodiments, the HVR may be an AbM HVR. AbM HVR represents a tradeoff between Kabat CDR and Chothia structural loops and is used by Oxford Molecular AbM antibody modeling software. In some embodiments, the HVR may be a "contact" HVR. The "contact" HVR is based on analysis of the complex crystal structure available. Residues of each of these HVRs are shown below.

The HVR may comprise the following "extended HVR": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102 or 95-102 (H3) in VH. For each of these extended-HVR definitions, the variable domain residues are numbered according to Kabat et al, supra.

"framework" or "FR" residues are those variable domain residues other than the HVR residues defined herein.

As used herein, a "recipient human framework" is a human framework comprising V derived from a human immunoglobulin framework or a human consensus framework _L Or V _H Framework of the amino acid sequence of the framework. The recipient human framework "derived from" a human immunoglobulin framework or human consensus framework may comprise its identical amino acid sequence, or it may comprise pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When pre-existing amino acid changes are present in VH, preferably those changes occur only at three, two or one of the 71H, 73H and 78H positions; for example, the amino acid residues at these positions may be 71A, 73T and/or 78A. In one embodiment, V _L Acceptor human frameworks are sequence-wise to V _L The human immunoglobulin framework sequences or human consensus framework sequences are identical.

"human consensus framework" is representative of selected human immunoglobulin V _L Or V _H The most frequently occurring amino acid residues in the framework sequence. In general, human immunoglobulin V _L Or V _H The selection of sequences is from a subset of the variable region sequences. Typically, the subgroup of sequences is Kabat et al Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, national Institutes of Health, bethesda, MD (1991). For V _L Examples of (a) include, as in Kabat et al, the subgroup kappa I, kappa II, kappa III or kappa IV as described above. In addition, for V _H The subgroup may be subgroup I, subgroup II or subgroup III as in Kabat et al (supra).

"amino acid modification" at a specified position, e.g., an anti-sortilin antibody of the present disclosure, refers to substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to the specified residue. Insertions "adjacent" to a given residue are referred to as insertions within one to two residues thereof. The insertion may be N-terminal or C-terminal to a particular residue. Preferred amino acid modifications herein are substitutions.

An "affinity matured" antibody, such as an anti-sortilin antibody of the disclosure, is an antibody having one or more alterations in one or more of its HVRs that result in an improvement in the affinity of the antibody for an antigen as compared to the parent antibody without those alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are prepared by methods known in the art. For example, marks et al, bio/Technology10:779-783 (1992) describe affinity maturation by VH-and VL-domain shuffling. Random mutagenesis of HVR and/or framework residues is described, for example, in the following: barbas et al, proc Nat. Acad. Sci. USA 91:3809-3813 (1994); schier et al, gene 169:147-155 (1995); yelton et al, J.Immunol 155:1994-2004 (1995); jackson et al, J.Immunol154 (7): 3310-9 (1995); and Hawkins et al, J.mol.biol 226:889-896 (1992).

As used herein, the term "specific recognition" or "specific binding" refers to a measurable and reproducible interaction, such as attraction or binding between a target and an antibody (such as an anti-sortilin antibody of the present disclosure), which determines the presence of the target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that specifically or preferentially binds to a target or epitope (e.g., an anti-sortilin antibody of the disclosure) is an antibody that binds to the target or epitope with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other targets or other epitopes of the target. It will also be appreciated by reading this definition that, for example, an antibody (or portion) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Antibodies that specifically bind to the target can have a specific binding capacity of at least about 10 ³ M ^-1 Or 10 ⁴ M ^-1 Sometimes about 10 ⁵ M ^-1 Or 10 ⁶ M ^-1 In other cases about 10 ⁶ M ^-1 Or 10 ⁷ M ^-1 About 10 ⁸ M ^-1 To 10 ⁹ M ^-1 Or about 10 ¹⁰ M ^-1 To 10 ¹¹ M ^-1 Or higher association constants. A variety of immunoassay formats may be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays are commonly used to select monoclonal antibodies that specifically immunoreact with a protein. See, e.g., harlow and Lane (1988) Antibodies, A Laboratory Manual, cold Spring Harbor Publications, new York for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, "interaction" between sortilin and a second protein includes, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits interaction between two proteins" when the antibody disrupts, reduces, or completely eliminates interaction between the two proteins. An antibody or fragment thereof of the present disclosure "inhibits the interaction between two proteins" when the antibody or fragment thereof binds to one of the two proteins.

Antibody "effector functions" refer to those biological activities attributable to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region) and vary with antibody isotype.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at Cys226 or Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of intact antibodies may comprise a population of antibodies that have all K447 residues removed, a population of antibodies that have no K447 residues removed, and a population of antibodies that have a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for antibodies of the present disclosure include human IgG1, igG2, igG3, and IgG4.

The "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of the native Fc region. Natural sequence human Fc regions include natural sequence human IgG1Fc regions (non-a and a allotypes); a native sequence human IgG2Fc region; a native sequence human IgG3Fc region; and the native sequence human IgG4Fc region and naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from the native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc-region has at least one amino acid substitution compared to the native sequence Fc-region or the Fc-region of the parent polypeptide, e.g., about 1 to about 10 amino acid substitutions, preferably about 1 to about 5 amino acid substitutions, in the native sequence Fc-region or the Fc-region of the parent polypeptide. The variant Fc-regions herein preferably have at least about 80% homology with the native sequence Fc-region and/or the Fc-region of the parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Furthermore, preferred fcrs are those that bind IgG antibodies (gamma receptors) and include receptors of the fcγri, fcγrii and fcγriii subclasses, including allelic variants and alternatively spliced forms of these receptors, fcγrii receptors including fcγrii ("activating receptors") and fcγrii ("inhibiting receptors") which have similar amino acid sequences differing primarily in their cytoplasmic domains. The activation receptor fcγriia contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. The inhibitory receptor fcyriib contains an immunoreceptor tyrosine-based inhibitory motif ("ITIM") in its cytoplasmic domain. The term "FcR" herein includes other fcrs, including those to be identified in the future. Fcrs can also increase the serum half-life of antibodies. As used herein, "percent (%) amino acid sequence identity" and "homology" with respect to a peptide, polypeptide, or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, and any conservative substitutions are not considered as part of the sequence identity after aligning the sequences and introducing gaps to achieve the maximum percent sequence identity, if desired. The alignment for determining the percent identity of amino acid sequences can be accomplished in a variety of ways known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ^TM (DNASTAR) software. The person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm known in the art to achieve maximum alignment over the full length of the sequences being compared.

An "isolated" cell is a molecule or cell identified and isolated from at least one contaminating cell with which it is normally associated in the environment in which it is produced. In some embodiments, the isolated cells do not bind to all components associated with the production environment. The form of the isolated cells is different from the form or environment found in nature. The isolated cells are different from the cells naturally occurring in the tissue, organ or individual. In some embodiments, the isolated cell is a host cell of the present disclosure.

An "isolated" nucleic acid molecule encoding an antibody (e.g., an anti-sortilin antibody of the disclosure) is a nucleic acid molecule identified and isolated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, the isolated nucleic acid does not bind to all components associated with the production environment. The form of the isolated nucleic acid molecules herein encoding polypeptides and antibodies differs from the form or environment found in nature. Thus, an isolated nucleic acid molecule is different from a nucleic acid encoding a polypeptide and an antibody naturally present in a cell herein.

The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to circular double stranded DNA, into which additional DNA fragments may be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors for recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

"Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotide may be a deoxynucleotide, a ribonucleotide, a modified nucleotide or base and/or an analogue thereof, or any substrate that may be incorporated into the polymer by a DNA or RNA polymerase or by a synthetic reaction.

"host cells" include single cells or cell cultures that may or may already be the recipient of the vector for incorporation of the polynucleotide insert. Host cells include progeny of a single host cell, and the progeny are not necessarily identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

As used herein, a "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals exposed thereto at the dosages and concentrations employed.

The term "about" as used herein refers to the usual error range for individual values as readily known to those of skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that relate to the value or parameter itself.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "an antibody" refers to one to a plurality of antibodies, e.g., molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

It is to be understood that the aspects and embodiments of the present disclosure described herein include, consist of, and consist essentially of the recited aspects and embodiments.

SUMMARY

The present disclosure relates to methods of treating and/or delaying progression of a disease, disorder or injury in an individual by administering an anti-sortilin antibody to the individual. Non-limiting examples of diseases, conditions or injuries that may be treated or delayed include frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic Lateral Sclerosis (ALS), traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis and osteoarthritis. In some embodiments, the disease or disorder that can be treated or delayed according to the methods provided herein is a neurodegenerative disease, such as alzheimer's disease, parkinson's disease, or frontotemporal dementia. In some embodiments, the disease or disorder that can be treated or delayed according to the methods provided herein is alzheimer's disease. In some embodiments, the disease or disorder that can be treated or delayed according to the methods provided herein is parkinson's disease. In some embodiments, the disease or disorder that can be treated or delayed according to the methods provided herein is frontotemporal dementia. In some embodiments, the disease or disorder that can be treated or delayed according to the methods provided herein is ALS. In any of the above embodiments, the individual is at risk of a disease or disorder. As described below, the methods of the present disclosure address the need in the art for methods of identifying patients to be treated with suitable doses of the anti-sortilin antibodies of the present disclosure and/or methods of administering such doses in a manner that promotes patient compliance.

Patients suffering from neurodegenerative diseases such as Alzheimer's disease, parkinson's disease, ALS and frontotemporal dementia are often chronically affected by these diseases and therefore require periodic treatment for many years. Since intravenous administration of therapeutic agents cannot be done at home, the patient must be transported to an infusion center, which can be a burden to the patient and caregivers. Furthermore, memory loss, mood swings, aggression, and other behavioral symptoms that may be associated with these diseases can make patient compliance difficult. Thus, for patients suffering from neurodegenerative diseases such as Alzheimer's disease, parkinson's disease, ALS and frontotemporal dementia, a therapeutic infrequent intravenous administration would be advantageous for patients suffering from these diseases. Furthermore, subcutaneous administration (e.g. by subcutaneous injection) is a convenient administration form for the treatment of patients suffering from neurodegenerative diseases (e.g. alzheimer's disease, parkinson's disease, ALS and frontotemporal dementia), as it can be done at home, e.g. by the caregivers or by the patient himself.

Advantageously, intravenous administration of a single dose of an anti-sortilin antibody of the present disclosure to a human according to the methods provided herein results in a Pharmacodynamic (PD) effect, e.g., an increase in the level of granulin precursors in plasma and cerebrospinal fluid, for an extended period of time (e.g., up to about 57 days or more after a single dose of the anti-sortilin antibody). See, e.g., examples 1-2.

Furthermore, advantageously, administration of multiple doses of the anti-sortilin antibodies of the present disclosure to cynomolgus monkeys results in a decrease in sortilin expression in leukocytes, as well as an increase in the levels of granulin precursors in serum and cerebrospinal fluid, which persists for several weeks (e.g., up to about 6 weeks or more) after the last dose of the anti-sortilin antibody, indicating long-term pharmacodynamic effects of the anti-sortilin antibodies of the present disclosure (see, e.g., example 4).

Thus, the methods provided herein allow for relatively infrequent administration of the anti-sortilin antibodies of the present disclosure by intravenous infusion (see, e.g., examples 1-3) or by subcutaneous administration (see, e.g., example 1), which is particularly beneficial for patients suffering from neurodegenerative diseases such as alzheimer's disease, parkinson's disease, ALS, and frontotemporal dementia.

Thus, in some embodiments, the present disclosure relates to methods of treating and/or delaying progression of a disease, disorder, or injury in an individual by administering an anti-sortilin antibody of the present disclosure to the individual at a dose of at least about 6 mg/kg. In some embodiments, the antibody is administered by intravenous infusion or by subcutaneous injection. In some embodiments, the methods provided herein comprise administering the anti-sortilin antibody about once every four weeks or less frequently. In some embodiments, the disease, disorder, or injury is alzheimer's disease. In some embodiments, the disease, disorder, or injury is parkinson's disease. In some embodiments, the disease, disorder, or injury is ALS. In some embodiments, the disease, disorder, or injury is frontotemporal dementia.

All references, including patents, patent applications, and publications, cited herein are hereby incorporated by reference in their entirety.

Therapeutic use

The present disclosure provides methods of treating and/or delaying progression of a disease, disorder, or injury in an individual comprising administering an anti-sortilin antibody to the individual. As disclosed herein, the anti-sortilin antibodies of the present disclosure may be used to treat frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophies, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis or osteoarthritis, and/or delay progression thereof. In some embodiments, the disease or disorder is frontotemporal dementia. In some embodiments, the disease or disorder is alzheimer's disease. In some embodiments, the disease or disorder is parkinson's disease.

Parkinson's disease

Parkinson's disease, which may be referred to as idiopathic or primary parkinsonism, hypokinesia ankylosing syndrome (HRS) or parkinsonism, is a neurodegenerative brain disorder that affects motor control. Progressive death of dopamine-producing cells in the brain leads to the major symptoms of parkinson's disease. Most commonly, parkinson's disease is diagnosed in people over 50 years of age. Symptoms of parkinson's disease include, but are not limited to tremors (e.g., tremors of hands, arms, legs, jaw, and face), muscle stiffness of the extremities and trunk, bradykinesia (bradykinesia), postural instability, walking difficulties, neuropsychiatric problems, speech or behavioral changes, depression, anxiety, pain, psychosis, dementia, hallucinations, and sleep problems.

Parkinson's disease is idiopathic in most people (no known cause). Most parkinson's disease cases occur in people in their families with no obvious history of the disorder and are thought to be caused by complex interactions between environmental and genetic factors. Cases of this parkinsonism are referred to as "sporadic" parkinsonism.

In addition, about 15% of parkinsonian patients have a known family history of parkinsonian, with some familial cases being associated with mutations in genes such as PARK2, LRRK2, PARK7, PINK1, PRKN or SNCA.

Mutations in the GBA1 and/or UCHL1 genes are also associated with parkinson's disease and are thought to alter the risk of progression of the disease. For example, about 5% of parkinson's disease patients carry mutations in the GBA1 gene encoding the lysosomal enzyme β -Glucocerebrosidase (GBA). Heterozygous or homozygous GBA1 mutations have been found to increase the risk of parkinson's disease by about 20-fold and about 30-fold. Furthermore, GBA1 mutations in parkinson's disease are associated with earlier age of onset and faster cognitive and motor decline. See, e.g., stoker et al Pathological Mechanisms and Clinical Aspects of GBA1 motion-Associated Parkinson's Disease, parkinson's Disease Pathogenesis and Clinical Aspects, codon Publications;2018, chapter 3; medlineplus [ dot ] gov/genetics/conditions/park-event/# resources; www [ dot ] ninds [ dot ] nih [ dot ] gov/Disorders/All-Disorders/Parkinsons-Disorders-changes-Progress-and Progress.

Granulin Precursors (PGRNs) encoded by the GRN gene are also associated with parkinson's disease. GRN Parkinson's disease risk alleles are associated with reduced levels of granulin precursor in plasma, cerebrospinal fluid and Brain, while loss of homozygosity of granulin precursor function causes neuronal ceroid lipofuscinosis, while loss of heterozygosity of granulin precursor function causes frontotemporal dementia (FTD), sometimes accompanied by Parkinson's manifestations (Smith et al, am J Hum Genet (2012) 90 (6): 1102-1107; boeve et al, brain (2006) 129 (Pt 11): 3103-3114; and Wauters et al, neurobiol imaging (2018) 67:84-94). In addition, reduced plasma granulin precursor levels have been associated with increased severity of Parkinson's disease (Yao et al, neurosci Lett (2020) 725:134873). The granule protein precursor deficiency also worsens the results of rodent parkinsonism models (Martens et al, J Clin Invest (2012) 122 (11): 3955-3959), whereas the delivery of granule protein precursor genes improved those results (Van Kampen et al, PLoS One (2014) 7;9 (5): e 97032).

The granulin precursor is a-88-kDa precursor protein of granulin. It is a highly conserved multifunctional secreted glycoprotein, forming a unique "beaded" structure. Its activity includes modulation of Central Nervous System (CNS) neuroinflammation, affecting cellular signaling pathways by controlling excitotoxicity, oxidative stress, synaptogenesis, inflammation and amyloid production (Hsiung et al, geneReview. University of Washington, seattle), acting as autocrine neuronal growth factors, promoting neurite proliferation in the brain (Gass et al, mol neurogenin (2012) 10 (7): 33), and promoting and enhancing survival of motor neurons and cortical neurons (De Muynck et al, neuromol imaging (2013) 34 (11): 2541-2547).

Recent genome-wide association studies (GWAS) have revealed that genetic determinants at risk of Parkinson's disease are rich in lysosomal genes, such as GBA, GPNMB, GALC, CTSB and GRN (Nalls et al, lancet neuron (2019) 18 (2): 1091-1102). Furthermore, it is known that after receptor-mediated uptake, granulin precursors are transported to lysosomes and their complete absence in humans causes early onset lysosomal storage disorders (Paushter et al Acta Neuropathol (2018) 136 (1): 1-17). These observations indicate that the granulin precursors play a role in lysosomal function. Emerging evidence also suggests that granulin precursors interact with lysosomal enzymes such as cathepsin D and glucocerebrosidase (GCase). Furthermore, it is believed that the granulin precursors together with their binding partners prosaposin (PSAP) regulate the activities of these enzymes (Arrant et al Acta Neuropathologica Comm (2019) 7 (1): 218; valdez et al, hum Mol Genet (2020) 29 (5): 716-726; zhou et al, PLoS One (2019) 14 (7): e0212382; valdez et al, hum Mol Genet (2017) 26 (24): 4861-4872; zhou et al, J Cell Biol (2015) 210 (6): 991-1002; and Zhou et al, mol neurogenin (2017) 23;12 (1): 62).

Thus, increasing the level of granulin precursors in the cerebrospinal fluid and/or plasma of a subject, e.g. suffering from parkinson's disease or at risk of suffering from parkinson's disease, treats parkinson's disease and/or delays progression of parkinson's disease, e.g. by increasing GCase activity, reducing inflammation and/or improving lysosomal function.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat parkinson's disease and/or delay progression in an individual. In some embodiments, administration of an anti-sortilin antibody of the disclosure to an individual having, or at risk of having, parkinson's disease may increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibiting the interaction between sortilin and progranulin, thereby treating parkinson's disease and/or delaying its progression.

In some embodiments, the disease or disorder to be treated according to the methods provided herein is parkinson's disease, e.g., sporadic parkinson's disease or parkinson's disease in an individual having at least one pathogenic mutation in the GBA1 gene or GBA1 gene product. In some embodiments, the parkinson's disease treated according to the methods provided herein is sporadic parkinson's disease. In some embodiments, the parkinson's disease to be treated according to the methods provided herein is parkinson's disease in an individual having at least one pathogenic mutation in the GBA1 gene or GBA1 gene product. In some embodiments, the individual is homozygous or heterozygous for one or more mutations in the GBA1 gene or GBA1 gene product. Pathogenic mutations in the GBA1 gene or GBA1 gene product are known in the art. See, e.g., avenali et al Front Aging Neurosci,2020; o' Reagan et al, J.Parkinsons Dis,7:411-422 (2017); zhang et al, front. Mol. Neurosci,11:43 (2018); and Zhang et al, parkisons Dis,3136415 (2018). Exemplary pathogenic mutations in the GBA1 gene or GBA1 gene product include, but are not limited to, N370S (c.1226a > G), L444P (c.1448t > C), R W, IVS2+1g > a, H255Q, D409H, recNcil, E326K, T369M, R496H, and 84insGG. The presence of mutations in the GBA1 gene or GBA1 gene product can be assessed using any method known in the art, such as sequencing, polymerase chain reaction, hybridization methods such as rayon light in situ hybridization, mass spectrometry, and immunoblotting.

In some embodiments, individuals treated according to the methods provided herein have Parkinson's disease classified as between stage I and stage III based on Hoehn and Yahr criteria, e.g., as described by Hoehn and Yahr, neurology (1967) 17:427-42 and Goetz et al, mov Disord (2004) 19:1020-8.

In some embodiments, an individual treated according to the methods provided herein receives one or more treatments for parkinson's disease prior to administration of an anti-sortilin antibody of the disclosure, such as a glutamate antagonist, an anticholinergic agent, a dopamine agonist, a levodopa (L-DOPA and decarboxylase [ DDC ] inhibitor), a monoamine oxidase B (MAO-B) inhibitor, a catechol-O-methyltransferase (COMT) inhibitor, a beta blocker, a selective serotonin uptake inhibitor (SSRI), a tricyclic antidepressant (TCA), or indomethacin. In some embodiments, the individual continues to receive one or more treatments for parkinson's disease after starting treatment with an anti-sortilin antibody of the disclosure. In some embodiments, the methods provided herein comprise administering an anti-sortilin antibody of the present disclosure in combination with one or more parkinson's disease treatments, such as glutamate antagonists, anticholinergic agents, dopamine agonists, levodopa (L-DOPA and decarboxylase [ DDC ] inhibitors), monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, beta blockers, selective serotonin uptake inhibitors (SSRI), tricyclic antidepressants (TCA), and indomethacin.

Treatment or delay of parkinson's disease may be assessed using any method known in the art.

In some embodiments, parkinson's disease treatment or delay in an individual treated according to the methods provided herein is assessed based on cognitive function before and after the individual receives one or more doses of an anti-sortilin antibody. Cognitive function may be assessed using any method known in the art, such as montreal cognitive assessment (MoCA). See, e.g., nareddine et al, J American Geriatrics Soc (2005) 53 (4): 695-9. In some embodiments, parkinson's disease treatment or delay is assessed based on one or more clinical assessments of parkinson's disease. Examples of clinical assessments of parkinson's disease that may be used to assess parkinson's disease treatment or delay in an individual treated according to the methods provided herein include, but are not limited to, the institute of Motion (MDS) -sponsored revised unified parkinsonism rating scale (UPDRS) third part (MDS-UPDRS third part) or the UPDRS total scale. See, e.g., goetz et al, (2008) Movement Disorders (15): 2129-2170.

In some embodiments, parkinson's disease treatment or delay in an individual treated according to the methods provided herein is assessed based on the level of one or more biomarkers of parkinson's disease (e.g., granulin precursors, GCase protein or activity, and/or alpha-synuclein). In some embodiments, the level of one or more biomarkers of parkinson's disease is assessed in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of an anti-sortilin antibody. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, the treatment or delay of parkinson's disease in an individual treated according to the methods provided herein is assessed based on the level of one or more biomarkers of lysosomal function. In some embodiments, the level of one or more biomarkers of lysosomal function is assessed in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of anti-sortilin antibody. In some embodiments, the biomarker of one or more lysosomal functions is GCase protein, GCase activity, glucosylceramine (lyso-Gb 1), and/or glucosylceramide. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA). In some embodiments, the level of lyso-Gb1 or glucosylceramide can be measured using any method known in the art, such as mass spectrometry, e.g., high pressure liquid chromatography-tandem mass spectrometry.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in an increase in the level of a granulin precursor protein in the plasma or cerebrospinal fluid of an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the level of a granulin precursor protein in the plasma or cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to methods provided herein results in an increase in the level of a granulin precursor in a subject's plasma or cerebrospinal fluid of at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, or more, compared to the level of the granulin precursor in the subject's plasma or cerebrospinal fluid prior to administration of the anti-sortilin antibody.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in an increase in the level of GCase protein in the plasma or cerebrospinal fluid of a subject of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the level of GCase protein in the plasma or cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, treatment with an anti-sortilin antibody of the present disclosure according to the methods provided herein results in an increase in the level of GCase protein in the plasma or cerebrospinal fluid of an individual of at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, or more, compared to the level of GCase protein in the plasma or cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in an increase in the level of GCase activity in the plasma or cerebrospinal fluid of a subject of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% compared to the level of GCase activity in the plasma or cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in an increase in the level of GCase activity in the plasma or cerebrospinal fluid of a subject of at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, at least about 300%, or more, compared to the level of GCase activity in the plasma or cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in a reduction in the level of α -synuclein in the plasma or cerebrospinal fluid of an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% compared to the level of α -synuclein in the plasma or cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in a reduction in glucosylceramine (lyso-Gb 1) level in the plasma or cerebrospinal fluid of a subject by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% compared to the lyso-Gb1 level in the plasma or cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

In some embodiments, treatment with an anti-sortilin antibody of the disclosure according to the methods provided herein results in a reduction in the level of glucosylceramide in the plasma or cerebrospinal fluid of an individual by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 100% compared to the level of glucosylceramide in the plasma or cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody.

In some embodiments, the treatment or delay of parkinson's disease in an individual treated according to the methods provided herein is assessed based on the level of one or more proteins in the cerebrospinal fluid proteome. In some embodiments, the level of one or more proteins in the cerebrospinal fluid proteome is assessed in cerebrospinal fluid samples obtained from the individual before and after the individual receives one or more doses of an anti-sortilin antibody. Non-limiting examples of methods that can be used to measure the level of one or more proteins in a cerebrospinal fluid proteome in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), mass spectrometry, and Western blotting.

Frontotemporal dementia

Frontotemporal dementia (FTD) is a condition caused by progressive deterioration of the frontal lobe of the brain. Over time, degeneration may progress to the temporal lobe. Next to the prevalence of Alzheimer's Disease (AD), FTD accounts for 20% of cases of alzheimer's disease. Clinical features of FTD include memory defects, behavioral abnormalities, personality changes and language impairment (Cruts, M. & Van Broeckhoven, C. & Trends Genet.24:186-194 (2008); news, D. Et al, neurology 51:1546-1554 (1998); ratnallli, E.; brayne, C.; dawson, K. & Hodges, J.R.; neurology58:1615-1621 (2002)).

Most cases of FTD are inherited in an autosomal dominant manner, but even in one family, symptoms can span the range from FTD with behavioral disorders to primary progressive aphasia, to degeneration of the cortical basal ganglia. As with most neurodegenerative diseases, FTD is characterized by the pathological presence of specific protein aggregates in the diseased brain. Historically, the initial description of FTD recognized the presence of an intra-neuronal accumulation of hyperphosphorylated Tau protein in the body of neurofibrillary tangles or Pick. The causal effects of microtubule-associated protein Tau are supported by identifying mutations in the gene encoding Tau protein in several families (Hutton, M. Et al, nature 393:702-705 (1998)). However, most FTD brains do not show accumulation of hyperphosphorylated Tau, but do show immunoreactivity to ubiquitin (Ub) and TARDNA binding protein (TDP 43) (Neumann, m. Et al, arch. Neurol 64:1388-1394 (2007)). Most cases of FTD with Ub content (FTD-U) showed mutations in the granulin-precursor gene.

Granulin precursor mutations result in a single dose deficiency and are known to be present in nearly 50% of familial FTD cases, making granulin precursor mutations the major genetic factor for FTD. Without wishing to be bound by theory, it is believed that the loss of function heterozygosity characteristic of the granulin precursor mutations suggests that granulin precursor expression plays a dose-dependent critical role in protecting healthy individuals from FTD. Thus, FTD progression may be treated and/or delayed by increasing the level of granulin precursors by inhibiting interactions between sortilin and granulin precursors.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat FTD and/or delay progression in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual having, or at risk of having, FTD can increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibiting interactions between sortilin and granulin precursors, thereby treating and/or slowing the progression of FTD.

In some embodiments, treatment and/or delay of FTD progression may be determined by changes from baseline in neurocognitive and/or functional testing or assessment (i.e., clinical outcome assessment). Non-limiting examples of neurocognitive and functional tests that may be used to evaluate treatment and/or delay of FTD progression include frontotemporal dementia clinical rating scale (FCRS), frontotemporal dementia rating scale (FRS), clinical global impression improvement (CGI-I) evaluation, neural inventory (NPI) evaluation, color Tail Test (CTT) part 2, repeatable combination for neuropsychiatric state evaluation (RBANS), delis-kaplan perform functional system color word disturbance test, interpersonal responsiveness index, winter laboratory speech evaluation (WLA), and summer laboratory speech evaluation (SLA). In some embodiments, treatment and/or delay of FTD progression may be determined by a change from baseline in a neurocognitive and/or functional test or assessment. In some embodiments, treating FTD and/or delaying progression thereof may be determined by changes from baseline in more than one neurocognitive and/or functional test or assessment (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more neurocognitive and/or functional tests or assessments).

In some embodiments, treating FTD and/or delaying its progression is determined by global and/or regional brain volume, white matter overstrain volume, brain perfusion, anisotropy fraction, average diffusivity, axial diffusivity and radial diffusivity, and/or changes in functional brain activity from baseline. In certain embodiments, cerebral perfusion is measured by arterial spin labeling MRI. In some embodiments, radial diffusivity is measured by diffusion tensor imaging. In certain embodiments, functional brain activity is measured by functional MRI.

In some embodiments, treating FTD and/or delaying its progression is determined by changes in the markers of neurodegeneration in whole blood, plasma, and CSF from baseline. Markers for neurodegeneration may include, but are not limited to, neurofilament light chain (Nfl), tau, and/or pTau. Neurofilament light chains can be measured by methods including, but not limited to, assays from Quanterix and/or Roche diagnostics. In some implementationsIn embodiments, treating FTD and/or delaying its progression is determined by a change in a marker of lysosomal function from baseline. The label for lysosomal function can be, but is not limited to, a cathepsin, such as cathepsin B (CTSB), NAGK (N-acetyl-D-glucosamine kinase), GCase protein, GCase activity, glucosylceramine (lyso-Gb 1), and/or glucosylceramide. In some embodiments, treatment of FTD and/or delay of progression thereof is determined by changes in the levels of inflammatory markers (e.g., osteopontin (SPP 1), YWHAE (14-3-3 protein ε), allograft inflammatory factor 1 (AIF 1), colony stimulating factor 1 (CSF 1), chitinase 1 (CHIT 1), lymphocyte antigen 86 (LY 86), and CD 86) relative to baseline. When PGRN is deficient, certain biomarkers of lysosomal function may be overexpressed. For example, cathepsin D (CTSD) and Lamp1 are overexpressed in PGRN-deficient mice (GRN knockout mice). See Huang et al, (2020) Acta Neuropath Comm 8:163; gotzl et al, (2014) Acta Neuropathol 127 (6): 845-60; and Lui et al, (2016) Cell165:921-935. Thus, restoration of PGRN function may reduce expression of a biomarker of lysosomal function, which is increased when PGRN is deficient. Certain biomarkers of complement function show overexpression in PGRN deficiency. For example, C1qb and C1qc (subunits constituting complement protein C1 q) show increased levels in PGRN-deficient (GRN knockout) mice. See Huang et al. (2020) Acta Neuropath Comm: 163; Etc. (2014) Acta Neuropathol 127 (6): 845-60; and Lui et al, (2016) Cell165:921-935. Thus, restoration of PGRN function may reduce expression of biomarkers of complement function that increase upon PGRN deficiency. In some embodiments, treating FTD and/or delaying progression thereof is determined by a change in a marker of microglial activity from baseline. Markers of microglial activity may be, but are not limited to, YKL-40 and/or interleukin-6. In some embodiments, treating FTD and/or delaying progression thereof is determined by a change in expression of messenger ribonucleic acid (mRNA) in peripheral cells relative to baseline. In some embodiments of the present invention, in some embodiments,treatment of FTD and/or delay of progression thereof is determined by changes in analytes related to FTD disease biology and/or response to anti-sortilin antibodies relative to baseline. In some embodiments, the level of one or more proteins (e.g., any of the markers described above) can be measured in a sample (e.g., whole blood, plasma, and/or CSF sample) obtained from the individual. Non-limiting examples of methods that can be used to measure the level of one or more proteins in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, treating FTD and/or delaying progression thereof is determined by neuroinflammation and/or a change in microglial activation from baseline. Neuroinflammation and/or microglial activation may be measured by any method known in the art. In certain embodiments, neuroinflammation and/or microglial activation may be measured using transporter-positron emission (TSPO-PET) imaging.

In some embodiments, an individual treated according to the methods provided herein is heterozygous for a mutation in GRN (granulin gene). In some embodiments, the mutation in the GRN is a loss-of-function mutation. In some embodiments, the individual is heterozygous for the C9orf72 hexanucleotide repeat amplification. In some embodiments, an individual treated according to the methods provided herein exhibits symptoms of FTD. In some embodiments, an individual treated according to the methods provided herein does not exhibit symptoms of FTD. In some such embodiments, the individual is symptomatic. In some such cases, the individual has been identified as heterozygous for the GRN gene mutation or as having a reduced PGRN level or function, but does not exhibit symptoms of FTD. In some such cases, the subject has FTD-GRN, or FTD caused by a mutation in the GRN gene. In some embodiments of the pre-symptomatic individual, the individual may exhibit an elevated level of one or more biomarkers relative to normal levels, such as an elevated level of Nfl, SPP1, ywae, AIF1, CSF1, CHIT1, or LY86, and/or may exhibit a reduced level of one or more biomarkers relative to normal levels, such as a reduced level of NAGK or CTSB.

Alzheimer's disease

Alzheimer's Disease (AD) is the most common form of dementia. There is no way to cure this disease, which worsens with disease progression and eventually leads to death. Most commonly, AD is diagnosed in people over 65 years old. However, less prevalent early-onset alzheimer's disease may occur earlier.

Common symptoms of alzheimer's disease include behavioral symptoms such as memory recent events difficulties, cognitive symptoms, confusion, irritability and aggression, mood swings, language disorders, and long-term memory loss. As the disease progresses, body function is lost, ultimately leading to death. Alzheimer's disease develops an unknown and variable amount of time before becoming fully apparent, and it can develop without diagnosis for many years.

Sortilin has been shown to bind to Amyloid Precursor Protein (APP) and APP processing enzyme BACE1. Without wishing to be bound by theory, it is believed that these interactions are related to alzheimer's disease. Thus, without wishing to be bound by theory, it is believed that the anti-sortilin antibodies of the present disclosure are useful for inhibiting such interactions and preventing, reducing the risk of, or treating alzheimer's disease in an individual in need thereof.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat and/or delay progression of alzheimer's disease in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual having, or at risk of having, alzheimer's disease can increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibit interactions between sortilin and granulin precursors, thereby treating and/or delaying progression of alzheimer's disease. In some embodiments, and without wishing to be bound by theory, it is believed that anti-sortilin antibodies of the present disclosure that inhibit interactions between sortilin and neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.), p75, amyloid Precursor Protein (APP) and/or aβ peptide, or inhibit one or more activities of sortilin, are useful for treating and/or delaying progression of alzheimer's disease in an individual in need thereof.

Vascular dementia

Vascular dementia (VaD) is a finely progressive deterioration of memory and other cognitive functions, which is thought to be due to cerebrovascular disease (vascular disease in the brain). Cerebrovascular disease is a progressive change in blood vessels (vasculature) in the brain (brain). The most common vascular change associated with age is the accumulation of cholesterol and other substances in the vessel wall. This results in thickening and hardening of the wall, as well as narrowing of the blood vessels, which may lead to a reduced or even complete cessation of blood flow to the brain region supplied by the affected artery. Vascular dementia patients often exhibit symptoms similar to those of Alzheimer's Disease (AD). However, the associated changes in the brain are not due to AD pathology but to chronic reduced blood flow in the brain, ultimately leading to dementia. VaD is considered one of the most common types of dementia in elderly people. Symptoms of VaD include memory difficulties, difficulties in organizing and solving complex problems, slow thinking, distraction or "inattention," difficulty retrieving words from memory, changes in emotion or behavior such as depression, irritability, or apathy, and hallucinations or delusions.

Without wishing to be bound by theory, it is believed that one or more activities of sortilin, or one or more interactions between sortilin and a granule protein precursor, the neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.), neurotensin, lipoprotein lipase, apolipoprotein AV, and/or receptor-related proteins are involved in vascular dementia. Thus, without wishing to be bound by theory, it is believed that anti-sortilin antibodies of the present disclosure that inhibit interactions between sortilin and neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.), neurotensin, p75, sortilin propeptide (Sort-pro), amyloid Precursor Protein (APP), aβ peptide, lipoprotein lipase (LpL), apolipoprotein AV (APOA 5), apolipoprotein E (APOE), and/or receptor-related protein (RAP); or inhibiting one or more activities of sortilin may be used to prevent vascular dementia, reduce the risk of vascular dementia, or treat vascular dementia in an individual in need thereof.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat vascular dementia and/or delay progression in an individual. In some embodiments, administration of an anti-sortilin antibody of the disclosure to an individual suffering from, or at risk of suffering from, vascular dementia may increase the level of granulin precursors in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibiting interactions between sortilin and granulin precursors, thereby treating and/or delaying progression of vascular dementia.

Dementia (dementia)

Dementia is a non-specific syndrome (i.e., a group of signs and symptoms) that manifests as a severe loss of overall cognitive ability in previously undamaged people, beyond that expected from normal aging. Dementia may be static, for example as a result of unique total brain damage. Alternatively, dementia may be progressive, resulting in long-term deterioration due to injury or disease of the body. Although dementia is more common in the elderly population, it may also occur before the age of 65. Areas of cognition affected by dementia include, but are not limited to, memory, attention scope, language, and problem solving. Typically, symptoms must be present for at least six months before an individual is diagnosed with dementia. Exemplary forms of dementia include, but are not limited to frontotemporal dementia, alzheimer's disease, vascular dementia, semantic dementia, and dementia with Lewy bodies.

Without wishing to be bound by theory, it is believed that administration of the anti-sortilin antibodies of the present disclosure may treat dementia and/or delay its progression. In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat dementia and/or delay progression in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual suffering from, or at risk of suffering from, dementia can increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibit interactions between sortilin and granulin precursors, thereby treating dementia and/or delaying its progression.

Seizure, retinal dystrophy, traumatic brain injury, and spinal cord injury

As used herein, retinal dystrophy refers to any disease or condition involving retinal degeneration. These diseases or conditions may lead to vision loss or complete blindness.

As used herein, epileptic seizures also include epileptic seizures, and refer to transient symptoms of abnormal overactivity or synchronized neuronal activity in the brain. External influences may be as severe as a violent beating motion, or may be as slight as a brief loss of consciousness. Seizures may manifest as mental state changes, tonic or clonogenic movements, convulsions, and various other mental symptoms.

Traumatic Brain Injury (TBI) may also be referred to as intracranial injury. Traumatic brain injury occurs when the brain is traumatic damaged by external forces. Traumatic brain injury may be classified based on severity, mechanism (closed or penetrating head injury), or other characteristics (e.g., occurring at a particular location or over a broad area).

Spinal Cord Injury (SCI) includes any spinal cord injury caused by trauma rather than disease. Symptoms can vary widely, from pain to paralysis to incontinence, depending on the location of the spinal cord and nerve root injury. Spinal cord injury is described as a different level of "incomplete", which can never affect a "complete" injury to a patient, meaning a complete loss of function.

Pre-neurotrophins (e.g., pre-neurotrophin-4/5, pre-NGF, pre-BDNF, etc.) have been shown to play a role in seizures, retinal dystrophy, traumatic brain injury, and spinal cord injury.

Thus, without wishing to be bound by theory, it is believed that anti-sortilin antibodies of the present disclosure inhibit interactions between sortilin and neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.); or inhibiting one or more activities of sortilin may be used to prevent, reduce the risk of, or treat seizures, retinal dystrophies, traumatic brain injury, and/or spinal cord injury in an individual in need thereof.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat and/or delay progression of retinal dystrophy, traumatic brain injury, and/or spinal cord injury in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to a subject having, or at risk of having, retinal dystrophy, traumatic brain injury, and/or spinal cord injury can increase the level of a granulin precursor in the subject, e.g., plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibiting the interaction between sortilin and progranulin, thereby treating and/or delaying progression of retinal dystrophy, traumatic brain injury, and/or spinal cord injury.

Adverse symptoms of aging

As used herein, undesirable aging symptoms include, but are not limited to, memory loss, behavioral changes, dementia, alzheimer's disease, retinal degeneration, atherosclerotic vascular disease, hearing loss and cell lysis.

In some embodiments, and without wishing to be bound by theory, it is believed that the anti-sortilin antibodies of the present disclosure that inhibit interactions between sortilin and granulin precursors, the neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.), neurotensin, p75, lipoprotein lipase (LpL), apolipoprotein AV (APOA 5), and/or receptor-related protein (RAP); or inhibiting one or more activities of sortilin may be used to prevent, reduce the risk of aging, or treat one or more adverse symptoms of aging.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat and/or delay progression of one or more adverse symptoms of aging in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual having, or at risk of having, one or more adverse symptoms of aging may increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibiting interactions between sortilin and progranulin, thereby treating and/or delaying progression of one or more adverse symptoms of aging.

Amyotrophic Lateral Sclerosis (ALS)

As used herein, amyotrophic Lateral Sclerosis (ALS), motor neuron disease, or rogridden disease are used interchangeably and refer to debilitating diseases with various etiologies characterized by rapid progressive weakness, muscle atrophy and fascicular tremor, muscle spasms, difficulty speaking (dysarthria), dysphagia, and dyspnea.

The single insulin deficiency of granulin precursors due to loss of heterozygosity mutations in the GRN gene results in reduced levels of CSF granulin precursors and is responsible for the pathology of frontotemporal dementia (FTD) and TDP-43 (Sleemers et al, (2009) Ann Neurol 65:603; smith et al, (2012) Am J Hum Genet 90:1102). TDP-43 was also identified as the major pathological protein in ALS, suggesting a similarity between ALS and FTD.

For example, more than twenty dominant mutations in TDP-43 have been identified in sporadic and familial ALS patients ((Lager-Tourene et al, (2009) Cell 136:1001) and TDP-43 positive aggregates (Prasad et al, (2019) Front Mol Neurosci 12:25) were found in about 95% of ALS cases furthermore, ALS risk genes such as MOBP, C9ORF72, MOBKL2B, NSF and FUS could also cause FTD (Karch et al, (2018) JAMA Neurol 75:860). Furthermore, granulin precursors and C9ORF72 mutations were associated with aberrant microglial activation, which seems to be another common pathology of FTD and ALS (Haukedal et al, (2019) J Mol Biol 431:1818). Other evidence also suggests that ALS and FTD are closely related disorders with overlapping genetic, neuropathological and clinical characteristics (Weishap et al, (Trends Mol 22:769; mc2:860). And that these two genetic agents could serve as a prompter of the two genetic agents, and that these two genetic agents could serve as prompter of the disease.

Furthermore, in addition to demonstrating that loss of granulin precursors is detrimental in various models of acute and chronic neurodegeneration (Boddaert et al, (2018) Methods Mol Biol 1806:233), over-expression of granulin precursors has been found to be protective in many animal models of ALS (Laird et al, (2010) PLoS One 5:el3368; tauffenberger et al, (2013) Hum Mol Genet 22:782; beel et al, (2018) Mol neurogenin 13:55; chang et al, (2017) J Exp Med 214:2611). Furthermore, in ALS patients, common variants in GRN are significantly associated with age reduction at onset and shorter survival after onset (Sleemers et al, (2008) Neurology 71:253).

In summary, both human genetics and data from disease models support the protective function of granulin precursors in reducing pathology in ALS cases associated with TDP-43 pathology.

In some embodiments, and without wishing to be bound by theory, it is believed that the anti-sortilin antibodies of the present disclosure that inhibit interactions between sortilin and granulin precursors, the neurotrophins of the present disclosure (e.g., pre-neurotrophin-3, pre-neurotrophin-4/5, pre-NGF, pre-BDNF, neurotrophin-3, neurotrophin-4/5, NGF, BDNF, etc.), neurotensin, p75, lipoprotein lipase (LpL), apolipoprotein AV (APOA 5), and/or receptor-related protein (RAP); or inhibiting one or more activities of sortilin may be used to prevent or treat one or more adverse symptoms of ALS.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat ALS and/or delay progression in an individual. In some embodiments, administration of an anti-sortilin antibody of the disclosure to an individual having or at risk of having ALS may increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibit interactions between sortilin and progranulin, thereby treating ALS and/or delaying its progression.

In some embodiments, the individual is heterozygous for the C9orf72 hexanucleotide repeat amplification.

In some embodiments, treating ALS and/or delaying progression thereof is determined by brain atrophy, brain connectivity, brain free water, and/or changes in encephalitis from baseline. Any method known in the art, including but not limited to MRI, may be used to measure brain atrophy, brain connectivity, brain free water, and/or encephalitis. In certain embodiments, brain atrophy is measured using structural MRI. In certain embodiments, brain free water and/or encephalitis is measured using Diffusion Tensor Imaging (DTI).

In some embodiments, treating ALS and/or delaying progression thereof is determined by changes in the granulin precursor, the neurodegenerative marker, the glial activation marker, and/or the TDP-43 pathological marker from baseline. In certain embodiments, the granulin precursors are measured using a lipogenic immunoassay. In certain embodiments, the marker of neurodegeneration includes, but is not limited to, a neurofilament light chain. The neurofilament light chain can be determined by any method known in the art, including but not limited to, assays from Quanterix and/or Roche diagnostics. In certain embodiments, markers of glial activation include, but are not limited to, YKL-40 (CHI 3L), IL-6, and/or GFAP. GFAP may be measured using any method known in the art including, but not limited to, assays from rocco diagnostics.

Multiple sclerosis (MULTIPLE SCLEROSIS)

Multiple Sclerosis (MS) may also be referred to as disseminated sclerosis or disseminated encephalomyelitis. MS is an inflammatory disease in which the myelin sheath of fat around brain and spinal cord axons is destroyed, leading to demyelination and scarring as well as a broad spectrum of signs and symptoms. See, e.g., www.ninds.nih.gov/identifiers/parameters-carrier-reduction/hop-through-res-research/multiple-scanner-hop-through-research.

Symptoms of MS include, but are not limited to, changes in sensation, such as loss of sensitivity or stinging; tingling or numbness, such as hypoesthesia and paresthesia; muscle weakness; clonic system; muscle cramps; difficulty in movement; difficulties in coordination and balance, such as ataxia; speech problems, such as dysarthria, or swallowing problems, such as dysphagia; vision problems such as nystagmus, optic neuritis (including phosphorus) and compound vision; fatigue; acute or chronic pain; bladder and bowel difficulties; cognitive impairment to varying degrees; emotional symptoms of depression or unstable emotion; the Uhthoff phenomenon, which is the worsening of existing symptoms caused by exposure to temperatures above normal ambient; and a lhemitte sign that is a rearwardly extending sensation of electricity when bending the neck.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat and/or delay progression of MS in an individual. In some embodiments, administration of an anti-sortilin antibody of the disclosure to an individual having, or at risk of having, MS may increase the level of a granulin precursor in the individual, e.g., plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibit interactions between sortilin and granulin precursors, thereby treating MS and/or delaying its progression.

Glaucoma and macular degeneration

Glaucoma describes, without limitation, a group of diseases characterized by damage to the optic nerve, resulting in vision loss and blindness. Glaucoma is typically caused by increased fluid pressure (e.g., intraocular pressure) in the anterior chamber below the cornea. Glaucoma results in a continuous loss of retinal ganglion cells that are important for vision. Age-related macular degeneration generally affects the elderly and mainly results in vision loss of the macula (central field of view). Macular degeneration causes, but is not limited to, drusen, changes in pigment, distortion of vision, eye bleeding, atrophy, decreased visual acuity, blurred vision, central dark spots, decreased color vision, and decreased contrast sensitivity.

In some embodiments, administration of an anti-sortilin antibody of the disclosure may treat and/or delay progression of glaucoma or macular degeneration in an individual. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual having or at risk of having glaucoma or macular degeneration may increase the level of a granulin precursor in the individual, e.g., in plasma and/or cerebrospinal fluid; and/or reduce sortilin levels; and/or inhibiting interactions between sortilin and one or more proteins; and/or inhibit interactions between sortilin and progranulin, thereby treating glaucoma or macular degeneration and/or delaying progression thereof.

Administration and drug dosage

The anti-sortilin antibodies provided herein (and any additional therapeutic agents) may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, intralesional, intraventricular, intracranial, intraspinal, intrasynovial, intrathecal, oral, topical, or inhalation routes. Parenteral administration includes intramuscular, intraarterial, intraarticular, intraperitoneal, subcutaneous, or intravenous administration. Intravenous administration includes administration as a bolus or by continuous infusion over a period of time. In some embodiments, the administration of the antibodies of the present disclosure is by intravenous administration. In some embodiments, the administration of the antibodies of the present disclosure is by subcutaneous injection. Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations at various points in time, bolus administrations, and pulse infusion.

The anti-sortilin antibodies provided herein are formulated, administered, and administered in a manner consistent with good medical practice. Factors considered herein include the particular disease or disorder being treated, the clinical condition of the particular mammal being treated, the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to the practitioner. Antibodies need not be, but are optionally formulated with one or more agents currently used to prevent or treat the disease or disorder. In some embodiments, the anti-sortilin antibodies of the present disclosure are formulated and/or administered in combination with one or more additional anti-sortilin agents, such as one or more anti-sortilin antibodies. The effective amount of such other agents will depend on the amount of antibody present in the formulation, the type of disease, disorder or treatment, and other factors described above.

In some embodiments, the dose of a particular anti-sortilin antibody of the present disclosure may be empirically determined in individuals who have been administered one or more administrations of the anti-sortilin antibody. In some embodiments, the individual is administered increasing doses of an anti-sortilin antibody of the disclosure. To assess the efficacy of an anti-sortilin antibody of the disclosure, the clinical symptoms of any disease, disorder, or injury of the disclosure (e.g., frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, edge dominant age-related TDP43 encephalopathy (LATE), acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, or osteoarthritis) may be monitored.

Intravenous administration

In some embodiments, the methods provided herein comprise administering an anti-sortilin antibody of the disclosure to an individual having, or at risk of having, a disease, disorder, or injury by intravenous infusion. In some embodiments, the disease, disorder, or injury is frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, or osteoarthritis. In some embodiments, the disease or disorder is alzheimer's disease. In some embodiments, the disease or disorder is frontotemporal dementia. In some embodiments, the disease or disorder is parkinson's disease.

In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of at least about 6 mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of about 6mg/kg and about 60mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of up to about 30 mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of up to about 60mg/kg. In some embodiments of the present invention, in some embodiments, the dosage is about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg about 33mg/kg, about 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg or about 60mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of about 6 mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of about 15 mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of about 30 mg/kg. In some embodiments, the anti-sortilin antibody is administered intravenously at a dose of about 60mg/kg.

Such doses may be administered intermittently. In certain embodiments, the dosing frequency is three times per day, twice per day, once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or less frequently. In certain embodiments, the dosing frequency is once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or less frequently.

In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously at any of the doses described above using any of the dosing regimens described below.

In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every four weeks or more frequently. In some embodiments, an anti-sortilin antibody of the disclosure is administered intravenously about once weekly (q 1 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every two weeks (q 2 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every three weeks (q 3 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every four weeks (q 4 w).

In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously at a frequency of about once every four weeks or less. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every four weeks (q 4 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every 5 weeks (q 5 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every six weeks (q 6 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every seven weeks (q 7 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every 8 weeks (q 8 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every 9 weeks (q 9 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every 10 weeks (q 10 w).

In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once a month or less frequently. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once a month. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every two months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every three months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every four months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every 5 months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered intravenously about once every six months.

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 6mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 6mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 6mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 6mg/kg about once every 8 weeks (q 8 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every four weeks (q 4 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 15mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 15mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 15mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 15mg/kg about once every 8 weeks (q 8 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every four weeks (q 4 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering to an individual an anti-sortilin antibody of the disclosure at a dose of up to about 30mg/kg, about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of up to about 30mg/kg about once a month.

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 30mg/kg about once every four weeks (q 4 w), i.e., once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 30mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 30mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 30mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 60mg/kg about once every four weeks (q 4 w), i.e., once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 60mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 60mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 60mg/kg about once every 8 weeks (q 8 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every four weeks (q 4 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 6mg/kg and about 60mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 6mg/kg and about 60mg/kg once a month.

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 6mg/kg and about 60mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 6mg/kg and about 60mg/kg about once every 8 weeks (q 8 w).

In some embodiments of the present invention, in some embodiments, the methods provided herein include administering a pharmaceutical composition in an amount of about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg about 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg or about 60mg/kg at a dose of about once every four weeks (q 4 w), that is, the anti-sortilin antibodies of the disclosure are administered intravenously to an individual about once every 28 days. In some embodiments of the present invention, in some embodiments, the methods provided herein include administering a pharmaceutical composition in an amount of about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about 34mg/kg the anti-sortilin antibody of the disclosure is administered intravenously to an individual at a dose of about once a month of about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg, or about 60 mg/kg. In some embodiments of the present invention, in some embodiments, the methods provided herein include administering a pharmaceutical composition in an amount of about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about 34mg/kg, about 35mg/kg a dose of about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg, or about 60mg/kg is administered intravenously (q 6 w) to an individual of the anti-sortilin antibody of the present disclosure about once every six weeks. In some embodiments of the present invention, in some embodiments, the methods provided herein include administering a pharmaceutical composition in an amount of about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg, about 33mg/kg, about 34mg/kg, about 35mg/kg a dose of about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg, or about 60mg/kg is administered intravenously (q 8 w) to an individual of the anti-sortilin antibody of the present disclosure about once every 8 weeks.

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 30mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 30mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 30mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 30mg/kg about once every 8 weeks (q 8 w).

In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every four weeks (q 4 w), i.e., about once every 28 days. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once a month. In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every six weeks (q 6 w). In some embodiments, the methods provided herein comprise intravenously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg about once every 8 weeks (q 8 w).

In certain embodiments, a dose of an anti-sortilin antibody of the disclosure is administered intravenously to an individual over about 60 minutes, for example using an infusion pump.

In certain embodiments, at least 1 dose, at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, at least 14 doses, at least 15 doses, at least 16 doses, at least 17 doses, at least 18 doses, at least 19 doses, at least 20 doses, at least 21 doses, at least 22 doses, at least 23 doses, at least 24 doses, at least 25 doses, at least 26 doses, at least 27 doses, at least 28 doses, at least 29 doses, at least 30 doses, at least 31 doses, at least 32 doses, at least 33 doses, at least 34 doses, at least 35 doses, at least 36 doses, or more of an anti-sortilin antibody of the present disclosure is administered intravenously to an individual. In certain embodiments, at least 6 doses of an anti-sortilin antibody are administered to an individual.

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues about once every four weeks, or more frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody is continued about once per week (q 1 w), about once every two weeks (q 2 w), about once every three weeks (q 3 w), or about once every four weeks (q 4 w).

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues approximately once every four weeks, or less frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody is continued about once every four weeks (q 4 w), about once every five weeks (q 5 w), about once every six weeks (q 6 w), about once every seven weeks (q 7 w), about once every eight weeks (q 8 w), about once every nine weeks (q 9 w), or about once every ten weeks (q 10 w).

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues at about once a month or less frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and the administration of the anti-sortilin antibody lasts about once a month, about once every two months, about once every three months, about once every four months, about once a month every five months, or about once every six months.

In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, or longer. In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 24 months, at least about 27 months, at least about 30 months, at least about 33 months, at least about 36 months, or longer. In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or more.

In some embodiments, an individual is administered an initial higher dose of an anti-sortilin antibody followed by one or more lower doses of an anti-sortilin antibody. In some embodiments, an initial dose of about 60mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more lower doses of anti-sortilin antibody of between about 6mg/kg and about 59mg/kg, or between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial dose of about 30mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more lower doses of anti-sortilin antibody between about 6mg/kg and about 29mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial dose of about 60mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more lower doses of anti-sortilin antibody between about 6mg/kg and about 59mg/kg, between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial lower dose of an anti-sortilin antibody is administered to an individual followed by one or more higher doses of an anti-sortilin antibody. In some embodiments, an initial dose of about 6mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more higher doses of anti-sortilin antibody between about 7mg/kg and about 30mg/kg, between about 15mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg. In some embodiments, an initial dose of about 15mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more higher doses of anti-sortilin antibody between about 16mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg. In some embodiments, an initial dose of about 30mg/kg of anti-sortilin antibody is administered to an individual followed by one or more higher doses of anti-sortilin antibody between about 31mg/kg and about 60 mg/kg.

As will be appreciated by one of ordinary skill in the art, other dosage regimens may be used according to the methods provided herein, e.g., based on monitoring an individual and/or a disease or disorder being treated according to methods known in the art (e.g., as described herein).

Subcutaneous administration

In some embodiments, the methods provided herein comprise administering an anti-sortilin antibody of the disclosure to an individual having, or at risk of having, a disease, disorder, or injury by subcutaneous administration. In some embodiments, the disease, disorder, or injury is frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, or osteoarthritis. In some embodiments, the disease or disorder is alzheimer's disease. In some embodiments, the disease or disorder is frontotemporal dementia. In some embodiments, the disease or disorder is parkinson's disease.

In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 150 mg. In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of at least about 260 mg. In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 260mg and about 4000 mg. In some embodiments of the present invention, in some embodiments, the methods provided herein include using about 260mg, about 280mg, about 300mg, about 320mg, about 340mg, about 360mg, about 380mg, about 400mg, about 420mg, about 440mg, about 460mg, about 480mg, about 500mg, about 520mg, about 540mg, about 560mg, about 580mg, about 600mg, about 620mg, about 640mg, about 660mg, about 680mg, about 700mg, about 720mg, about 740mg, about 760mg, about 780mg, about 800mg, about 820mg, about 840mg, about 860mg, about 880mg, about 900mg, about 920mg, about 940mg, about 960mg, about 980mg, about 1000mg, about 1020mg, about 1040mg, about 1060mg, about 1080mg about 1100mg, about 1120mg, about 1140mg, about 1160mg, about 1180mg, about 1200mg, about 1220mg, about 1240mg, about 1260mg, about 1280mg, about 1300mg, about 1320mg, about 1340mg, about 1360mg, about 1380mg, about 1400mg, about 1420mg, about 1440mg, about 1460mg, about 1480mg, about 1500mg, about 1520mg, about 1540mg, about 1560mg, about 1580mg, about 1600mg, about 1620mg, about 1640mg, about 1660mg, about 1680mg, about 1700mg, about 1720mg, about 1740mg, about 1760mg, about 1780mg, about 1800mg, about 1820mg, about 1840mg, about 1860mg, about 1880mg, about 1900mg, about 1640mg about 1100mg, about 1120mg, about 1140mg, about 1160mg, about 1180mg, about 1200mg, about 1220mg, about 1240mg, about 1260mg, about 1280mg, about 1300mg, about 1320mg, about 1340mg, about 1360mg, about 1380mg, about 1400mg, about 1420mg, about 1440mg, about 1460mg, about 1480mg, about 1500mg, about about 1520mg, about 1540mg, about 1560mg, about 1580mg, about 1600mg, about 1620mg, about 1640mg, about 1660mg, about 1680mg, about 1700mg, about 1720mg, about 1740mg, about 1760mg, about 1780mg, about 1800mg, about 1820mg, about 1840mg, about 1860mg, about 1880mg, about 1900mg, about, A dose of any of about 3560mg, about 3580mg, about 3600mg, about 3620mg, about 3640mg, about 3660mg, about 3680mg, about 3700mg, about 3720mg, about 3740mg, about 3760mg, about 3780mg, about 3800mg, about 3820mg, about 3840mg, about 3860mg, about 3880mg, about 3900mg, about 3920mg, about 3940mg, about 3960mg, about 3980mg, or about 4000mg of an anti-sortilin antibody of the present disclosure is subcutaneously administered to an individual.

In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 270mg and about 600mg, between about 600mg and about 675mg, between about 675mg and about 720mg, between about 720mg and about 1350mg, between about 1350mg and about 1600mg, between about 1600mg and about 1800mg, or between about 1800mg and about 3600 mg. In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of about 270mg, about 675mg, about 720mg, about 1350mg, about 1800mg, or about 3600 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 150 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 270 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 300 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 600 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 675 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 720 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 1350 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 1600 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 1800 mg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 3600 mg.

In some embodiments, the methods provided herein comprise subcutaneously administering an anti-sortilin antibody of the disclosure to an individual at a dose of between about 6mg/kg and about 60mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of up to about 30 mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of up to about 60mg/kg. In some embodiments of the present invention, in some embodiments, the dosage is about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg, about 11mg/kg, about 12mg/kg, about 13mg/kg, about 13.3mg/kg, about 14mg/kg, about 15mg/kg, about 16mg/kg, about 17mg/kg, about 18mg/kg, about 19mg/kg, about 20mg/kg, about 21mg/kg, about 22mg/kg, about 23mg/kg, about 24mg/kg, about 25mg/kg, about 26mg/kg, about 27mg/kg, about 28mg/kg, about 29mg/kg, about 30mg/kg, about 31mg/kg, about 32mg/kg about 33mg/kg, about 34mg/kg, about 35mg/kg, about 36mg/kg, about 37mg/kg, about 38mg/kg, about 39mg/kg, about 40mg/kg, about 41mg/kg, about 42mg/kg, about 43mg/kg, about 44mg/kg, about 45mg/kg, about 46mg/kg, about 47mg/kg, about 48mg/kg, about 49mg/kg, about 50mg/kg, about 51mg/kg, about 52mg/kg, about 53mg/kg, about 54mg/kg, about 55mg/kg, about 56mg/kg, about 57mg/kg, about 58mg/kg, about 59mg/kg or about 60mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 6 mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 13.3 mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 15 mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 30 mg/kg. In some embodiments, the anti-sortilin antibody is administered subcutaneously at a dose of about 60mg/kg.

Such doses may be administered intermittently. In certain embodiments, the dosing frequency is three times per day, twice per day, once per week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or less frequently. In certain embodiments, the dosing frequency is once a month, once every two months, once every three months, once every four months, once every five months, once every six months, or less frequently.

In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously at any of the doses described above using any of the dosing regimens described below.

In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every four weeks (q 4 w) or more frequently. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once a week (q 1 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every two weeks (q 2 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every three weeks (q 3 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every four weeks (q 4 w).

In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously at a frequency of about once every four weeks (q 4 w) or less. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every four weeks (q 4 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every 5 weeks (q 5 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every six weeks (q 6 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every seven weeks (q 7 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every 8 weeks (q 8 w). In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every 9 weeks (q 9 w). In some embodiments, an anti-sortilin antibody of the disclosure is administered subcutaneously about once every 10 weeks (q 10 w).

In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously at a frequency of about once a month or less. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once per month. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every two months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every three months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every four months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about every 5 months. In some embodiments, the anti-sortilin antibodies of the disclosure are administered subcutaneously about once every six months.

In certain embodiments, a dose of an anti-sortilin antibody is subcutaneously administered to an individual as a subcutaneous injection. In certain embodiments, a dose of an anti-sortilin antibody is subcutaneously administered to an individual as a slow subcutaneous injection within about 15 minutes.

In certain embodiments, at least 1 dose, at least 2 doses, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, at least 10 doses, at least 11 doses, at least 12 doses, at least 13 doses, at least 14 doses, at least 15 doses, at least 16 doses, at least 17 doses, at least 18 doses, at least 19 doses, at least 20 doses, at least 21 doses, at least 22 doses, at least 23 doses, at least 24 doses, at least 25 doses, at least 26 doses, at least 27 doses, at least 28 doses, at least 29 doses, at least 30 doses, at least 31 doses, at least 32 doses, at least 33 doses, at least 34 doses, at least 35 doses, at least 36 doses, or more of the anti-sortilin antibody is administered subcutaneously to an individual. In certain embodiments, at least 6 doses of an anti-sortilin antibody are administered to an individual.

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues about once every four weeks (q 4 w) or more frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody is continued about once per week (q 1 w), about once every two weeks (q 2 w), about once every three weeks (q 3 w), or about once every four weeks (q 4 w).

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues about once every four weeks (q 4 w), or less frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody is continued about once every four weeks (q 4 w), about once every five weeks (q 5 w), about once every six weeks (q 6 w), about once every seven weeks (q 7 w), about once every eight weeks (q 8 w), about once every nine weeks (q 9 w), or about once every ten weeks (q 10 w).

In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and thereafter the administration of the anti-sortilin antibody continues at about once a month or less frequently. In some embodiments, the first dose of the anti-sortilin antibody is administered on the first day of the treatment period, and the administration of the anti-sortilin antibody lasts about once a month, about once every two months, about once every three months, about once every four months, about once a month every five months, or about once every six months.

In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 12 months, or longer. In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 24 months, at least about 27 months, at least about 30 months, at least about 33 months, at least about 36 months, or longer. In some embodiments, the individual is treated with an anti-sortilin antibody of the disclosure for a treatment period of at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or more.

In some embodiments, an individual is administered an initial higher dose of an anti-sortilin antibody followed by one or more lower doses of an anti-sortilin antibody. In some embodiments, an initial dose of about 60mg/kg of anti-sortilin antibody is administered to an individual followed by one or more lower doses of anti-sortilin antibody between about 6mg/kg and about 59mg/kg, or between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial dose of about 30mg/kg of anti-sortilin antibody is administered to an individual followed by one or more lower doses of anti-sortilin antibody between about 6mg/kg and about 29mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial dose of about 60mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more lower doses of anti-sortilin antibody between about 6mg/kg and about 59mg/kg, between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg. In some embodiments, an initial lower dose of an anti-sortilin antibody is administered to an individual followed by one or more higher doses of an anti-sortilin antibody. In some embodiments, an initial dose of about 6mg/kg of anti-sortilin antibody is administered to an individual, followed by one or more higher doses of anti-sortilin antibody between about 7mg/kg and about 30mg/kg, between about 15mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg.

As will be appreciated by one of ordinary skill in the art, other dosage regimens may be used according to the methods provided herein, e.g., based on monitoring an individual and/or a disease or disorder being treated according to methods known in the art (e.g., as described herein).

Sortilin antibodies

In some aspects, the present disclosure provides anti-sortilin antibodies that bind sortilin, e.g., human sortilin or mammalian sortilin.

Sortilin protein

In some aspects, the disclosure provides anti-sortilin antibodies that bind sortilin. Sortilin is variously referred to as sortilin 1, SORT1, 100kDa NT receptor, glycoprotein 95 (GP 95), granulin precursor receptor (PGRN-R), and neurotensin receptor 3 (NT-3 or NTR-3). Sortilin is a 831 amino acid protein that encodes a type I membrane receptor. A variety of sortilin homologs are known, including, but not limited to, human sortilin, rat sortilin, cynomolgus sortilin, and mouse sortilin. The amino acid sequence of human sortilin is as set forth in SEQ ID NO:1 (bold indicates the key amino acid residues predicted to be involved in granulin precursor binding, underlined indicates the predicted pre-NGF binding region):

In addition, the amino acid sequence of the mouse sortilin is shown as SEQ ID NO:2 is shown as follows:

in addition, the amino acid sequence of the rat sortilin is shown as SEQ ID NO:3, shown in the following:

in addition, the amino acid sequence of the cynomolgus monkey sortilin is shown in SEQ ID NO:27, as shown in:

in some embodiments, the antibodies of the disclosure bind to mammalian sortilin, human sortilin, primate sortilin (e.g., cynomolgus sortilin), mouse sortilin, and/or rat sortilin. In some embodiments, antibodies of the present disclosure can bind to an epitope within one or more of mammalian sortilin, human sortilin, primate sortilin (e.g., cynomolgus sortilin), mouse sortilin, and rat sortilin. In some embodiments, the antibodies of the disclosure bind to human sortilin. In some embodiments, the antibodies of the disclosure bind to mouse sortilin. In some embodiments, the antibodies of the disclosure bind cynomolgus sortilin. In some embodiments, the antibodies of the disclosure bind to human sortilin and mouse sortilin. In some embodiments, the antibodies of the disclosure bind to human sortilin, mouse sortilin, and cynomolgus monkey.

In some embodiments, sortilin is a preprotein that includes a signal sequence. In some embodiments, the sortilin is a mature protein. In some embodiments, the mature sortilin does not include a signal sequence. In some embodiments, the mature sortilin is expressed on a cell.

Sortilins of the present disclosure include several domains including, but not limited to, signal sequences, propeptides, luminal domains, vps10p domains (e.g., including Asp-box motifs), 10-leaf β -propeller structures and hydrophobic loops, 10CC domains, transmembrane domains, and cytoplasmic domains. In addition, sortilin of the present disclosure is expressed at high levels in many tissues, including but not limited to brain, spinal cord, heart and skeletal muscle, thyroid, placenta and testes. Sortilin is a member of the Vps10p family of sortilin receptors, which also includes, but is not limited to, sortilin-related receptors with type a repeats (SorLA), sortilin-related receptor CNS expression 1 (SorCS 1), sortilin-related receptor CNS expression 2 (SorCS 2), and sortilin-related receptor CNS expression 3 (SorCS 3). The luminal region of sortilin is aligned with each of the two luminal domains (Vps 10p domains) in yeast Vps10 p. The markers for the Vps10p domain are an amino-terminal propeptide and a carboxy-terminal fragment containing 10 conserved cysteine (10 CC) residues. Other receptors of the Vps10p family share Vps10p domains at the amino terminus and contain other extracellular domains.

The Vps10p family of sortable receptors has different functions within the nervous system and elsewhere. Receptors have been shown to be multifunctional, binding to several different ligands, including but not limited to granulin Precursors (PGRN), pre-nerve growth factor (Pro-NGF), nerve Growth Factor (NGF), PCSK9, pre-neurotrophin, pre-neurotrophin-3 (Pro-NT 3), pre-neurotrophin-4/5, brain-derived neurotrophic factor (Pro-BDNF), brain-derived neurotrophin (BDNF), neurotrophin-3 (NT 3), neurotrophin-4/5, neurotensin, p75NTR, sortilin pre-peptide (Sort-Pro), amyloid Precursor Protein (APP), lipoprotein lipase (LpL), apolipoprotein AV (APOA 5), apolipoprotein E (APOE 2,3, 4), receptor-related proteins (RAP) and plasminogen activators and involved in intracellular sorting, endocytosis and signal transduction. Sortilins of the present disclosure have been shown to mediate rapid endocytosis of lipoprotein lipase, neurotensin, and the precursor form of nerve growth factor; and targeting proteins for transport from the golgi to late endosomes. Furthermore, sortilins of the present disclosure have been shown to form complexes with p75 on cell membranes and are essential for pre-Nerve Growth Factor (NGF) -induced neuronal death. It has also recently been shown that members of the Vps10p receptor family interact with members of the neurotrophin family, including NGF, brain derived neurotrophic factors, neurotrophin-3 and neurotrophin-4/5 or the pro domain forms of neurotrophins (pre-neurotrophins). Sortilin of the present disclosure has also been shown to bind and modulate extracellular levels of PCSK9, PCSK9 targeting low density lipoprotein receptor for degradation in lysosomes, resulting in increased LDL cholesterol levels.

As disclosed herein, the interaction between sortilin and pre-neurotrophins or neurotrophins of the present disclosure is mediated by a Vps10p domain comprising a 10-leaf β -propeller structure and an Asp-box motif. In certain embodiments, sortilins of the present disclosure contain a Vps10p domain comprising a 10-bladed β -propeller structure and located at amino acid residues 78-611 of human sortilin (SEQ ID NO: 1) or at amino acid residues of mammalian sortilin corresponding to SEQ ID NO:1 within amino acid residues 78-611. In certain embodiments, amino acid residues 190-220 of human sortilin (SEQ ID NO: 1) or a sequence corresponding to SEQ ID NO:1 and amino acid residues 190-220 are located within the Vps10p domain.

The Vps10p domains of the present disclosure can include Asp-box motifs. As used herein, an Asp-cassette motif has the following sequence: (S/T) -X- (D/N) -X-X-X-X- (W/F/Y) (SEQ ID NO: 22), or X-X- (S/T) -X- (D/N) -X-G-X- (T/S) - (W/F/Y) -X (SEQ ID NO: 23), wherein X represents any amino acid. In human sortilin, the Asp-box motif is located at amino acid residues 200-207 (SSDFAKNF (SEQ ID NO: 24)). Thus, in certain embodiments, the Asp-box motif is located at amino acid residues 200-207 of human sortilin (SEQ ID NO: 1) or at amino acid residues of mammalian sortilin corresponding to SEQ ID NO:1 from amino acid residues 200 to 207.

As disclosed herein, the interaction between sortilin of the present disclosure and p75 is mediated by the 10CC domain of the hydrophobic loop of the Vps10p domain.

In certain embodiments, sortilins of the present disclosure contain amino acid residues 610-757 located in human sortilin (SEQ ID NO: 1) or a sortilin of a mammal corresponding to SEQ ID NO:1 from amino acid residues 610-757. In some embodiments, amino acid residues 592-593, 610-660, and/or 667-749 of human sortilin (SEQ ID NO: 1) or a sequence corresponding to SEQ ID NO:1, amino acid residues 592-593, 610-660, and/or 667-749 are located within the 10CC domain of sortilin.

In other embodiments, sortilins of the present disclosure contain a hydrophobic loop within the Vps10p domain located at amino acid residues 130-141 of human sortilin (SEQ ID NO: 1) or at amino acid residues of mammalian sortilin corresponding to SEQ ID NO:1 from amino acid residues 130 to 141.

As will be appreciated by those of skill in the art, the beginning and ending residues of the domains of the present disclosure may vary depending on the computer modeling program used or the method used to determine the domain.

Exemplary anti-sortilin antibodies

In some embodiments, the anti-sortilin antibodies used in the methods of the present disclosure are described in WO 2016164637A1, which is incorporated herein by reference.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises one or more of: (a) HVR-H1 comprising a sequence identical to SEQ ID NO:6 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; (b) HVR-H2 comprising a sequence identical to SEQ ID NO:7 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and (c) HVR-H3 comprising a sequence identical to SEQ ID NO:8 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and/or wherein the light chain variable domain comprises one or more of the following: (a) HVR-L1 comprising a nucleotide sequence identical to SEQ ID NO:9 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; (b) HVR-L2 comprising a nucleotide sequence identical to SEQ ID NO:10 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and (c) HVR-L3 comprising a nucleotide sequence identical to SEQ ID NO:11 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3, and wherein the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3. In some embodiments, an anti-sortilin antibody comprises an HVR-H1 comprising an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to amino acid sequence YTFTKYYMS (SEQ ID NO: 6), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in amino acid sequence YTFTKYYMS (SEQ ID NO: 6) have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-H2 comprising an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to amino acid sequence IINPIGGSTSYAQKFQG (SEQ ID NO: 7), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in amino acid sequence IINPIGGSTSYAQKFQG (SEQ ID NO: 7) have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-H3 comprising an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to amino acid sequence ARDPSGIALAGPASRGYQGMDV (SEQ ID NO: 8), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in amino acid sequence ARDPSGIALAGPASRGYQGMDV (SEQ ID NO: 8) have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L1, which HVR-L1 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to amino acid sequence RASQSVSSNLA (SEQ ID NO: 9), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in amino acid sequence RASQSVSSNLA (SEQ ID NO: 9) have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L2 comprising an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the amino acid sequence GASTRAT (SEQ ID NO: 10), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the amino acid sequence GASTRAT (SEQ ID NO: 10) have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L3 comprising an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to amino acid sequence QQARLGPWT (SEQ ID NO: 11), but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in amino acid sequence QQARLGPWT (SEQ ID NO: 11) have been substituted, inserted, and/or deleted.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3, HVR-H1 comprises amino acid sequence YTFTKYYMS (SEQ ID NO: 6), HVR-H2 comprises amino acid sequence IINPIGGSTSYAQKFQG (SEQ ID NO: 7), HVR-H3 comprises amino acid sequence ARDPSGIALAGPASRGYQGMDV (SEQ ID NO: 8), the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3, HVR-L1 comprises amino acid sequence RASQSVSSNLA (SEQ ID NO: 9), HVR-L2 comprises amino acid sequence gastat (SEQ ID NO: 10), and HVR-L3 comprises amino acid sequence QQARLGPWT (SEQ ID NO: 11).

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises one or more of: (a) HVR-H1 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-H1 amino acid sequence of antibody S-15-10-7; (b) HVR-H2 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-H2 amino acid sequence of antibody S-15-10-7; and (c) HVR-H3 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-H3 amino acid sequence of antibody S-15-10-7; and/or wherein the light chain variable domain comprises one or more of the following: (a) HVR-L1 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-L1 amino acid sequence of antibody S-15-10-7; (b) HVR-L2 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-L2 amino acid sequence of antibody S-15-10-7; and (c) HVR-L3 comprising an amino acid sequence that has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the HVR-L3 amino acid sequence of antibody S-15-10-7.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3, and wherein the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3. In some embodiments, an anti-sortilin antibody comprises an HVR-H1, which HVR-H1 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-H1 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-H1 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-H2, which HVR-H2 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-H2 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-H2 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-H3, which HVR-H3 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-H3 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-H3 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L1, which HVR-L1 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-L1 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-L1 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L2, which HVR-L2 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-L2 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-L2 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted. In some embodiments, an anti-sortilin antibody comprises an HVR-L3, which HVR-L3 comprises an amino acid sequence comprising a substitution (e.g., a conservative substitution, insertion, or deletion) relative to the HVR-L3 amino acid sequence of antibody S-15-10-7, but retains the ability to bind sortilin. In certain embodiments, up to 1, up to 2, up to 3, up to 4, up to 5, or up to 6 amino acids in the HVR-L3 amino acid sequence of antibody S-15-10-7 have been substituted, inserted, and/or deleted.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3, HVR-H1 comprises the HVR-H1 amino acid sequence of antibody S-15-10-7, HVR-H2 comprises the HVR-H2 amino acid sequence of antibody S-15-10-7, HVR-H3 comprises the HVR-L1 amino acid sequence of antibody S-15-10-7, HVR-L2 comprises the HVR-L2 amino acid sequence of antibody S-15-10-7, and HVR-L3 comprises the HVR-L3 amino acid sequence of antibody S-15-10-7.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises one, two, three, or four Framework Regions (FR) selected from the group consisting of VHFR1, VHFR2, VHFR3, and VHFR4, wherein: VH FR1 comprises SEQ ID NO:12, and VHFR2 comprises the amino acid sequence of SEQ ID NO:13, and VHFR3 comprises the amino acid sequence of SEQ ID NO:14, and VHFR4 comprises the amino acid sequence of SEQ ID NO:15, an amino acid sequence of seq id no; and/or the light chain variable domain comprises one, two, three or four framework regions selected from the group consisting of VLFR1, VLFR2, VLFR3 and VLFR4, wherein: VL FR1 comprises SEQ ID NO:16, and VLFR2 comprises the amino acid sequence of SEQ ID NO:17, and VLFR3 comprises the amino acid sequence of SEQ ID NO:18, and VLFR4 comprises the amino acid sequence of SEQ ID NO:19, and a sequence of amino acids.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a sequence that hybridizes to SEQ ID NO:20 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical; and/or wherein the light chain variable domain comprises a sequence identical to SEQ ID NO:21 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a sequence that hybridizes to SEQ ID NO:20, wherein the heavy chain variable domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, HVR-H3 of the amino acid sequence of seq id no; and/or wherein the light chain variable domain comprises a sequence identical to SEQ ID NO:21, wherein the light chain variable domain comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a sequence that hybridizes to SEQ ID NO:20, wherein the heavy chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to an amino acid sequence of HVR-H1 amino acid sequence of antibody S-15-10-7, HVR-H2 amino acid sequence of antibody S-15-10-7, and HVR-H3 amino acid sequence of HVR-H3 of antibody S-15-10-7; and/or wherein the light chain variable domain comprises a sequence identical to SEQ ID NO:21, wherein the light chain variable domain comprises an HVR-L1 amino acid sequence of antibody S-15-10-7, and an HVR-L3 amino acid sequence of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the heavy chain variable domain amino acid sequence of antibody S-15-10-7; and/or wherein the light chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the light chain variable domain amino acid sequence of antibody S-15-10-7.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a heavy chain variable domain amino acid sequence of antibody S-15-10-7, an HVR-H2 comprising an HVR-H1 amino acid sequence of antibody S-15-10-7, and an HVR-H3 comprising an HVR-H3 amino acid sequence of antibody S-15-10-7; and/or wherein the light chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the light chain variable domain amino acid sequence of antibody S-15-10-7, wherein the light chain variable domain comprises HVR-L1, HVR-L2 and HVR-L3, the HVR-L1 comprising the HVR-L1 amino acid sequence of antibody S-15-10-7, and the HVR-L2 comprising the HVR-L3 amino acid sequence of antibody S-15-10-7.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a sequence that hybridizes to SEQ ID NO:20 has an amino acid sequence of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity and contains substitutions (e.g., conservative substitutions, insertions or deletions relative to SEQ ID NO: 20), but an anti-sortilin antibody comprising the sequence retains the ability to bind sortilin. In certain embodiments, the sequence set forth in SEQ ID NO:20, and 1 to 10 amino acids in total are substituted, inserted and/or deleted in the amino acid sequence. In certain embodiments, the sequence set forth in SEQ ID NO:20, and 1 to 6 amino acids in total are substituted, inserted and/or deleted in the amino acid sequence. In certain embodiments, the substitution, insertion, or deletion occurs in a region other than the HVR (i.e., in the FR region). In some embodiments, the substitution, insertion, or deletion occurs in one or more FR regions (i.e., in VHFR1, VHFR2, VHFR3, and/or VHFR 4). In certain embodiments, substitutions, insertions, or deletions occur in one or more HVRs (i.e., in HVR-H1, HVR-H2, and/or HVR-H3). In some embodiments, the anti-sortilin antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, including post-translational modifications of the sequence.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises a sequence that hybridizes to SEQ ID NO:21 has an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and contains substitutions (e.g., conservative substitutions, insertions, or deletions relative to SEQ ID NO: 21), but an anti-sortilin antibody comprising said sequence retains the ability to bind sortilin. In certain embodiments, the sequence set forth in SEQ ID NO:21, and 1 to 10 amino acids in the amino acid sequence are substituted, inserted and/or deleted. In certain embodiments, the sequence set forth in SEQ ID NO:21, and 1-6 amino acids in the amino acid sequence are substituted, inserted and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region other than the HVR (i.e., in the FR region). In some embodiments, the substitution, insertion, or deletion occurs in one or more FR regions (i.e., in VLFR1, VLFR2, VLFR3, and/or VLFR 4). In certain embodiments, substitutions, insertions, or deletions occur in one or more HVRs (i.e., in HVR-L1, HVR-L2, and/or HVR-L3). In some embodiments, the anti-sortilin antibody comprises a light chain variable domain comprising the amino acid sequence of SEQ ID NO:21, including post-translational modifications of the sequence.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a heavy chain variable domain amino acid sequence of antibody S-15-10-7, and comprises a substitution (e.g., a conservative substitution, insertion, or deletion relative to the heavy chain variable domain amino acid sequence of antibody S-15-10-7). But an anti-sortilin antibody comprising this sequence retains the ability to bind sortilin. In certain embodiments, 1 to 10 amino acids in total are substituted, inserted, and/or deleted in the heavy chain variable region amino acid sequence of antibody S-15-10-7. In certain embodiments, 1 to 6 total amino acids in the heavy chain variable region amino acid sequence of antibody S-15-10-7 are substituted, inserted, and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region other than the HVR (i.e., in the FR region). In some embodiments, the substitution, insertion, or deletion occurs in one or more FR regions (i.e., in VHFR1, VHFR2, VHFR3, and/or VHFR 4). In certain embodiments, substitutions, insertions, or deletions occur in one or more HVRs (i.e., in HVR-H1, HVR-H2, and/or HVR-H3). In some embodiments, an anti-sortilin antibody comprises a heavy chain variable domain comprising the heavy chain variable domain amino acid sequence of antibody S-15-10-7, including post-translational modifications of the sequence.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the light chain variable domain amino acid sequence of antibody S-15-10-7, and comprises a substitution (e.g., a conservative substitution, insertion, or deletion relative to the light chain variable domain amino acid sequence of antibody S-15-10-7). But an anti-sortilin antibody comprising this sequence retains the ability to bind sortilin. In certain embodiments, 1 to 10 amino acids total in the light chain variable region amino acid sequence of antibody S-15-10-7 are substituted, inserted, and/or deleted. In certain embodiments, 1 to 6 total amino acids in the light chain variable region amino acid sequence of antibody S-15-10-7 are substituted, inserted, and/or deleted. In certain embodiments, the substitution, insertion, or deletion occurs in a region other than the HVR (i.e., in the FR region). In some embodiments, the substitution, insertion, or deletion occurs in one or more FR regions (i.e., in VLFR1, VLFR2, VLFR3, and/or VLFR 4). In certain embodiments, substitutions, insertions, or deletions occur in one or more HVRs (i.e., in HVR-L1, HVR-L2, and/or HVR-L3). In some embodiments, an anti-sortilin antibody comprises a light chain variable domain comprising the light chain variable domain amino acid sequence of antibody S-15-10-7, including post-translational modifications of the sequence.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:20, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 21.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a heavy chain variable domain comprising a heavy chain variable domain amino acid sequence of antibody S-15-10-7 and a light chain variable domain comprising a light chain variable domain amino acid sequence of antibody S-15-10-7.

In some embodiments, an anti-sortilin antibody of the disclosure comprises a polypeptide comprising SEQ ID NO:31 or 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no. In some embodiments, an anti-sortilin antibody of the disclosure comprises a polypeptide comprising SEQ ID NO:31 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no. In some embodiments, an anti-sortilin antibody of the disclosure comprises a polypeptide comprising SEQ ID NO:32 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no.

In some embodiments, the anti-sortilin antibody of the present disclosure is anti-sortilin antibody S-15-10-7, e.g., as described in WO 2016164637A1, which is incorporated herein by reference in its entirety.

Table 1: anti-sortilin antibody sequences.

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In some aspects, the disclosure also provides antibodies that bind sortilin. In some embodiments, the antibodies of the present disclosure are any of the anti-sortilin antibodies described in the "exemplary anti-sortilin antibodies" section herein. In some embodiments, the antibody binds to human sortilin and/or mammalian sortilin. In some embodiments, the antibody comprises a polypeptide comprising SEQ ID NO:20 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable domain of an amino acid sequence of seq id no. In some embodiments, the antibody has a human IgG1 isotype. In some embodiments, the antibody comprises an Fc region comprising the amino acid substitutions L234A, L235A and P331S, wherein the numbering of the residue positions is according to EU numbering. In some embodiments, the antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:32, and a heavy chain of an amino acid sequence of seq id no.

Anti-sortilin antibody binding regions

In some embodiments, an anti-sortilin antibody of the disclosure binds to one or more amino acids within amino acid residues 237-247 of human sortilin, or amino acid residues on sortilin corresponding to amino acid residues 237-247 of human sortilin. In some embodiments, the anti-sortilin antibodies of the disclosure bind to SEQ ID NO:1, or one or more amino acids within amino acid residues 237-247 corresponding to SEQ ID NO:1 from amino acid residues 237 to 247.

In some embodiments, an anti-sortilin antibody of the disclosure binds to one or more amino acids within amino acid residues 314-338 of human sortilin, or amino acid residues on sortilin corresponding to amino acid residues 314-338 of human sortilin. In some embodiments, the anti-sortilin antibodies of the disclosure bind to SEQ ID NO:1 or to one or more amino acids within amino acid residues 314-338 of SEQ ID NO: amino acid residues 314-338 of 1.

In some embodiments, an anti-sortilin antibody of the disclosure binds to one or more amino acids within amino acid residues 237-247 and 314-338 of human sortilin, or to amino acid residues on sortilin corresponding to amino acid residues 237-247 and 314-338 of human sortilin. In some embodiments, the anti-sortilin antibodies of the disclosure bind to SEQ ID NO:1 and 314-338, or to one or more amino acids within amino acid residues 237-247 and 314-338, or to amino acids corresponding to SEQ ID NO:1 and amino acid residues 237-247 and 314-338.

In some embodiments, an anti-sortilin antibody of the present disclosure binds to one or more amino acids within amino acid sequence NGLWVSKNFGG (SEQ ID NO: 4) of sortilin (e.g., human sortilin). In some embodiments, an anti-sortilin antibody of the present disclosure binds to one or more amino acids within amino acid sequence FASVMADKDTTRRIHVSTDQGDTWS (SEQ ID NO: 5) of sortilin (e.g., human sortilin). In some embodiments, an anti-sortilin antibody of the present disclosure binds to one or more amino acids within amino acid sequence NGLWVSKNFGG (SEQ ID NO: 4) and within amino acid sequence FASVMADKDTTRRIHVSTDQGDTWS (SEQ ID NO: 5) of sortilin, e.g., human sortilin.

In some embodiments, an anti-sortilin antibody of the disclosure binds to one or more amino acids within the beta-push domain of sortilin (e.g., human sortilin).

Anti-sortilin antibody binding affinity

The anti-sortilin antibodies of the present disclosure may have micromolar, nanomolar, or picomolar affinity for a target antigen (e.g., human sortilin or mammalian sortilin).

In certain embodiments, the binding affinity of an anti-sortilin antibody of the disclosure to a target antigen (e.g., human sortilin or mammalian sortilin) is determined by a dissociation constant KD is measured. Dissociation constants can be determined by any analytical technique known in the art, including biochemical or biophysical techniques such as rayon photo-activated cell sorting (FACS), flow cytometry, enzyme-linked immunosorbent assay (ELISA), surface Plasmon Resonance (SPR), biolayer interferometry (see, e.g., the Octet system of ForteBio), mesoscale discovery assays (see, e.g., MSD-SET), isothermal Titration Calorimetry (ITC), differential Scanning Calorimetry (DSC), circular Dichroism (CD), stop-stream analysis, and colorimetric or rayon photoprotein solubilization analysis; or a cell binding assay. In some embodiments, the dissociation constant (K) of sortilin (e.g., human sortilin or mammalian sortilin) _D ) Measured at a temperature of about 25 ℃. In some embodiments, the dissociation constant (K) of sortilin (e.g., human sortilin or mammalian sortilin) _D ) Measured at a temperature of about 4 ℃. In some embodiments, the dissociation constant (K) of sortilin (e.g., human sortilin or mammalian sortilin) _D ) Measured at 4℃or room temperature using, for example, FACS or biological layer interferometry. In some embodiments, the dissociation constant (K) of sortilin (e.g., human sortilin or mammalian sortilin) _D ) Measured at 4℃or room temperature using, for example, a cell binding assay, a ForteBio assay or an MSD-SET assay.

In some embodiments of any of the antibodies provided herein, the antibody has a binding activity to a target antigen (e.g., human sortilin or mammalian sortilin)<1M、<100nM、<10nM、<1nM、<0.1nM、<0.01nM or<0.001nM (e.g. 10 ^-8 M or less, e.g. 10 ^-8 M to 10 ^-13 M, e.g. 10-9M to 10 ^-13 Dissociation constant (K) of M) _D )。

In certain embodiments, an anti-sortilin antibody of the disclosure is directed against K of human sortilin and/or mammalian sortilin (e.g., mouse sortilin or cynomolgus sortilin) _D Less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less thanAt 2nM, less than 1nM, less than 0.5nM, less than 0.1nM, less than 0.09nM, less than 0.08nM, less than 0.07nM, less than 0.06nM. Less than 0.05nM, less than 0.04nM, less than 0.03nM, less than 0.02nM, less than 0.01nM, less than 0.009nM, less than 0.008nM, less than 0.007nM, less than 0.006nM, less than 0.005nM, less than 0.004nM, less than 0.003nM, less than 0.002nM, less than 0.001nM or less than 0.001nM.

In some embodiments, the antibodies of the disclosure have a dissociation constant (K) ranging from about 0.005nM to about 100nM _D ) Binding to human sortilin. In some embodiments, the antibodies of the disclosure exhibit a dissociation constant (K) in the range of about 0.005nM to about 1nM (e.g., about 0.005nM, about 0.006nM, about 0.007nM, about 0.008nM, about 0.009nM, about 0.01nM, about 0.02nM, about 0.03nM, about 0.04nM, about 0.05nM, about 0.06nM, about 0.07nM, about 0.08nM, about 0.09nM, about 0.1nM, about 0.2nM, about 0.3nM, about 0.4nM, about 0.5nM, about 0.6nM, about 0.7nM, about 0.8nM, about 0.9nM, or about 1nM _D ) Binding to human sortilin. In some embodiments of the present invention, in some embodiments, the antibodies of the disclosure can range from about 1nM to about 10nM (e.g., about 1nM, about 1.2nM, about 1.4nM, about 1.6nM, about 1.8nM, about 2nM, about 2.2nM, about 2.4nM, about 2.6nM, about 2.8nM, about 3nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.4nM, about 4.6nM, about 4.8nM, about 5nM, about 5.2nM, about about 5.4nM, about 5.6nM, about 5.8nM, about 6nM, about 6.2nM, about 6.8nM, about 7nM, any of about 7.2nM, about 7.4nM, about 7.6nM, about 7.8nM, about 8nM, about 8.2nM, about 8.4nM, about 8.6nM, about 8.8nM, about 9nM, about 9.2nM, about 9.4nM, about 9.6nM, about 9.8nM or any of about 10 nM) of the enzyme conjugate _D ) Binding to human sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 0.029nM _D ) Binding to human sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) And (5) combining. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to human sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to human sortilin. In some embodimentsIn the antibodies of the present disclosure, the dissociation constant (K _D ) And (5) combining. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to human sortilin.

In some embodiments, the antibodies of the disclosure have a dissociation constant (K) ranging from about 0.005nM to about 100nM _D ) Binding to mouse sortilin. In some embodiments of the present invention, in some embodiments, the antibodies of the disclosure can range from about 1nM to about 10nM (e.g., about 1nM, about 1.2nM, about 1.4nM, about 1.6nM, about 1.8nM, about 2nM, about 2.2nM, about 2.4nM, about 2.6nM, about 2.8nM, about 3nM, about 3.2nM, about 3.4nM, about 3.6nM, about 3.8nM, about 4nM, about 4.2nM, about 4.4nM, about 4.6nM, about 4.8nM, about 5nM, about about 5.2nM, about 5.4nM, about 5.6nM, about 5.8nM, about 6nM, about 6.2nM, about 6.8nM, about 7nM, about 7.2nM, about 7.4nM, about 7.6nM, about 7.8nM, about 8nM, about 8.2nM, about 8.4nM, about 8.6nM, about 8.8nM, about 9nM, about 9.2nM, about 9.4nM, about 9.6nM, about 9.8nM or about 10 nM) _D ) Binding to mouse sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to mouse sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to mouse sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to mouse sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to mouse sortilin. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to mouse sortilin.

In some embodiments, an anti-sortilin antibody of the disclosure has a dissociation constant (K) for sortilin (e.g., human sortilin or mammalian sortilin _D ) Was determined using the ForteBio binding assay. In an exemplary vertebrate binding assay, affinity measurements are generally performed as previously described (Estep et al, MAbs.2013Mar-Apr;5 (2): 270-8). Briefly, vertebrate affinity measurements were performed by online loading of anti-sortilin antibodies onto AHQ sensors. Will senseThe device was equilibrated offline in assay buffer for 30 minutes and then monitored online for 60 seconds to establish a baseline. For an anti-binding assay, a sensor loaded with an anti-sortilin antibody is exposed to 100nM antigen (e.g., human sortilin or mammalian sortilin, such as a human or mouse sortilin Fc fusion protein), and then transferred into an assay buffer for dissociation rate assay. Additional anti-binding can be determined by loading biotinylated sortilin (e.g., human or mouse sortilin monomers) onto an SA sensor and exposing to about 100nM anti-sortilin antibody in solution. Monovalent binding assays can be obtained by loading sortilin, e.g., a human or mouse sortilin Fc fusion protein, onto an AHQ sensor and then exposing to about 100nM of an anti-sortilin antibody Fab. Additional monovalent measurements can be made by loading biotinylated sortilin (e.g., human or mouse sortilin monomers) onto an SA sensor and then exposing to about 100nM of anti-sortilin antibody Fab in solution. Kinetic data can be fitted using 1:1 binding, for example using a model in data analysis software provided by ForteBio.

In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of between about 0.5nM and about 2nM _D ) Human sortilin is bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Human sortilin is bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 0.9nM, about 0.96nM, about 0.98nM, about 1nM, about 1.18nM, about 1.2nM, or about 1.24nM _D ) Human sortilin is bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 2.3nM or about 3.3nM _D ) Human sortilin is bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of between about 0.5nM and about 2nM _D ) Mouse sortilin was bound, for example, as measured using the ForteBio assay as described herein. In one placeIn some embodiments, the antibodies of the disclosure have a dissociation constant (K) of between about 1nM and about 8nM _D ) Mouse sortilin was bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 0.6nM, about 0.68nM, about 0.7nM, about 0.76nM, about 0.77nM, about 0.8nM, about 0.9nM, about 1nM, about 1.11nM, or about 1.2nM _D ) Mouse sortilin was bound, for example, as measured using the ForteBio assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 1.4nM or about 6.4nM _D ) Mouse sortilin was bound, for example, as measured using the ForteBio assay as described herein.

In some embodiments, an anti-sortilin antibody of the disclosure has a dissociation constant (K) for sortilin (e.g., human sortilin or mammalian sortilin _D ) Is determined using a mesoscale discovery assay (see, e.g., MSD-SET). In an exemplary MSD-SET assay, equilibrium affinity measurements (Estep et al, (2013) MAbs 270-8) were performed as previously described. Solution Equilibrium Titration (SET) was performed in pbs+0.1% IgG-free BSA (PBSF), with antigen (e.g., human sortilin or mammalian sortilin, such as biotinylated human sortilin or mouse sortilin) kept constant at 50pM, and incubated with 3-5-fold serial dilutions of anti-sortilin antibodies starting at 10 nM. Anti-sortilin antibodies (20 nM in PBS) were coated onto standard binding MSD-ECL plates at 4℃overnight or at room temperature for 30 min. Plates were then blocked for 30 minutes with shaking at 700rpm, followed by three washes with wash buffer (pbsf+0.05% Tween 20). SET samples were applied and incubated on the plate for 150 seconds, shaken at 700rpm, and then washed once. Antigens captured on the plates (e.g., human sortilin or mammalian sortilin, such as biotinylated human sortilin or mouse sortilin) were detected by incubation on the plates for 3 minutes with 250ng/mL sulfuric acid labeled streptavidin in PBSF. Plates were washed three times with wash buffer and then read on MSD sector imager 2400 instrument using 1x read buffer with surfactant tdead. The percentage of free antigen was plotted in Prism as a function of titrated antibody and fitted to two Sub-equation to extract K _D 。

In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of between about 0.02nM and about 0.3nM _D ) Human sortilin is bound, for example, as measured using the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) _D ) Human sortilin is bound, for example, as measured using, for example, the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 0.023nM, about 0.032nM, about 0.076nM, about 0.19nM, about 0.23nM, or about 0.26nM _D ) Human sortilin is bound, for example, as measured using, for example, the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 2.3nM or about 3.3nM _D ) Human sortilin is bound, for example, as measured using, for example, the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of between about 0.05nM and about 0.2nM _D ) Mouse sortilin is bound, for example, as measured using the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) The binding to mouse sortilin is, for example, as measured using an MSD-SET assay, e.g., as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of about 0.07nM, about 0.1nM, or about 0.11nM _D ) Mouse sortilin is bound, for example, as measured using, for example, the MSD-SET assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K) of, for example, about 1.4nM or about 6.4nM _D ) Mouse sortilin is bound as measured using, for example, the MSD-SET assay as described herein.

In some embodiments, an anti-sortilin antibody of the disclosure has a dissociation constant (K) for sortilin (e.g., human sortilin or mammalian sortilin _D ) Determined using a cell-based assay, such as a cell binding assay. In an exemplary cell binding assay, cells expressing human or mouse sortilin are used, e.g., with mouse sortilinHEK293T cells transiently transfected or stably expressing human sortilin were subjected to cell binding affinity measurements at 4 ℃. Cells were collected, washed in PBS and incubated with an amount of antibody close to antibody KD. Antibodies were diluted in FACS buffer (pbs+2% fbs+0.01% sodium azide). After 1 hour incubation on ice, the cells were washed three times in FACS buffer and incubated with anti-human PE conjugated secondary antibodies (BD Biosciences,1:100 dilution) for 30 minutes on ice. Cells were then washed twice in 200 μl FACS buffer and subsequently analyzed on a FACS Canto or iQE FACS screening instrument (intelllicyt Corp). Binding to human and/or mouse sortilin was measured as median rayon light intensity (MFI) of PE. To determine apparent affinity for sortilin expressed by cells, antibodies are added to cells in a titration, e.g., 0.16-40nM for human sortilin, 0.39-50nM for mouse sortilin, and their binding K is determined by non-linear curve fitting (e.g., using a modified single total pad prism) _D 。

In some embodiments, an antibody of the disclosure has a dissociation constant (K) of between about 1nM and about 10nM (e.g., any of about 1nM, about 2nM, about 3nM, about 4nM, about 5nM, about 6nM, about 7nM, about 8nM, about 9nM, or about 10 nM) _D ) Binding to human sortilin expressed on cells, e.g., as measured using, e.g., a cell binding assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to human sortilin expressed on cells, e.g., as measured using, e.g., the cell binding assays described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) Binding to human sortilin expressed on cells, e.g., as measured using, e.g., a cell binding assay as described herein. In some embodiments, an antibody of the disclosure has a dissociation constant (K) of between about 1nM and about 24nM (e.g., any of about 1nM, about 2nM, about 3nM, about 4nM, about 5nM, about 6nM, about 7nM, about 8nM, about 9nM, about 10nM, about 11nM, about 12nM, about 13nM, about 14nM, about 15nM, about 16nM, about 17nM, about 18nM, about 20nM, about 21nM, about 22nM, about 23nM, or about 24 nM) _D ) Binding to expression on cellsMouse sortilin, for example, as measured using, for example, a cell binding assay as described herein. In some embodiments, the antibodies of the disclosure have a dissociation constant (K _D ) The mouse sortilin expressed on the cells is bound, e.g., as measured using, e.g., a cell binding assay as described herein.

In some embodiments, a monovalent antibody (e.g., fab) or monovalent form of a full length antibody (e.g., as described herein) is used to determine the K of an anti-sortilin antibody of the disclosure to sortilin _D . In some embodiments, K is determined using, for example, a bivalent antibody and monomeric recombinant sortilin as described herein _D 。

Anti-sortilin antibody Activity

In certain aspects, an anti-sortilin antibody of the disclosure reduces cellular levels of sortilin (e.g., cell surface levels of sortilin, intracellular levels of sortilin, and/or total levels of sortilin); increasing the level of the granulin precursor (e.g., extracellular level of granulin precursor and/or cellular level of granulin precursor); and/or inhibit interactions (e.g., binding) between the granulin precursor and sortilin. In certain aspects, the anti-sortilin antibodies of the disclosure increase extracellular levels of progranulin and decrease cellular levels of sortilin. In some embodiments, the decrease in cellular level of sortilin is a decrease in cellular surface level of sortilin, a decrease in intracellular level of sortilin, a decrease in total level of sortilin, or any combination thereof. In some embodiments, an anti-sortilin antibody of the disclosure (a) induces sortilin degradation, sortilin cleavage, sortilin internalization, sortilin down-regulation, or any combination thereof; (b) Inhibiting interactions between sortilin and one or more proteins by: decreasing the effective level of sortilin available for interaction with one or more proteins, inducing degradation of sortilin, or both; (c) Inhibiting interactions between sortilin and granulin precursors; (d) Inhibiting the interaction between sortilin and progranulin (pro-NGF); (e) specifically binds human sortilin; (f) Specifically binds to human sortilin and mouse sortilin or to human sortilin, mouse sortilin, and cynomolgus sortilin; (g) inducing one or more granulin precursor activities; (h) Reducing internalization of the granulin precursor or fragment thereof; (i) increasing the effective concentration of the granulin precursor; (j) Inhibiting interactions between sortilin expressed on cells and granulin precursors; (k) reducing expression of one or more pro-inflammatory mediators; and any combination thereof.

As contemplated herein, the anti-sortilin antibodies of the present disclosure may also inhibit the interaction (e.g., binding) of sortilin with one or more proteins, including, but not limited to, neurotrophins, such as pre-neurotrophin, pre-neurotrophin-3, pre-neurotrophin-4/5, pro-nerve growth factor (Pro-NGF), nerve Growth Factor (NGF), pro-brain-derived neurotrophic factor (Pro-BDNF), brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4), neurotensin, low affinity Nerve Growth Factor (NGF) receptor (p 75), lipoprotein lipase (LpL), apolipoprotein AV (APOA 5), apolipoprotein E (APOE), such as APOE2, APOE3 and APOE4, amyloid precursor protein

The anti-sortilin antibodies of the present disclosure may also reduce the expression of one or more pro-inflammatory mediators including, but not limited to, cytokines (such as type I and type II interferons), IL-6, IL12p70, IL12p40, IL-1β, TNF- α, IL-8, crp, IL-20 family members, IL-33, lif, osm, cntf, gm-CSF, IL-11, IL-12, IL-17, IL-18, crp) and chemokines (such as CXCL1, CCL2, CCL3, CCL4, and CCL 5).

Inhibiting interactions between sortilin and granulin precursors

Sortilin has been shown to interact directly with (e.g., bind to) and mediate degradation of the granulin precursor (e.g., zheng, Y, etc., (2011) PLoS ONE 6 (6): e 21023). In some embodiments, the antibodies of the present disclosure reduce, block, or inhibit interactions between sortilin and granulin precursors.

In some embodiments, an anti-sortilin antibody of the disclosure inhibits interactions (e.g., binding) between sortilin and a sortilin precursor, if it reduces binding of a sortilin precursor to sortilin by 20% or more at a saturated antibody concentration, as assessed using any method known in the art, e.g., any in vitro assay or cell-based assay described herein or known in the art. In some embodiments, an anti-sortilin antibody of the disclosure inhibits interactions (e.g., binding) between sortilin and a sortilin precursor, if it reduces binding of a sortilin precursor to sortilin at a saturated antibody concentration relative to a control antibody, as assessed using any in vitro assay or cell-based assay described herein or known in the art.

In some embodiments, an anti-sortilin antibody of the disclosure reduces binding of a progranulin to sortilin by at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%. At least about 47%, at least about 48%, at least about 49%, or at least about 50%. In some embodiments, an anti-sortilin antibody of the disclosure reduces binding of a granulin precursor to sortilin by at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%. In some embodiments, an anti-sortilin antibody of the disclosure reduces binding of a human granulin precursor to human sortilin by between about 20% and about 50%, e.g., any of about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%. In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of human granulin precursors to human sortilin by about 21%. In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of human granulin precursors to human sortilin by about 36%. In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of a mouse granulin precursor to a mouse sortilin by between about 20% and about 50%, e.g., any of about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%. In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of a mouse granule protein precursor to mouse sortilin by about 21%. In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of a mouse granule protein precursor to mouse sortilin by about 24%. In some embodiments, the binding of the granulin precursor to sortilin, e.g., the binding of human granulin precursor to human sortilin or the binding of mouse granulin precursor to mouse sortilin, may be assessed using any method known in the art, e.g., any in vitro assay or cell-based assay described herein or known in the art.

In some embodiments, interactions, e.g., binding, between sortilin and granulin precursors can be assessed using a cell-based assay. In an exemplary cell-based assay, recombinant human or mouse granule protein precursors (adiogen) were biotinylated using the EZ-Link Micro NHS-PEG4 kit from ThermoScientific/Pierce according to the manufacturer's instructions. Cells expressing human or mouse sortilin may be generated as known in the art. Sortilin expressing cells or control cells were harvested and washed in PBS. Biotinylated human or mouse granulin precursors are added to pbs+2% fbs with or without anti-sortilin antibodies (10 μg/ml), e.g., anti-sortilin antibodies of the disclosure or control isotype antibodies, and incubated on ice for 2 hours. After washing the cells 3 times in pbs+2% fbs, the cells were incubated on ice for 30 minutes in streptavidin-apc (BD Biosciences, 1:100). The cells were then washed again, resuspended in pbs+2% fbs, and analyzed by flow cytometry, e.g., ON FACS canto (tm) flow cytometer (BD Biosciences, misssiuga, ON). Granulin precursor binding was measured as the median rayon light intensity of APCs of the sortilin-expressing cell population. In some embodiments of the cell-based assays described herein, the human or mouse biotinylated granulin precursor is used at increasing concentrations of 0nM and about 100 nM. In some embodiments of the cell-based assays described herein, cells expressing human or mouse sortilin are incubated with 15nM biotinylated human or mouse granulin precursor and 67nM anti-sortilin antibody (e.g., an anti-sortilin antibody of the disclosure).

In some embodiments, an anti-sortilin antibody of the disclosure reduces binding of a human granulin precursor to human sortilin by between about 20% and about 50%, e.g., any of about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%, as assessed using a cell-based assay (e.g., a cell-based assay described herein). In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of human granulin precursors to human sortilin by about 21%, assessed using a cell-based assay (as described herein). In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of human granulin precursors to human sortilin by about 36%, assessed using a cell-based assay (such as the cell-based assays described herein). In some embodiments, an anti-sortilin antibody of the disclosure reduces binding of a mouse granulin precursor to a mouse sortilin by between about 20% and about 50%, e.g., by between about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%, as assessed using a cell-based assay (e.g., a cell-based assay described herein). In some embodiments, the anti-sortilin antibodies of the disclosure reduce the binding of a mouse granulin precursor to mouse sortilin by about 21%, assessed using a cell-based assay (such as the cell-based assays described herein). In some embodiments, the anti-sortilin antibodies of the disclosure reduce binding of a mouse granulin precursor to mouse sortilin by about 24%, assessed using a cell-based assay (such as the cell-based assays described herein).

In some embodiments, interactions between sortilin and granulin precursors may be assessed using an in vitro assay. For example, interactions between sortilin and granulin precursors can be assessed using ForteBio and surface plasmon resonance analysis (e.g., skeldal, S, et al, (2012) J Biol chem.,287:43798; and Andersen, OS, et al, (2010) THE JOURNAL OF BIOLOGICAL CHEMISTRY,285,12210-12222). In an exemplary assay, surface Plasmon Resonance (SPR) analysis is used to characterize interactions between sortilin and particle protein precursors. Can be performed on a Biacore2000 instrument (Biacore, sweden) using CaHBS as standard running buffer (10mM HEPES,pH 7.4, 140mM NaCl,2mM CaCl ₂ 1mM EGTA and 0.005% Tween 20), and the binding of human or mouse granulin precursors to immobilized sortilin, and the binding of human or mouse sortilin to immobilized granulin precursors. The biosensor chip from Biacore (CM 5, catalog number BR-1000-14) may be activated using NHS/EDC, for example, the method described by the supplier, followed by coating with sortilin to a protein density of 79fmol/mm ² And for use with natural particulate protein precursors Affinity measurement of proteins. Regeneration of the flow cell after each cycle of the ligand binding assay was performed by two 10 μl pulses of regeneration buffer (10 mM glycine-HCl, pH4.0, 500mM NaCl,20mM EDTA and 0.005% Tween 20) and a single injection of 0.001% SDS. Fitting of the sensorgram for affinity estimation can be done using BIA evaluation version 3.1. Immobilization of His-granulin precursors was also performed on CM5 biosensor plates using NHS/EDC coupling kit according to the manufacturer's instructions (Biacore, sweden) to obtain similar surface densities of immobilized proteins (. About.300 fmol/mm) ² ). The biosensor chip with immobilized granulin precursors may also be used to examine binding of sortilin in the absence or presence of the anti-sortilin antibodies of the disclosure.

In some embodiments, the interaction between sortilin and granulin precursors may be assessed using an immunoassay, such as an ELISA assay. In an exemplary assay, human or mouse sortilin (R & D Systems) was immobilized on ELISA plates (2 μg/ml in PBS) overnight. Plates were washed in wash buffer (pbs+0.05% TWEEN 20) and blocked with binding buffer (pbs+1% bsa) for 1 hour at 37 ℃. Recombinant human or mouse granulin precursors (adiogen) were biotinylated using the EZ-Link Micro NHS-PEG4 kit from thermo scientific/Pierce. Different concentrations of biotinylated granulin precursors were added to the immobilized sortilin and incubated for 30 minutes at room temperature. Plates were washed three times in wash buffer and incubated with streptavidin-HRP (1:200,R&D Systems in binding buffer) for 20 minutes. Plates were washed three more times and incubated with TMB substrate solution until developed. The reaction was quenched by the addition of 50 μl of 2N sulfuric acid and the color was quantified using a Biotek Synergy H1 plate reader. Data were analyzed and fitted in Prism.

In some embodiments, inhibition of the interaction between a granulin precursor and sortilin by an anti-sortilin antibody of the disclosure is assessed using a competition assay (e.g., an in vitro competition assay). In an exemplary competition assay, a Forte Bio Octet Red384 system (Pall Forte Bio Corporation, menlo Park, CA) boxed in a standard sandwich format was used. Control anti-target IgG was loaded onto the AHQ sensor and unoccupied Fc binding sites on the sensor were blocked with irrelevant IgG1 antibodies. The sensor was then exposed to 100nM target antigen followed by a second anti-target antibody. Data processing was performed using data analysis software 7.0 for ForteBio. Additional binding of the secondary antibody following antigen binding is indicative of an unoccupied epitope (non-competitor), while no binding is indicative of epitope blocking (competitor).

In some embodiments, an anti-sortilin antibody of the disclosure inhibits or reduces interactions (e.g., binding) between sortilin and a granulin precursor by reducing sortilin levels or expression (e.g., as described herein).

In some embodiments, inhibition of the interaction (e.g., binding) between sortilin and a granulin precursor by an antibody of the disclosure results in an increase in the level of the granulin precursor, e.g., as described below. In some embodiments, inhibition of the interaction (e.g., binding) between sortilin and granulin precursors by an antibody of the disclosure results in an increase in extracellular granulin precursor levels, e.g., as described below.

Reducing sortilin levels

In some embodiments, the anti-sortilin antibodies of the disclosure reduce cellular levels of sortilin. In some embodiments, an anti-sortilin antibody of the disclosure reduces a cellular level of sortilin by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as assessed using any in vitro, cell-based, or in vivo method known in the art or described herein. In some embodiments, the decrease in cellular level of sortilin is a decrease in cellular surface level of sortilin, a decrease in intracellular level of sortilin, a decrease in total level of sortilin, or any combination thereof.

In some embodiments, the anti-sortilin antibodies of the disclosure reduce cell surface levels of sortilin. The cell surface level of sortilin may be measured using any method known in the art, such as an in vitro cell-based assay or a suitable in vivo method. In some embodiments, an anti-sortilin antibody of the disclosure reduces a cell surface level of sortilin if it induces a 20% or more reduction in the cell surface level of sortilin, e.g., as measured by any in vitro cell-based assay or suitable in vivo method described herein or known in the art. In some embodiments, an anti-sortilin antibody of the disclosure reduces cell surface levels of sortilin, if it induces a reduction in cell surface levels of sortilin at saturated antibody concentrations and/or relative to control antibodies, as measured by any in vitro cell-based assay or suitable in vivo method described herein or known in the art.

In an exemplary cell-based assay, sortilin-expressing cells, such as human U-251 cells or murine neuro-2A (N2A) cells, are incubated with 50nM of anti-sortilin antibody for 72 hours. Cells were then harvested with trypsin, washed in PBS and labeled with another primary anti-sortilin antibody (e.g., at a concentration of about 5 μg/ml). After incubating the cells with primary antibody on ice for 1 hour, the cells are washed three times in pbs+2% fbs and then incubated with secondary antibody, e.g. 5 μg/ml anti-human PE secondary antibody (Southern Biotech) or another suitable secondary antibody. The cells are then washed again using flow cytometry, e.g., using FACS Canto ^TM The system quantitates cell surface sortilin levels and measures as median rayon light intensity of PE.

In some embodiments, an antibody of the disclosure induces a decrease in cell surface level of sortilin of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as assessed using an in vitro cell-based assay (e.g., as described herein). In some embodiments, the antibodies of the disclosure induce a reduction in cell surface level of sortilin of at least about 51.6%, e.g., assessed using an in vitro cell-based assay (e.g., as described herein). In some embodiments, the antibodies of the disclosure induce a decrease in cell surface level of sortilin of at least about 87.82%, e.g., assessed using an in vitro cell-based assay (e.g., as described herein). In some embodiments, the antibodies of the present disclosure induce a decrease in cell surface level of sortilin of at least about 89%, e.g., as assessed using an in vitro cell-based assay, e.g., using an anti-sortilin antibody concentration of about 0.63nM as described herein.

In some embodiments, the cell surface level of sortilin, e.g., in macrophages, dendritic cells, and/or leukocytes obtained from an individual treated with an anti-sortilin antibody of the disclosure. In some cases, the cell surface level of human sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from a human treated with an anti-sortilin antibody of the present disclosure. In some cases, the cell surface level of mouse sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from mice treated with an anti-sortilin antibody of the present disclosure. In some cases, the cell surface level of cynomolgus sortilin may be assessed in vivo, for example in macrophages, dendritic cells and/or leukocytes obtained from cynomolgus monkeys treated with the anti-sortilin antibodies of the present disclosure. In some embodiments, an anti-sortilin antibody of the disclosure induces a decrease in cell surface level of sortilin of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as measured by any in vivo method described herein or known in the art.

In some embodiments, the anti-sortilin antibodies of the disclosure reduce intracellular levels of sortilin. Intracellular levels of sortilin may be measured using any method known in the art, for example, cell-based in vitro assays or suitable in vivo methods. In some embodiments, an anti-sortilin antibody of the disclosure reduces intracellular levels of sortilin if it induces a 20% or more reduction in intracellular levels of sortilin, as measured by any in vitro cell-based assay or suitable in vivo method described herein or known in the art. In certain embodiments, an anti-sortilin antibody of the disclosure reduces the intracellular level of sortilin if it induces a reduction in the concentration of saturated antibodies and/or a reduction in the intracellular level of sortilin relative to a control antibody, as measured by any in vitro cell-based assay or suitable in vivo method described herein or known in the art. For example, intracellular levels of sortilin may be assessed by an in vitro cell-based assay using sortilin-expressing cells (e.g., human U251 cells expressing human sortilin or mouse neural 2A cells expressing mouse sortilin). In some cases, intracellular levels of sortilin may be assessed by cell-based in vitro assays, for example using macrophages, dendritic cells, and/or leukocytes. In some cases, the intracellular level of human sortilin may be assessed by in vitro cell-based assays, e.g., using human macrophages, human dendritic cells, and/or human leukocytes. In some cases, intracellular levels of sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from an individual treated with an anti-sortilin antibody of the disclosure. In some cases, the intracellular level of human sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from a human treated with an anti-sortilin antibody of the present disclosure. In some cases, intracellular levels of mouse sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from mice treated with an anti-sortilin antibody of the present disclosure. In some cases, intracellular levels of cynomolgus sortilin may be assessed in vivo, for example in macrophages, dendritic cells and/or leukocytes obtained from cynomolgus monkeys treated with the anti-sortilin antibodies of the present disclosure. In some embodiments, an antibody of the disclosure induces a decrease in intracellular level of sortilin of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as measured by any in vitro or in vivo method described herein or known in the art. In some embodiments, the antibodies of the present disclosure induce a reduction in intracellular levels of sortilin of at least about 51.6%, e.g., as measured by an in vitro cell-based assay as described herein or known in the art. In some embodiments, the antibodies of the present disclosure induce at least about 89% decrease in intracellular levels of sortilin, e.g., as measured by an in vitro cell-based assay as described herein or known in the art.

In some embodiments, the anti-sortilin antibodies of the disclosure reduce the overall level of sortilin. The total level of sortilin may be measured using any method known in the art, such as an in vitro cell-based assay or a suitable in vivo method. In some embodiments, an anti-sortilin antibody of the disclosure reduces the total level of sortilin if it induces a 20% or more reduction in the total level of sortilin, as measured by any in vitro cell-based assay or suitable in vivo method described herein or known in the art. In certain embodiments, an anti-sortilin antibody of the present disclosure reduces the total level of sortilin if it induces a reduction at a saturated antibody concentration and/or at the total level of sortilin relative to a control antibody, as measured by any in vitro cell-based assay described herein or known in the art, or a suitable in vivo method. For example, the total level of sortilin may be assessed by an in vitro cell-based assay using sortilin-expressing cells (e.g., human U251 cells expressing human sortilin or mouse neural 2A cells expressing mouse sortilin). In some cases, the total level of sortilin may be assessed by in vitro cell-based assays, for example using macrophages, dendritic cells, and/or leukocytes. In some cases, the total level of human sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from a human treated with an anti-sortilin antibody of the present disclosure. In some cases, the total level of mouse sortilin may be assessed in vivo, for example in macrophages, dendritic cells, and/or leukocytes obtained from mice treated with an anti-sortilin antibody of the present disclosure. In some cases, the total level of cynomolgus sortilin may be assessed in vivo, for example in macrophages, dendritic cells and/or leukocytes obtained from cynomolgus monkeys treated with the anti-sortilin antibodies of the present disclosure. In some embodiments, the antibodies of the present disclosure induce a reduction in the total level of sortilin of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as measured by any in vitro or in vivo method described herein or known in the art. In some embodiments, the antibodies of the present disclosure induce a reduction in total level of sortilin of at least about 51.6%, e.g., as measured by an in vitro cell-based assay as described herein or known in the art. In some embodiments, the antibodies of the present disclosure induce a reduction in total levels of sortilin of at least about 89%, e.g., as measured by an in vitro cell-based assay as described herein or known in the art.

In some embodiments, the total level of sortilin may be assessed by an in vitro cell-based assay. In an exemplary assay, white blood cells, e.g., primary monocytes, e.g., human primary monocytes, are isolated from blood, e.g., heparinized human blood (Blood Centers of the Pacific) using a rose-shaped human monocyte enrichment cocktail (STEMCELL Technologies) according to manufacturer's protocol. Monocytes were inoculated in RPMI (Invitrogen) containing 10% fetal bovine serum (Hyclone) and 50. Mu.g/ml M-CSF (Peprotech) to induce differentiation into macrophages, or in 100. Mu.g/ml IL-4+100. Mu.g/ml GM-CSF (Peprotech) to induce differentiation into dendritic cells. After 5 days, the cells were harvested. For macrophages, cells attached to the plate were harvested using only a cell scraper. For dendritic cells, cells in suspension are collected. After washing in PBS, the cells were seeded at 0.4. Mu.l/well in 12-well plates. A 50 nanomolar concentration of a control antibody or Fab, or full length anti-sortilin antibody (IgG) or anti-sortilin antibody Fab, e.g., an anti-sortilin antibody of the present disclosure or an anti-sortilin antibody Fab of the present disclosure, was added to each well and incubated for 48 hours. Cells were then lysed on ice using RIPA buffer (Thermo Fisher Scientific) and protease inhibitor (Life Technologies). Lysates were collected and centrifuged at 10,000Xg for 10 min at 4 ℃. The supernatant was collected and protein concentration was measured using BCA kit according to the manufacturer's instructions (Thermo Fisher Pierce). Cell lysates were then analyzed by immunoblotting. In some embodiments, an anti-sortilin antibody of the disclosure reduces the total level of sortilin in white blood cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100%, e.g., as assessed using an in vitro cell-based assay (e.g., as described herein). In some embodiments, the antibodies of the present disclosure induce a reduction in total levels of sortilin in leukocytes of at least about 51.6%, e.g., assessed using a cell-based in vitro assay (e.g., as described herein). In some embodiments, the antibodies of the present disclosure induce a reduction in total sortilin levels in leukocytes of at least about 89%, e.g., assessed using an in vitro cell-based assay (e.g., as described herein).

As used herein, the level of sortilin may refer to the level of expression of a gene encoding sortilin; expression levels of one or more transcripts encoding sortilin; expression levels of the protein isolate; and/or the amount of sortilin present in and/or on the cell surface. In some embodiments, the level of sortilin refers to an effective concentration of sortilin. Any method known in the art for determining the level of gene expression, transcription, translation and/or protein abundance or localization can be used to determine the level of sortilin. In some embodiments, the cellular level of sortilin, e.g., the surface level of sortilin, the intracellular level of sortilin, and/or the total level of sortilin, is measured using any method known in the art, e.g., flow cytometry-based methods, immunoblotting methods, e.g., western blotting, mass spectrometry, immunohistochemistry, imaging methods, e.g., fluorescence microscopy, and immunoassays, e.g., ELISA.

In certain embodiments, an anti-sortilin antibody of the disclosure may reduce cellular levels of sortilin (e.g., cell surface levels of sortilin, intracellular levels of sortilin, and/or total levels of sortilin) by inducing sortilin degradation. Thus, in some embodiments, the anti-sortilin antibodies of the disclosure induce sortilin degradation. In certain embodiments, an anti-sortilin antibody of the disclosure may reduce cellular levels of sortilin (e.g., cell surface levels of sortilin, intracellular levels of sortilin, and/or total levels of sortilin) by inducing down-regulation of sortilin. Thus, in some embodiments, an anti-sortilin antibody of the disclosure induces sortilin down-regulation. In certain embodiments, an anti-sortilin antibody of the disclosure may reduce a cellular level of sortilin (e.g., a cell surface level of sortilin, an intracellular level of sortilin, and/or a total level of sortilin) by inducing cleavage of sortilin. Thus, in some embodiments, an anti-sortilin antibody of the disclosure induces sortilin cleavage. In certain embodiments, an anti-sortilin antibody of the disclosure may reduce cellular levels of sortilin (e.g., cell surface levels of sortilin, intracellular levels of sortilin, and/or total levels of sortilin) by inducing internalization of sortilin. Thus, in some embodiments, the anti-sortilin antibodies of the disclosure induce sortilin internalization. In certain embodiments, an anti-sortilin antibody of the disclosure may reduce a cellular level of sortilin (e.g., a cell surface level of sortilin, an intracellular level of sortilin, and/or a total level of sortilin) by inducing shedding of sortilin. Thus, in some embodiments, an anti-sortilin antibody of the disclosure induces sortilin shedding.

In some embodiments, the anti-sortilin antibodies of the disclosure induce desensitization of sortilin. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics that transiently activate sortilin. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to inducing a reduction in cellular levels of sortilin and/or inhibiting interactions (e.g., binding) between sortilin and one or more sortilin ligands. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to inducing sortilin degradation. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to inducing cleavage of sortilin. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to induction of sortilin internalization. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to inducing sortilin shedding. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to inducing down-regulation of sortilin expression. In some embodiments, the anti-sortilin antibodies of the present disclosure act as ligand mimics and transiently activate sortilin prior to induction of sortilin desensitization.

The anti-sortilin antibodies of the present disclosure may reduce the cellular level of sortilin at a half-maximal effective concentration (EC 50) in the micromolar, nanomolar, or picomolar range (e.g., when measured in vitro). In certain embodiments, the EC50 of the antibody is any of ∈1 μM, ∈100nM, +.10 nM, +.1 nM, +.0.1 nM, +.0.01 nM, or +.0.001 nM. In certain embodiments, the EC50 of the antibody is less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 60nM, less than 50nM, less than 40nM, less than 30nM, less than 20nM, less than 10nM, less than 9nM, less than 8nM, less than 7nM, less than 6nM, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 0.5nM, less than 0.1nM, less than 0.09nM, less than 0.08nM, less than 0.07nM, less than 0.05nM, less than 0.04nM, less than 0.03nM, less than 0.02nM, less than 0.01nM. Less than 0.009nM, less than 0.008nM, less than 0.007nM, less than 0.006nM, less than 0.005nM, less than 0.004nM, less than 0.003nM, less than 0.002nM, less than 0.001nM or less than 0.0009nM. In certain embodiments, for example, the EC50 of the antibody is about 0.086nM when measured in vitro. In certain embodiments, the EC50 of the antibody is less than any of about 1000pM, about 950pM, about 900pM, about 850pM, about 800pM, about 750pM, about 700pM, about 650pM, about 600pM, about 550pM, about 500pM, about 450pM, about 400pM, about 350pM, about 300pM, about 250pM, about 200pM, about 150pM, about 100pM, about 50pM, about 40pM, about 30pM, about 20pM, about 10pM, about 1pM, about 0.5pM, about 0.1pM, or about 0.05 pM. In some embodiments of the present invention, in some embodiments, the EC50 of an antibody may be any one of about 1000pM, about 950pM, about 900pM, about 850pM, about 800pM, about 750pM, about 700pM, about 600pM, about 550pM, about 500pM, about 450pM, about 400pM, about 350pM, about 300pM, about 250pM, about 200pM, about 150pM, about 100pM, about 50pM, about 40pM, about 30pM, about 20pM, about 10pM, about 1pM, about 0.5pM, or about 0.1pM, and an upper limit of about 0.05pM, about 0.1pM, about 0.5pM, about 10pM, about 20pM, about 30pM, about 40pM, about 50pM, about 100pM, about 150pM, about 200pM, about 250pM, about 300pM, about 350pM, about 400pM, about 450pM, about 550pM, about 500pM, about 600pM, about 500pM, about 600pM, about 900pM, or about 900pM independently selected, wherein the lower limit is less than the upper limit. In some embodiments, the EC50 of the antibody is any one of about 1pM, about 2pM, about 3pM, about 4pM, about 5pM, about 6pM, about 7pM, about 8pM, about 9pM, about 10pM, about 15pM, about 25pM, about 30pM, about 35pM, about 40pM, about 45pM, about 50pM, about 55pM, about 60pM, about 65pM, about 70pM, about 75pM, about 80pM, about 90pM, about 95pM, about 100pM, about 105pM, about 110pM, about 115pM, about 120pM, about 125pM, about 140pM, about 145pM, about 150pM, about 155pM, about 160pM, about 165pM, about 170pM, about 175pM, about 180pM, about 185pM, about 190, about 195pM, or about 200 pM. In certain embodiments, the EC50 of the antibody is about 86pM.

Various methods of measuring antibody EC50 values are known in the art, including, for example, by flow cytometry. In some embodiments, EC50 is measured in vitro using cells expressing sortilin, e.g., cells engineered to express sortilin, e.g., human sortilin or mammalian sortilin. In some embodiments, the EC50 is measured at a temperature of about 4 ℃. In some embodiments, the EC50 is measured at a temperature of about 25 ℃. In some embodiments, the EC50 is measured at a temperature of about 35 ℃. In some embodiments, the EC50 is measured at a temperature of about 37 ℃. In some embodiments, the EC50 is determined using a monovalent antibody (e.g., fab) or a monovalent form of a full length antibody. In some embodiments, EC50 is determined using antibodies that contain constant regions that demonstrate enhanced Fc receptor binding. In some embodiments, EC50 is determined using antibodies that contain constant regions that exhibit reduced Fc receptor binding.

Increasing the level of granulin precursors

In some embodiments, the anti-sortilin antibodies of the disclosure increase the level of granulin precursors. In some embodiments, the granulin precursor level may refer to, but is not limited to, an extracellular level of granulin precursor, an intracellular level of granulin precursor, and a total level of granulin precursor. In some embodiments, the increase in the level of the granulin precursor comprises an increase in the extracellular level of the granulin precursor. In some embodiments, the increase in the level of the granulin precursor comprises an increase in the level of the granulin precursor in the cell. In some embodiments, the increase in the level of the granulin precursor comprises an increase in the total level of granulin precursor.

In some embodiments, an anti-sortilin antibody of the disclosure increases a granulin precursor level in vitro or in vivo (e.g., in the brain, blood, and/or peripheral organs of an individual). In some embodiments, an anti-sortilin antibody of the disclosure increases a granulin precursor level if it induces a 20% or more increase in granulin precursor level, e.g., as measured by any in vitro cell-based assay or tissue-based (e.g., brain tissue-based) assay described herein or known in the art. In some embodiments, an anti-sortilin antibody of the disclosure increases a granulin precursor level if it induces an increase in the granulin precursor level measured at a saturated antibody concentration and/or relative to a control antibody in any in vitro cell-based assay or tissue-based (e.g., brain tissue-based) assay as described herein or known in the art.

In some embodiments, the anti-sortilin antibodies of the disclosure increase extracellular levels of progranulin in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases extracellular levels of a progranulin by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases extracellular levels of a progranulin by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% in vitro.

In some embodiments, an anti-sortilin antibody of the disclosure increases extracellular levels of progranulin in vivo (e.g., in the brain, blood, and/or peripheral organs of an individual). In some embodiments, an anti-sortilin antibody of the disclosure increases extracellular levels of progranulin in vivo by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, an anti-sortilin antibody of the disclosure increases extracellular levels of progranulin in vivo by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%.

In some embodiments, the anti-sortilin antibodies of the disclosure increase intracellular levels of progranulin in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases intracellular levels of a progranulin by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases intracellular levels of a progranulin by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% in vitro.

In some embodiments, an anti-sortilin antibody of the disclosure increases intracellular levels of a granulin precursor in vivo (e.g., in the brain, blood, and/or peripheral organs of an individual). In some embodiments, an anti-sortilin antibody of the disclosure increases intracellular levels of a progranulin in vivo by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, an anti-sortilin antibody of the disclosure increases intracellular levels of a progranulin in vivo by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%.

In some embodiments, the anti-sortilin antibodies of the disclosure increase the total level of progranulin in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases the total level of progranulin in vitro by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, the anti-sortilin antibodies of the disclosure increase the total level of progranulin by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% in vitro.

In some embodiments, an anti-sortilin antibody of the disclosure increases the total level of granulin precursors in vivo (e.g., in the brain, blood, and/or peripheral organs of an individual). In some embodiments, an anti-sortilin antibody of the disclosure increases the total level of progranulin in vivo by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, an anti-sortilin antibody of the disclosure increases the total level of progranulin in vivo by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%.

As used herein, granulin precursor level may refer to the expression level of a gene encoding granulin precursor; expression levels of one or more transcripts encoding granulin precursors; to the expression level of the granulin precursor protein; and/or the amount of granulin precursor protein secreted by the cell and/or present in the cell. In some embodiments, the granulin precursor level refers to an effective concentration of granulin precursor. Any method known in the art for measuring gene expression, transcription, translation, protein abundance, protein secretion, and/or protein localization levels can be used to determine the level of the particulate protein precursor.

In some embodiments, an anti-sortilin antibody of the disclosure increases the secretion of a progranulin by any of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, an anti-sortilin antibody of the disclosure increases the secretion of a progranulin by any of at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%. In some embodiments, an anti-sortilin antibody of the disclosure increases the secretion of a progranulin by any of at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3-fold, at least about 3.1-fold, at least about 3.2-fold. At least about 3.3 times, at least about 3.4 times, at least about 3.5 times, at least about 3.6 times, at least about 3.7 times, at least about 3.8 times, at least about 3.9 times, or at least about 4 times. In some embodiments, an anti-sortilin antibody of the disclosure increases granulin precursor secretion by about 1.9-fold.

Various methods of measuring the secretion of granulin precursors are known in the art, including for example by ELISA. In an exemplary assay, U-251 human astrocytoma cells were seeded in 96-well plates and incubated overnight. The next morning, anti-sortilin antibodies (e.g., anti-sortilin antibodies of the disclosure) and control antibodies (e.g., positive control antibodies, such as goat anti-human sortilin (gtSort) from R & D Systems, AF3154, and/or isotype control antibodies, such as goat IgG, ADI-88 (human IgG 1), and ADI-89 (human IgG 1)) were added at a final dilution of 50nM or 5nM, and the cells were incubated for about 72 hours. Cell culture medium was then collected and the concentration of granulin precursors in the culture medium samples was determined using the R & D Systems human granulin precursors Duoset ELISA kit. In some embodiments, EC50 is measured in vitro using cells expressing human sortilin. In some embodiments, the granule protein precursor secretion is determined using a monovalent antibody (e.g., fab) or a monovalent form of a full length antibody. In some embodiments, the granule protein precursor secretion is determined using an antibody comprising a constant region that exhibits enhanced Fc receptor binding. In some embodiments, the granule protein precursor secretion is determined using antibodies that contain constant regions that exhibit reduced Fc receptor binding.

In some embodiments, the anti-sortilin antibodies of the disclosure increase the effective concentration of a granulin precursor in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases the effective concentration of a progranulin by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases the effective concentration of a progranulin by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% in vitro. In some embodiments, an anti-sortilin antibody of the disclosure increases an effective concentration of a granulin precursor in vivo (e.g., in the brain, blood, and/or peripheral organs of an individual). In some embodiments, an anti-sortilin antibody of the disclosure increases the effective concentration of a progranulin in vivo by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%. In some embodiments, an anti-sortilin antibody of the disclosure increases the effective concentration of a progranulin in vivo by at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300%. In some embodiments, the anti-sortilin antibodies of the disclosure increase the in vitro or in vivo effective concentration of a granulin precursor by increasing the extracellular level of the granulin precursor, the intracellular level of the granulin precursor, the total level of granulin precursor, the expression level of a gene encoding the granulin precursor, the expression level of one or more transcripts encoding the granulin precursor, the expression level of granulin precursor protein, the amount of granulin precursor protein secreted from a cell, and/or the amount of granulin precursor protein present in a cell. Any method known in the art for measuring the effective concentration of a granulin precursor may be used, such as any in vitro, in vivo, or cell-based assay described herein, such as ELISA, western blotting, flow cytometry, and mass spectrometry.

In some embodiments, the anti-sortilin antibodies of the disclosure reduce internalization of a granulin precursor or fragment thereof. In some embodiments, the anti-sortilin antibodies of the disclosure reduce internalization and degradation of a granulin precursor or fragment thereof. In some embodiments, the anti-sortilin antibodies of the present disclosure reduce internalization of a granulin precursor or fragment thereof by reducing, inhibiting, or blocking the interaction between sortilin and granulin precursor. In some embodiments, an anti-sortilin antibody of the disclosure reduces internalization of a granulin precursor or a fragment thereof, and increases granulin precursor levels, e.g., extracellular levels of granulin precursor. Methods and reagents for measuring internalization (e.g., internalization) of proteins such as granulin precursors are known in the art, including but not limited to imaging methods such as rayon light microscopy, confocal microscopy or total internal reflection imaging (TIRF) or flow cytometry.

In some embodiments, an anti-sortilin antibody of the disclosure increases one or more granulin precursor activities. In some embodiments, an anti-sortilin antibody of the disclosure increases one or more granulin precursor activities, such as an effective concentration of granulin precursor, an extracellular level of granulin precursor, an intracellular level of granulin precursor, a total level of granulin precursor, an expression level of a gene encoding granulin precursor, an expression level of one or more transcripts encoding granulin precursor, an expression level of granulin precursor protein, an amount of granulin precursor protein secreted from a cell, and/or an amount of granulin precursor protein present in a cell, by increasing granulin precursor levels (e.g., as described above).

In some embodiments, an anti-sortilin antibody according to any of the above embodiments may incorporate any of the features, alone or in combination.

Antibody fragments

In some embodiments of any of the antibodies provided herein, the antibody is an antibody fragment. Antibody fragments include, but are not limited to, fab '-SH, F (ab') ₂ Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al, nat. Med.9:129-134 (2003). For reviews of scFv fragments, see for example WO93/16185; and U.S. patent nos. 5571894 and 5587458. Fab and F (ab') which contain salvage receptor binding epitope residues and have increased in vivo half-life ₂ See U.S. patent No. 5869046 for a discussion of fragments.

Dimeric antibodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404097; WO 1993/01161; hudson et al, nat. Med.9:129-134 (2003). Tri-and tetra-antibodies are also described in Hudson et al, nat. Med.9:129-134 (2003). A single domain antibody is an antibody fragment comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (see, e.g., U.S. patent No. 6248516).

Antibody fragments may be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and by recombinant host cells (e.g., E.coli or phage), as described herein.

In some embodiments, the antibody fragment is used in combination with a second sortilin antibody and/or with one or more antibodies that specifically bind to a pathogenic protein selected from the group consisting of: amyloid beta or fragments thereof, tau, IAPP, alpha-synuclein, TDP-43, FUS protein, prion protein, prPSc, huntingtin, calcitonin, superoxide dismutase, ataxin, lewy body, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein AI, serum amyloid a, medullasin, prolactin, transthyretin, lysozyme, beta 2 microglobulin, gelsolin, cutin, cysteine protease inhibitors, immunoglobulin light chain AL, S-IBM protein, repeat related non-ATG (RAN) translation products, dipeptide repeat (DPR) peptides, glycine-alanine (GA) repeat peptides, glycine-proline (GP) repeat peptides, glycine-arginine (GR) repeat peptides, proline-alanine (PA) repeat peptides, proline-arginine (PR) repeat peptides, and any combination thereof.

Chimeric and humanized antibodies

In some embodiments of any of the antibodies provided herein, the antibody is a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. patent No. 4816567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another example, the chimeric antibody is a "class switch" antibody, wherein the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In some embodiments of any of the antibodies provided herein, the antibody is a humanized antibody. Typically, the non-human antibodies are humanized to reduce immunogenicity to humans while maintaining the specificity and affinity of the parent non-human antibody. In certain embodiments, the humanized antibody is substantially non-immunogenic in humans. In certain embodiments, the humanized antibody has substantially the same affinity for the target as an antibody of another species from which the humanized antibody is derived. See, for example, U.S. patent No. 5530101, 5693761;5693762; and 5585089. In certain embodiments, amino acids of antibody variable domains are identified that can be modified without reducing the natural affinity of the antigen binding domain while reducing its immunogenicity. See, for example, U.S. patent nos. 5766886 and 5869619. Typically, a humanized antibody comprises one or more variable domains in which the HVRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally further comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues of a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed in, for example, almagro et al, front. Biosci.13:1619-1633 (2008), and are further described, for example, in U.S. Pat. Nos. 5821337, 7527791, 6982321 and 7087409. Human framework regions that may be used for humanization include, but are not limited to: the framework regions were selected using the "best fit" method (see, e.g., sims et al, J. Immunol.151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subsets of light or heavy chain variable regions (see, e.g., carter et al, proc. Natl. Acad. Sci. USA 89:4285 (1992); and Prestad et al, J. Immunol.151:2623 (1993)); human mature (somatic mutation) framework regions or human germline framework regions (see, e.g., almagro and Franson front. Biosci.13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., baca et al, J. Biol. Chem.272:10678-10684 (1997), and Rosok et al, J. Biol. Chem.271:22611-22618 (1996)).

Human antibodies

In some embodiments of any of the antibodies provided herein, the antibody is a human antibody. Various techniques known in the art may be used to produce human antibodies. van Dijk et al, curr.Opin.Phacol.5:368-74 (2001) and Lonberg Curr.Opin.Immunol.20:450-459 (2008) generally describe human antibodies.

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a fully human antibody or a fully antibody having human variable regions that respond to antigen challenge. Mouse strains with defective production of mouse antibodies with large fragments of the human Ig locus can be designed, and it is expected that such mice will produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can maintain large variable gene diversity and appropriate regulation of antibody production and expression. By utilizing the mouse mechanism for antibody diversification and selection and lack of immune tolerance to human proteins, the human antibody repertoire replicated in these mouse strains can produce high affinity fully human antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human MAbs with the desired specificity can be generated and selected. Some exemplary methods are described in U.S. patent No. 5545807,EP 546073 and EP 546073. See, e.g., XENOMOUSE ^TM Technical U.S. patent nos. 6075181 and 6150584; describesTechnical U.S. patent No. 5770429; describe->Technical U.S. patent No. 7041870; describes Patent application publication No. US2007/0061900 of the technology. The human variable regions of the whole antibodies produced by these animals may be further modified, for example by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (see, e.g., kozbor J. Immunol.133:3001 (1984) and Boerner et al, J. Immunol.147:86 (1991)). Li et al, proc.Natl.Acad.Sci.USA, 1:3557-3562 (2006) also describe human antibodies produced by human B cell hybridoma technology. Other methods include, for example, those described in U.S. patent No. 7189826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (Trioma technology) is also described in Vollmers et al Histology and Histopathology (3): 927-937 (2005) and Vollmers et al Methods and Findings in Experimental and Clinical Pharmacology (3): 185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with the desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

In some embodiments of any of the antibodies provided herein, the antibodies are human antibodies isolated by in vitro methods and/or the combinatorial library is screened for antibodies having the desired activity. Suitable examples include, but are not limited to, phage display (CAT, morphosys, dyax, biosite/Medarex, XOma, symphogen, alexion (previously referred to as Proliferon), affimed), ribosome display (CAT), yeast-based platform (Adimab), and the like. In certain phage display methods, all components of the VH and VL genes are cloned separately by Polymerase Chain Reaction (PCR) and randomly recombined in phage libraries, and then screened for antigen binding phage as described by Winter et al, ann.rev.immunol.12:433-455 (1994), for example, a variety of methods are known in the art for generating phage display libraries and screening for antibodies in these libraries with the desired binding characteristics. See also Sidhu et al, J.mol. Biol.338 (2): 299-310,2004; lee et al, J.mol.biol.340 (5): 1073-1093,2004; fellouse Proc. Natl. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al, J.Immunol. Methods 284 (2): 1.19-132 (2004). Phage typically display antibody fragments in the form of single chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the original repertoire (e.g., from a human) can be cloned to provide a single source of antibodies against multiple non-self and self antigens without any immunization, as described by Griffiths et al, EMBO J.12:725-734 (1993). Finally, natural libraries can also be prepared synthetically by cloning unrearranged V gene fragments from stem cells, as described by Hoogenboom et al, J.mol.biol.,227:381-388,1992, and encoding highly variable HVR3 regions using PCR primers comprising random sequences and completing the rearrangement in vitro. Patent publications describing human antibody phage libraries include, for example: us patent No. 5750373 and us patent publication nos. 2007/0292936 and 2009/0002360. Antibodies isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

Constant region comprising Fc region

In some embodiments of any of the antibodies provided herein, the antibody comprises an Fc region. In some embodiments, the Fc region is a human IgG1, igG2, igG3, and/or IgG4 isotype. In some embodiments, the antibody is of the IgG class, igM class, or IgA class.

In certain embodiments of any of the antibodies provided herein, the antibody has an IgG2 isotype. In some embodiments, the antibody comprises a human IgG2 constant region. In some embodiments, the human IgG2 constant region comprises an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (fcγiib).

In certain embodiments of any of the antibodies provided herein, the antibody has an IgG1 isotype. In some embodiments, the antibody comprises a mouse IgG1 constant region. In some embodiments, the antibody comprises a human IgG1 constant region. In some embodiments, the human IgG1 constant region comprises an Fc region. SEQ ID NO:28 or SEQ ID NO:29 provides an exemplary human IgG1 heavy chain constant region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (fcγiib).

In certain embodiments of any of the antibodies provided herein, the antibody has an IgG4 isotype. In some embodiments, the antibody comprises a human IgG4 constant region. In some embodiments, the human IgG4 constant region comprises an Fc region. In some embodiments, the antibody binds to an inhibitory Fc receptor. In certain embodiments, the inhibitory Fc receptor is inhibitory Fc-gamma receptor IIB (fcγiib).

In certain embodiments of any of the antibodies provided herein, the antibody has a hybrid IgG2/4 isotype. In some embodiments, the antibodies comprise amino acid sequences comprising amino acids 118-260 according to the EU numbering of human IgG2 and amino acids 261-447 according to the EU numbering of human IgG4 (WO 1997/11971; WO 2007/106585).

In some embodiments, the Fc region increases aggregation without activating complement as compared to a corresponding antibody comprising an Fc region that does not comprise an amino acid substitution.

It may also be desirable to modify the anti-sortilin antibodies of the present disclosure to modify effector function and/or increase the serum half-life of the antibodies. For example, the Fc receptor binding sites on the constant region can be modified or mutated to remove or reduce binding affinity for certain Fc receptors, such as fcγri, fcγrii, and/or fcγriii, thereby reducing antibody-dependent cell-mediated cytotoxicity. In some embodiments, effector function is impaired by removing N-glycosylation of the Fc region of the antibody (e.g., the CH2 domain of IgG). In some embodiments, effector function is impaired by modifying regions of human IgG such as 233-236, 297 and/or 327-331, such as WO 99/58372 and Armour et al, molecular Immunology 40:585-593 (2003); reddy et al, J.Immunology164:1925-1933 (2000). In other embodiments, it may also be desirable to modify the anti-sortilin antibodies of the present disclosure to modify effector function, thereby increasing binding selectivity to FcgRIIb (CD 32 b) containing ITIM, e.g., increasing aggregation of the anti-sortilin antibody on adjacent cells, without activating humoral responses, including antibody-dependent cell-mediated cytotoxicity and antibody-dependent cellular phagocytosis.

To increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an antibody fragment), for example, as described in us patent 5739277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., igG1, igG2, igG3, or IgG 4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

Multispecific antibodies

A multispecific antibody is an antibody that has binding specificity for at least two different epitopes, including epitopes on the same or another polypeptide (e.g., one or more sortilin polypeptides of the disclosure). In some embodiments, the multispecific antibody may be a bispecific antibody. In some embodiments, the multispecific antibody may be a trispecific antibody. In some embodiments, the multispecific antibody may be a tetraspecific antibody. Such antibodies may be derived from full length antibodies or antibody fragments (e.g., F (ab') ₂ Bispecific antibodies). In some embodiments, the multispecific antibody comprises a first antigen-binding region that binds to a first site on sortilin and comprises a second antigen-binding region that binds to a second site on sortilin. In some embodiments, the multispecific antibody comprises a first antigen-binding region that binds sortilin and a second antigen-binding region that binds a second polypeptide.

Provided herein are multispecific antibodies comprising a first antigen-binding region comprising six HVRs of an antibody described herein (e.g., antibody S-15-10-7) that bind sortilin, and a second antigen-binding region that binds to a second polypeptide. In some embodiments, the first antigen binding region comprises V of an antibody described herein (e.g., antibody S-15-10-7) _H Or V _L 。

In some embodiments of any of the multispecific antibodies provided herein, the second polypeptide is a) an antigen that facilitates transport across the blood-brain barrier; (b) An antigen that facilitates transport across the blood brain barrier selected from Transferrin Receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, CRM197, alpaca single domain antibody, TMEM 30 (a), protein transduction domain, TAT, syn-B, penetratin, polyarginine peptide, vascular peptide and ANG1005; (c) A pathogenic protein selected from the group consisting of amyloid β, oligomeric amyloid β, amyloid β plaques, amyloid precursor protein or fragments thereof, tau, IAPP, α -synuclein, TDP-43, FUS protein, C9orf72 (chromosome 9 open reading frame 72), C9RAN protein, prion protein, prPSc, huntingtin, calcitonin, superoxide dismutase, ataxin 1, ataxin 2, ataxin 3, ataxin 7, ataxin 8, ataxin 10, lewis, atrial natriuretic factor, islet amyloid polypeptide, insulin, apolipoprotein, serum amyloid a, medullarin, prolactin, thyroxine carrier, lysozyme, β2 microglobulin, gelsolin, (d) a ligand and/or protein expressed on immune cells, wherein said ligand and/or protein is selected from the group consisting of CD40, OX40, icosa 28, CD137, CD/BB, CTLA-27, gil-7, g-PD 3, g-3, H-3-PD, H-3, g-3, and/or a fragment thereof; and/or (e) a protein, lipid, polysaccharide or glycolipid expressed on one or more tumor cells, and any combination thereof.

Many antigens are known in the art to facilitate transport across the blood brain barrier (see, e.g., gabothule r. Neurobiol. Dis.37:48-57 (2010)). Such secondary antigens include, but are not limited to, transferrin Receptor (TR), insulin receptor (HIR), insulin-like growth factor receptor (IGFR), low density lipoprotein receptor-related proteins 1 and 2 (LPR-1 and 2), diphtheria toxin receptor, including CRM197 (non-toxic mutant of diphtheria toxin), llama single domain antibodies such as TMEM30 (a) (flip), protein transduction domains such as TAT, syn-B or penetratin, polyarginine or normally positively charged peptides, vascular peptide peptides such as ANG1005 (see, e.g., gabothule, 2010), and other cell surface proteins enriched on blood brain barrier endothelial cells (see, e.g., daneman et al, PLoS One 5 (10): el3741 (2010)).

Multivalent antibodies can recognize sortilin antigens as well as, without limitation, additional antigens such as peptide a or alpha-synuclein antigens, tau protein antigens, TDP-43 protein antigens, prion protein antigens, huntingtin antigens, RAN translation product antigens, including dipeptide repeats (DPR peptides) consisting of glycine-alanine (GA), glycine-proline (GP), glycine-arginine (GR), proline-alanine (PA), or proline-arginine (PR), insulin receptor antigens, or insulin-like growth factor receptor antigens, transferrin receptor antigens, or any other antigen that facilitates transfer of antibodies across the blood brain barrier. In some embodiments, the second polypeptide is transferrin. In some embodiments, the second polypeptide is Tau. In some embodiments, the second polypeptide is aβ. In some embodiments, the second polypeptide is TREM2. In some embodiments, the second polypeptide is an α -synuclein.

Multivalent antibodies comprise at least one polypeptide chain (preferably two polypeptide chains), wherein one or more polypeptide chains comprise two or more variable domains. For example, the polypeptide chain can comprise VD1- (X1) _n -VD2-(X2) _n -Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, fc is a polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, one or more polypeptide chains may comprise V _H -C _H 1-Flexible Joint-V _H -C _H 1-Fc region chain; or V _H -C _H 1-V _H -C _H 1-Fc region chain. The multivalent antibodies herein preferably further comprise at least 2 (preferably 4) light chain variable region polypeptides. Multivalent antibodies herein may, for example, comprise about 2 and about 8 light chain variable region polypeptides. The light chain variable domain polypeptides contemplated herein comprise a light chain variable domain, and optionally further comprise a CL domain.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello Nature 305:537 (1983), WO 93/08829, and Traunecker et al, EMBO J.10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5731168). See also WO2013/026833 (CrossMab). Multispecific antibodies may also be prepared by engineering electrostatic control effects for the preparation of antibody Fc-heterodimer molecules (WO 2009/089004 A1); crosslinking two or more antibodies (see, e.g., U.S. patent No. 4676980); leucine was used; bispecific antibody fragments are prepared using "diabody" techniques (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and the use of single chain Fv (scFv) dimers (see, e.g., gruber et al, J. Immunol.152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al, J.Immunol.147:60 (1991).

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576). Antibodies herein also include "dual action FAb" or "DAF" comprising antigen binding sites that bind to a variety of sortilin antigens (see, e.g., US 2008/0069820).

Antibodies with improved stability

Amino acid sequence modifications of the anti-sortilin antibodies of the disclosure, or antibody fragments thereof, are also contemplated to improve stability during manufacture, storage, and in vivo administration. For example, it may be desirable to reduce degradation of antibodies or antibody fragments of the present disclosure by a variety of pathways, including, but not limited to, oxidation and deamidation. Amino acid sequence variants of antibodies or antibody fragments are prepared by introducing appropriate nucleotide changes into nucleic acids encoding the antibodies or antibody fragments, or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the amino acid sequence of an antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics (i.e., reduced sensitivity to degradation, and one or more of the characteristics of the anti-sortilin antibodies of the present disclosure described herein).

Antibody variants

In some embodiments of any of the antibodies provided herein, amino acid sequence variants of the antibodies are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies.

Substitution, insertion and deletion variants

In some embodiments of any of the antibodies provided herein, antibody variants having one or more amino acid substitutions are provided. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues from the amino acid sequence of the antibody.

Table 2: amino acid substitutions

Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that have a significant difference in the size of the (a) structure of the polypeptide backbone in the region of the substitution (e.g., as a sheet or helical conformation), (b) charge or hydrophobicity of the molecule at the target site, or (c) side chains. Naturally occurring residues are grouped based on common side chain properties:

(1) Hydrophobic: norleucine, met, ala, val, leu, ile;

(2) Neutral hydrophilicity: cys, ser, thr, asn, gin;

(3) Acid: asp, glu;

(4) Base: his, lys, arg;

(5) Residues that affect chain orientation: gly, pro; and

(6) Fragrance: TRP, tyr, phe.

For example, a non-conservative substitution may involve exchanging a member of one of these classes for a member of another class. Such substituted residues may be introduced, for example, in regions of human antibodies that are homologous to non-human antibodies, or in non-homologous regions of the molecule.

In making changes to the polypeptides or antibodies described herein, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index according to its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydrophilic amino acid index in conferring interactive biological function on proteins is known in the art. Kyte et al, J.mol.biol.,157:105-131 (1982). It is known that certain amino acids may be substituted with other amino acids having similar hydropathic indices or scores and still retain similar biological activity. In making the change based on the hydropathic index, in certain embodiments, substitution of amino acids within ±2 of the hydropathic index is included. In certain embodiments, those included within ±1, and in certain embodiments, those included within ±0.5.

It will also be appreciated in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly when the resulting biofunctional proteins or peptides are used in immunological embodiments. In certain embodiments, the maximum local average hydrophilicity of an amino acid (determined by the hydrophilicity of its adjacent amino acids) is related to its immunogenicity and antigenicity, i.e., to the biological properties of the protein.

The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0±1); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based on similar hydrophilicity values, in certain embodiments, substitutions of amino acids having hydrophilicity values within ±2 are included, in certain embodiments, those within ±1 are included, and in certain embodiments, those within ±0.5 are included. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity. These regions are also referred to as "epitope core regions".

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such alterations do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the HVR that do not substantially reduce binding affinity. Such alterations may be, for example, outside of antigen-contacting residues in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, three, four, five, or six amino acid substitutions.

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusion of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

Any cysteine residues that do not participate in maintaining the correct conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to increase its stability (particularly when the antibody is an antibody fragment, such as an Fv fragment).

Glycosylation variants

In some embodiments of any of the antibodies provided herein, the antibody is altered to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of an antibody glycosylation site can be conveniently accomplished by altering the amino acid sequence so as to create or remove one or more glycosylation sites.

Glycosylation of antibodies is typically N-linked or O-linked. N-linkage refers to the attachment of the carbohydrate moiety to the side chain of the asparagine residue. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic attachment of a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to antibodies of the present disclosure can be conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above tripeptide sequences (for an N-linked glycosylation site). Alterations (for O-linked glycosylation sites) can also be made by adding or replacing one or more serine or threonine residues in the sequence of the original antibody.

When an antibody of the present disclosure comprises an Fc region, the carbohydrates attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched double-antennary oligosaccharides, which are linked to Asn297, typically by an N-bond, according to the Kabat numbering of the CH2 domain of the Fc region. Oligosaccharides may include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to GlcNAc in the "stem" of a double-antennary oligosaccharide structure. In some embodiments, oligosaccharides in antibodies of the present disclosure may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. See, for example, U.S. patent publication nos. 2003/0157108 and 2004/0093621. Examples of publications related to "defucosylation" or "fucose deficiency" antibody variants include: US 2003/0157108; US 2003/015614; US 2002/0164328; US 2004/0093621; US 2004/013321; US 2004/010704; US 2004/0110282; US 2004/0109865; okazaki et al, J.mol.biol.336:1239-1249 (2004); yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Led3CHO cells lacking protein fucosylation (Ripka et al, arch. Biochem. Biophysis. 249:533-545 (1986); US 2003/0157108), and knockout cell lines, such as the α -1, 6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., yamane-Ohnuki et al, biotech. Bioeng.87:614 (2004), and Kanda et al, biotechnol. Bioeng.94 (4): 680-688 (2006)).

Modified constant regions

In some embodiments of any of the antibodies provided herein, the antibody comprises a wild-type or unmodified Fc region comprising one or more modifications, e.g., relative to the same isotype. In some embodiments, the Fc region is capable of binding to an fcγ receptor.

In some embodiments of any of the antibodies provided herein, the modified antibody Fc region is an IgG1 modified Fc region. In some embodiments, the IgG1 modified Fc region comprises one or more modifications. For example, in some embodiments, the IgG1 modified Fc region comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments, the one or more amino acid substitutions are selected from (Bolt S et al, (1993) Eur J Immunol 23:403-411), D265A (Shields et al, (2001) R.J.biol.chem.276, 6591-6604), L234A, L235A (Hutchins et al, (1995) Proc Natl Acad Sci USA,92:11980-11984; alegre et al, (1994) transition 57:1537-1543.31; xu et al, (2000) Cell Immunol, 200:16-26), G237A (Alegre et al, (1994) transition 57:1537-1543.31; xu et al, (2000) C226S, C229S, E233P, L234V, L234F, L235E (Mcware et al, (2007) 118:1195-109), and/1192), G237A (Alegre et al, (1994) transition 57:1537-1543.31; xu et al, (2000) Cell Immunol, 200:16-26), C226S, C229S, E233P, L234V, L234F, L235E (Mcware et al, (2007) and (1996) amino acid positions 328/328, 35S, 35E, 35S, and so forth.

In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises an N297A mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises D265A and N297A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises a D270A mutation according to EU numbering. In some embodiments, the IgG1 modified Fc region provided herein comprises L234A and L235A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises L234A and G237A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises L234A, L235A and G237A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises one or more (including all) of the P238D, L328E, E233, G237D, H268D, P271G and a330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises one or more S267E/L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises P238D, L328E, E233D, G237D, H268D, P271G and a330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises P238D, L328E, G237D, H268D, P271G and a330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises P238D, S267E, L328E, E233D, G237D, H268D, P271G and a330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises P238D, S267E, L328E, G237D, H268D, P271G and a330R mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises C226S, C229S, E233P, L234V and L235A mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises L234F, L235E and P331S mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises S267E and L328F mutations according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises an S267E mutation according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises replacing the constant heavy chain 1 (CH 1) and hinge region of IgG1 with the CH1 and hinge region of IgG2 having a kappa light chain (amino acids 118-230 of IgG2 according to EU numbering).

In some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc comprises two or more amino acid substitutions that increase antibody aggregation without activating complement as compared to a corresponding antibody having an Fc region that does not comprise the two or more amino acid substitutions. Thus, in some embodiments of any of the IgG1 modified Fc regions provided herein, the Fc region comprises an amino acid substitution at position E430G and one or more amino acid substitutions at residue positions in the Fc region selected from the group consisting of: L234F, L235A, L E, S267E, K322A, L328F, A330S, P331S according to EU numbering and any combination thereof. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G, L243A, L235A and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G, a330S, and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G, K322A, A S and P331S according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G, K322A and a330S according to EU numbering. In some embodiments, the IgG1 modified Fc region comprises amino acid substitutions at positions E430G, K322A and P331S according to EU numbering.

In some embodiments of any of the IgG1 modified Fc regions provided herein, the IgG1 modified Fc may further comprise an a330L mutation (Lazar et al Proc Natl Acad Sci USA,103:4005-4010 (2006)), or one or more of the L234F, L235E and/or P331S mutations (Sazinsky et al Proc Natl Acad Sci USA,105:20167-20172 (2008)), according to EU numbering convention, to eliminate complement activation. In some embodiments of any of the IgG1 modified Fc regions provided herein, the IgG1 modified Fc can further comprise one or more of a330L, A330S, L234F, L235E and/or P331S amino acid substitutions according to EU numbering. In some embodiments of any of the IgG1 modified Fc regions provided herein, the IgG1 modified Fc can further comprise one or more mutations to increase antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S T and T256E mutations according to EU numbering convention). In some embodiments of any of the IgG1 modified Fc regions provided herein, the IgG1 modified Fc may further comprise one or more of E430G, E430S, E430F, E430T, E345K, E Q, E345R, E345Y, S440Y and/or S440W amino acid substitutions according to EU numbering.

In some embodiments of any of the antibodies provided herein, the antibody has a human IgG1 isotype (huIgG 1) and the Fc region comprises one or more amino acid substitutions selected from the group consisting of N297A, N297Q, D265A, L234A, L235A, C226S, C229S, P238S, E P, L234V, P238A, A327Q, A327G, P329A, K A, L234F, L235E, P S, T394D, A330L, M Q, A252Y, S T or T256E and any combination thereof, wherein the numbering of residues is according to EU numbering. In some embodiments of any of the antibodies provided herein, the antibody comprising an IgG1Fc region further comprises one or more amino acid substitutions selected from a330L, L234F, L235E or P331S, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments of any of the antibodies provided herein, the antibody comprising an IgG1Fc region further comprises one or more amino acid substitutions selected from the group consisting of M252Y, S T or T256E, and any combination thereof, wherein the numbering of the residues is according to EU numbering.

In some embodiments of any of the antibodies provided herein, the antibody has a human IgG4 isotype (huIgG 4) and the Fc region comprises one or more amino acid substitutions selected from the group consisting of E233P, F234V, L a/F234A, L235A, G237A, E318A, S228P, L236E, S241P, L248E, T394D, M252Y, S254T, T256E, N297A, N297Q and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments of any of the antibodies provided herein, the antibody comprising an IgG4Fc region further comprises one or more amino acid substitutions selected from the group consisting of M252Y, S T or T256E, and any combination thereof, wherein the numbering of the residues is according to EU numbering. In some embodiments of any of the antibodies provided herein, the antibody comprising an IgG4Fc region further comprises an S228P amino acid substitution, wherein the numbering of the residues is according to EU numbering.

In some embodiments of any of the antibodies provided herein, the antibody has a human IgG1 isotype (huIgG 1) and the Fc region comprises amino acid substitutions at positions L234A, L235A and P331S, wherein numbering of the residue positions is according to EU numbering. In some embodiments, the antibody comprises a huIgG1Fc region comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody comprises a huIgG1Fc region comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:26, and a sequence of amino acids. In some embodiments, the antibody comprises a polypeptide comprising SEQ ID NO:31 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no. In some embodiments, the antibody comprises a polypeptide comprising SEQ ID NO:32 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no.

In some embodiments of any of the antibodies provided herein, the modified antibody Fc region is an IgG2 modified Fc region. In some embodiments, the IgG2 modified Fc region comprises one or more modifications. For example, in some embodiments, the IgG2 modified Fc region comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments of any one of the IgG2 modified Fc regions provided herein, the one or more amino acid substitutions is selected from the group consisting of V234A (Alegre et al, transformation 57:1537-1543 (1994); xu et al, cell Immunol,200:16-26 (2000)); G237A (Cole et al, transformation, 68:563-571 (1999)); H268Q, V309L, A330S, P331S (US 2007/0148167; armour et al, eur J Immunol 29:2613-2624 (1999); armour et al The Haematology Journal (suppl.1): 27 (2000); armour et al The Haematology Journal (suppl.1): 27 (2000)); C219S, and/or C220S (White et al, cancer Cell 27,138-148 (2015)); S267E, L328F (Chu et al, mol Immunol,45:3926-3933 (2008)); or M252Y, S254T, and/or T256E, according to EU numbering convention. In some embodiments of any of the IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions V234A and G237A according to EU numbering. In some embodiments of any of the IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions C219S or C220S according to EU numbering. In some embodiments of any of the IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions a330S and P331S according to EU numbering. In some embodiments of any of the IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering.

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc comprises a C127S amino acid substitution according to EU numbering convention (White et al, (2015) Cancer cell27,138-148; light et al, protein Sci.19:753-762 (2010), and WO 2008/079246). In some embodiments of any of the IgG2 modified Fc regions, the antibodies have an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to the EU numbering convention (White et al, cancer Cell 27:138-148 (2015); light et al, protein Sci.19:753-762 (2010); and WO 2008/079246).

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc comprises a C220S amino acid substitution according to EU numbering convention. In some embodiments of any IgG2 modified Fc region, the antibody has an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to EU numbering convention.

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc comprises a C219S amino acid substitution according to EU numbering convention. In some embodiments of any IgG2 modified Fc region, the antibody has an IgG2 isotype with a kappa light chain constant domain comprising a C214S amino acid substitution according to EU numbering convention.

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc comprises IgG2 isotype heavy chain constant domain 1 (CH 1) and a hinge region (White et al, cancer Cell27:138-148 (2015)). In certain embodiments of any of the IgG2 modified Fc regions provided herein, the IgG2 isotype CH1 and the hinge region comprise amino acid sequences according to EU numbering of amino acids 118-230. In some embodiments of any IgG2 modified Fc region, the antibody Fc region comprises an S267E amino acid substitution, an L328F amino acid substitution, or both, and/or an N297A or N297Q amino acid substitution according to EU numbering convention.

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc further comprises one or more amino acid substitutions at positions E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S Y and S440W according to EU numbering. In some embodiments of any IgG2 modified Fc region, the Fc may further comprise one or more mutations to enhance the half-life of the antibody in human serum (e.g., one or more (including all) of the M252Y, S T and T256E mutations according to the EU numbering convention). In some embodiments of any IgG2 modified Fc region, the Fc may further comprise a330S and P331S amino acid substitutions.

In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc is an IgG2/4 hybrid Fc. In some embodiments, the IgG2/4 hybrid Fc comprises IgG2 amino acids 118-260 and IgG4 amino acids 261-447. In some embodiments of any of the IgG2 modified Fc regions provided herein, the Fc comprises one or more amino acid substitutions at positions H268Q, V309L, A S and P331S according to EU numbering.

In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions provided herein, the Fc comprises one or more additional amino acid substitutions according to EU numbering selected from a330L, L234F, L235E or P331S, and any combination thereof.

In certain embodiments of any of the IgG1 and/or IgG2 modified Fc regions provided herein, the Fc comprises one or more amino acid substitutions at residue positions selected from the group consisting of C127S, L234A, L234F, L235A, L235E, S267E, K A, L328 82348 330S, P331S, E345R, E430G, S Y, and any combination thereof, according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A and P331S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, A S and P331S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, K322A, A S and P331S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, K322A and a330S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, K322A and P331S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some embodiments of any of the IgG1 and/or IgG2 modified Fc regions, the Fc comprises amino acid substitutions at positions E345R, E430G and S440Y according to EU numbering.

In some embodiments of any of the antibodies provided herein, the antibody has an IgG4 isotype and comprises a modified Fc region. In some embodiments, the IgG4 modified Fc region comprises one or more modifications. For example, in some embodiments, the IgG4 modified Fc region comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc region of the same isotype). In some embodiments of any of the IgG4 modified Fc regions provided herein, the one or more amino acid substitutions are selected from L235A, G237A, S229P, L236E (Reddy et al, J Immunol164:1925-1933 (2000)), S267E, E318A, L328F, M252Y, S254T, and/or T256E, according to the EU numbering convention. In some embodiments of any IgG4 modified Fc region, the Fc may further comprise L235A, G237A and E318A amino acid substitutions according to the EU numbering convention. In some embodiments of any IgG4 modified Fc region, the Fc may further comprise S228P and L235E amino acid substitutions according to EU numbering convention. In some embodiments of any IgG4 modified Fc region, the IgG4 modified Fc may further comprise S267E and L328F amino acid substitutions according to EU numbering convention.

In some embodiments of any of the IgG4 modified Fc regions provided herein, the IgG4 modified Fc comprises an S228P mutation according to EU numbering convention (Angal et al, mol immunol.30:105-108 (1993)) and/or Peters et al, J Biol chem.287 (29): 24525-33 (2012) to enhance antibody stability.

In some embodiments of any of the IgG4 modified Fc regions provided herein, the IgG4 modified Fc can further comprise one or more mutations to increase antibody half-life in human serum (e.g., one or more (including all) of the M252Y, S T and T256E mutations according to EU numbering convention).

In some embodiments of any of the IgG4 modified Fc regions provided herein, the Fc comprises an L235E amino acid substitution according to EU numbering. In certain embodiments of any of the IgG4 modified Fc regions, the Fc comprises one or more amino acid substitutions according to EU numbering at a residue position selected from the group consisting of C127S, F234A, L235A, L235E, S267E, K322A, L F, E345R, E430G, S440Y and any combination thereof. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G, L243A, L235A and P331S according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G and P331S according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises an amino acid substitution at position E430 according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc region comprises amino acid substitutions at positions E430G and K322A according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises amino acid substitutions at positions S267E and L328F according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises an amino acid substitution at position C127S according to EU numbering. In some embodiments of any of the IgG4 modified Fc regions, the Fc comprises amino acid substitutions at positions E345R, E430G and S440Y according to EU numbering.

Nucleic acids, vectors and host cells

The anti-sortilin antibodies of the present disclosure may be produced using recombinant methods and compositions, for example, as described in U.S. patent No. 4817567. In some embodiments, there is provided a polypeptide having any anti-sortilin encoding the disclosureAn isolated nucleic acid of the nucleotide sequence of an antibody. Such nucleic acids may encode a light chain variable domain comprising an anti-sortilin antibody (V _L ) And/or comprising the heavy chain variable domain of an anti-sortilin antibody (V _H ) (e.g., the light chain and/or the heavy chain of an antibody). In some embodiments, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In some embodiments, host cells comprising such nucleic acids or vectors are also provided. In some embodiments, the host cell comprises (e.g., has been transduced with): (1) Comprising encoding a polypeptide comprising antibody V _L A vector comprising (2) a nucleic acid encoding an amino acid sequence comprising antibody V, or (2) a nucleic acid comprising an amino acid sequence comprising antibody VH _L First vector comprising a nucleic acid encoding an amino acid sequence comprising antibody V _H A second vector of a nucleic acid of an amino acid sequence of (a). In some embodiments, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, sp20 cell). Host cells of the present disclosure also include, but are not limited to, isolated cells, in vitro cultured cells, and ex vivo cultured cells.

Methods of making the anti-sortilin antibodies of the disclosure are provided. In some embodiments, the methods comprise culturing a host cell of the present disclosure comprising a nucleic acid encoding an anti-sortilin antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

For recombinant production of the anti-sortilin antibodies of the disclosure, nucleic acids encoding the anti-sortilin antibodies are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of antibodies).

Suitable vectors comprising nucleic acid sequences encoding any of the anti-sortilin antibodies of the disclosure include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors may be constructed according to standard techniques or may be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell to be used, useful cloning vectors generally have the ability to self-replicate, may have a single target for a particular restriction endonuclease, and/or may carry a marker gene that may be used to select for clones comprising the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, bluescript (e.g., pBSSK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, colE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, strategene and Invitrogen.

Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells. For example, the anti-sortilin antibodies of the present disclosure may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5648237, 5789199 and 5840523. After expression, the antibodies may be isolated from the bacterial cell paste in soluble fractions and may be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of antibodies with a partially or fully human glycosylation pattern (e.g., gerngross Nat. Biotech.22:1409-1414 (2004); and Li et al, nat. Biotech.24:210-215 (2006)).

Suitable host cells for expressing glycosylated antibodies may also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda cells. Plant cell cultures may also be used as hosts (e.g., U.S. Pat. nos. 5959177, 6040498, 6420548, 7125978 and 6417429, describing PLANTIBODIES for antibody production in transgenic plants) ^TM Technology).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed with SV40 (COS-7); human embryonic kidney cell lines (293 or 293 cells, e.g., as described by Graham et al, J.Gen. Virol.36:59 (1977); hamster kidney cells (BHK); mouse support cells (TM 4 cells, e.g., as described by Mather, biol. Reprod.23:243-251 (1980)); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (HepG 2); mouse mammary tumor (MMT 060562) cells; TRI cells, for example as described by Mather et al, annals N.Y. Acad.Sci.383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, proc.Natl. Acad. Sci.USA 77:4216 (1980)); myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., yazaki and Wu, methods in Molecular Biology, volume 248 (b.k.c.lo, ed., humana Press, totowa, NJ), pp.255-268 (2003).

Diagnostic use

The anti-sortilin antibodies of the present disclosure also have diagnostic utility. Thus, the present disclosure provides methods of using the antibodies of the present disclosure, or functional fragments thereof, for diagnostic purposes, e.g., detecting sortilin in an individual or a tissue sample from an individual.

In some embodiments, the individual is a human. In some embodiments, the individual is a human patient suffering from or at risk of developing a disease, disorder, or injury of the present disclosure. In some embodiments, the diagnostic method comprises detecting sortilin in a biological sample, such as a biopsy sample, tissue, or cell. In some embodiments, an anti-sortilin antibody described herein is contacted with a biological sample and antigen-bound antibodies are detected. For example, in order to detect and/or quantify the levels of disease-associated cells or sortilins, biopsy samples may be stained with an anti-sortilin antibody as described herein. The detection method may comprise quantification of antigen-binding antibodies. Detection of antibodies in biological samples can be performed by any method known in the art, including immunofluorescence microscopy, immunocytochemistry, immunohistochemistry, ELISA, FACS analysis, immunoprecipitation, or micro-positron emission tomography. In certain embodiments, the antibody is radiolabeled, e.g., with 18F, and subsequently detected using a micro-positron emission tomography assay. Antibody binding can also be quantified in individuals by non-invasive techniques such as Positron Emission Tomography (PET), X-ray computed tomography, single Photon Emission Computed Tomography (SPECT), computed Tomography (CT), and Computed Axial Tomography (CAT).

In other embodiments, the anti-sortilin antibodies of the disclosure may be used to detect and/or quantify microglial cells in brain samples taken, for example, from a preclinical disease model (e.g., a non-human disease model, such as a mouse or cynomolgus monkey disease model). Thus, for example, the anti-sortilin antibodies of the present disclosure may be used to evaluate a therapeutic response following treatment in a model of a disease, disorder or injury, such as frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophies, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, or osteoarthritis, as compared to a control. In some embodiments, the anti-sortilin antibodies of the disclosure may be used to assess post-treatment therapeutic response in a model of a neurodegenerative or neurological disease or disorder (such as frontotemporal dementia, alzheimer's disease, or parkinson's disease), e.g., as compared to a control.

Biomarkers

Particulate protein precursors

In some embodiments, the methods of treating or slowing the progression of a disease or disorder provided herein further comprise measuring the level of granulin precursor protein in a blood (e.g., plasma or serum) or cerebrospinal fluid sample obtained from the subject before and after the subject receives one or more doses of an anti-sortilin antibody of the disclosure.

In some embodiments, administering an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein results in an increase in the level of a granulin precursor protein in the plasma and/or cerebrospinal fluid of the individual as compared to prior to administration of the anti-sortilin antibody. In some embodiments, administration of an anti-sortilin antibody of the present disclosure to an individual according to the methods provided herein results in an increase in the level of a granulin precursor protein in the plasma and/or cerebrospinal fluid of the individual of at least about 1.4 fold, at least about 1.8 fold, at least about 2 fold, at least about 2.2 fold, at least about 2.4 fold, at least about 2.6 fold, at least about 2.8 fold, at least about 3 fold, at least about 3.2 fold, at least about 3.4 fold, at least about 3.6 fold, at least about 3.8 fold, at least about 4 fold, at least about 4.2 fold, at least about 4.8 fold, at least about 5 fold, at least about 5.2 fold, at least about 5.4 fold, at least about 5.6 fold. At least about 5.8 fold, at least about 6 fold, at least about 6.2 fold, at least about 6.4 fold, at least about 6.6 fold, at least about 6.8 fold, at least about 7 fold, at least about 7.2 fold, at least about 7.4 fold, at least about 7.6 fold, at least about 7.8 fold, at least about 8.2 fold, at least about 8.4 fold, at least about 8.6 fold, at least about 8.8 fold, at least about 9 fold, at least about 9.2 fold, at least about 9.4 fold, at least about 9.6 fold, at least about 9.8 fold, at least about 10 fold or more as compared to prior to administration of the anti-sortilin antibody.

In some embodiments, administering an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein results in an increase in the level of a granulin precursor protein in the plasma and/or cerebrospinal fluid of the individual compared to before the administration of the anti-sortilin antibody of at least about any one of: 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more. In some embodiments, administering an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein results in an increase in the level of a granulin precursor protein in the plasma and/or cerebrospinal fluid of the individual compared to before the administration of the anti-sortilin antibody of at least about any one of: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more.

In some embodiments, the increase in the level of granulin precursor protein in the plasma and/or cerebrospinal fluid of the subject is present at any one of about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma and/or cerebrospinal fluid of the subject is present at any one of about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more after administration of the anti-sortilin antibody.

In some embodiments, the increase in the level of granulin precursor protein in the plasma and/or cerebrospinal fluid of the subject is at any of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days after administration of the anti-sortilin antibody. About 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 70, about 71, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 98, about 101, about 102, about 103, about 101, about 100, about 104, about about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 1280, about 129, about 131, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 168, about 170, about 172, about 169, about 172, about 146, any one of about 173, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 191, about 192, about 193, about 194, about 198, about 199, about 200, about 201 or more days.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual at a dose of about 6mg/kg according to the methods provided herein results in a decrease in plasma protein levels of the individual compared to the plasma protein levels of the individual prior to administration of the anti-sortilin antibody, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.2 fold, or more than about 4.5 fold. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of a granulin precursor protein in the plasma of the individual by at least about 2-fold or more as compared to the level of the granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 3-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to a method provided herein at a dose of about 6mg/kg results in an increase in the level of a granulin precursor protein in the plasma of the individual of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250% or more compared to the level of the granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject is present at any one of about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 38 days, about 39 days, at any one of about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 42 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 28 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual at a dose of about 15mg/kg according to the methods provided herein results in a decrease in plasma protein levels in the individual compared to the plasma protein levels in the individual prior to administration of the anti-sortilin antibody, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.2 fold, or more than about 4.5 fold. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 15mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 2-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 15mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 3-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to a method provided herein at a dose of about 15mg/kg results in an increase in the level of a granulin precursor protein in the plasma of the individual of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250% or more compared to the level of the granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, an increase in the level of granulin precursor protein in the plasma of the subject occurs at any one of about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 59 days, about 60 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 42 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual at a dose of about 30mg/kg according to the methods provided herein results in a decrease in plasma protein levels in the individual compared to the plasma protein levels in the individual prior to administration of the anti-sortilin antibody, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.2 fold, or more than about 4.5 fold. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 2-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 3-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of a granulin precursor protein in the plasma of the individual compared to the level of the granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody of at least about any one of: 5%, 10%, 15%, 20%, 25%, 30%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more. In some embodiments, an increase in the level of granulin precursor protein in the plasma of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 18 days, about 21 days, about 23 days, about 24 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 59 days, about 60 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 56 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 57 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual at a dose of about 60mg/kg according to the methods provided herein results in a decrease in plasma protein levels in the individual compared to the plasma protein levels in the individual prior to administration of the anti-sortilin antibody, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 1.6 fold, at least about 1.7 fold, at least about 1.8 fold, at least about 1.9 fold, at least about 2 fold, at least about 2.1 fold, at least about 2.2 fold, at least about 2.4 fold, at least about 2.5 fold, at least about 2.6 fold, at least about 2.7 fold, at least about 2.8 fold, at least about 2.9 fold, at least about 3 fold, at least about 3.1 fold, at least about 3.2 fold, at least about 3.3 fold, at least about 3.4 fold, at least about 3.5 fold, at least about 3.6 fold, at least about 3.7 fold, at least about 3.8 fold, at least about 3.9 fold, at least about 4.1 fold, at least about 4.2 fold, at least about 4.3 fold, at least about 4.4 fold, at least about 4.5 fold, at least about 4.6 fold, at least about 4.2 fold, or more than about 4.5 fold. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 2-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of granulin precursor protein in the plasma of the individual of at least about 3-fold or more as compared to the level of granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of a granulin precursor protein in the plasma of the individual compared to the level of the granulin precursor protein in the plasma of the individual prior to administration of the anti-sortilin antibody of at least about any one of: 5%, 10%, 15%, 20%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more. In some embodiments of the present invention, in some embodiments, an increase in the plasma granulin level of the subject after administration of the anti-sortilin antibody is about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 58 days, about 59 days, about 60 days, about 61 days, about about 62, about 63, about 66, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 95, about 98, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 119, about 120 or more days. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 84 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 84 days or more after administration of the anti-sortilin antibody.

In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 150mg results in an increase in the level of a granulin precursor protein in the plasma of the individual by at least about any one of: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 28 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 28 days or more after administration of the anti-sortilin antibody.

In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 300mg results in an increase in the level of a granulin precursor protein in the plasma of the individual by at least about any one of: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 28 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 28 days or more after administration of the anti-sortilin antibody.

In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure according to the methods provided herein to an individual at a dose of about 600mg results in an increase in the level of a granulin precursor protein in the plasma of the individual by at least about any one of: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody to about 28 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the plasma of the individual occurs about 28 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the present disclosure to an individual according to a method provided herein at a dose of about 6mg/kg results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25% or more as compared to the level of a granulin precursor protein in cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 5% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 10% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 12% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 15% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 6mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 18% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, an increase in the level of granulin precursor protein in cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 59 days, about 60 days, or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 25 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 57 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to a method provided herein results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% or more as compared to the level of the granulin precursor protein in cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 15mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 15% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 15mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 60% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 15mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 80% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, an increase in the level of granulin precursor protein in cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 59 days, about 60 days, or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 25 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 57 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to a method provided herein results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% or more as compared to the level of the granulin precursor protein in cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 15% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 60% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 30mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 80% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, an increase in the level of granulin precursor protein in cerebrospinal fluid of an individual occurs at about 1 day, about 2 days, about 3 days, about 6 days, about 7 days, about 8 days, about 13 days, about 14 days, about 18 days, about 21 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 58 days, about 59 days, about 60 days, about 61 days, about 62 days, about 63 days, about 66 days, about 68 days, about 69 days, about 70 days, about 73 days, about 82 days, about 73 days, about 80 days, about 73 days, about 72 days, about 82 days, about 73 days, or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 25 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 57 days or more after administration of the anti-sortilin antibody.

In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to a method provided herein results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100% or more as compared to the level of the granulin precursor protein in cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 15% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 60% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments, intravenous administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein at a dose of about 60mg/kg results in an increase in the level of granulin precursor protein in the cerebrospinal fluid of the individual of at least about 80% or more as compared to the level of granulin precursor protein in the cerebrospinal fluid of the individual prior to administration of the anti-sortilin antibody. In some embodiments of the present invention, in some embodiments, an increase in the level of granulin precursor protein in cerebrospinal fluid of an individual after administration of an anti-sortilin antibody is about 1 day, about 2 days, about 3 days, about 6 days, about 8 days, about 13 days, about 18 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 39 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, about 60 days, about 61 days, about 62 days, about 63 days, about 64 days, about 51 days, about 52 days, about 55 days, about 57 days, about 58 days, about 56 days, about about 65, about 67, about 69, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120 or more. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 25 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 43 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 57 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 84 days or more after administration of the anti-sortilin antibody.

In some embodiments, subcutaneously administering a dose of an anti-sortilin antibody of the disclosure according to a method provided herein to an individual results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% or more as compared to the level of a granulin precursor protein in cerebrospinal fluid of the individual prior to administering the anti-sortilin antibody. In some embodiments, subcutaneously administering a dose of an anti-sortilin antibody of the disclosure according to a method provided herein to an individual results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% or more as compared to the level of a granulin precursor protein in cerebrospinal fluid of the individual prior to administering the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 8 days, about 13 days, about 18 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 39 days, about 41 days, about 42 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 21 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 42 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 7 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 14 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 27 days or more after administration of the anti-sortilin antibody.

In some embodiments, subcutaneously administering a dose of an anti-sortilin antibody of the disclosure according to a method provided herein to an individual results in an increase in the level of a granulin precursor protein in cerebrospinal fluid of the individual by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70% or more as compared to the level of a granulin precursor protein in cerebrospinal fluid of the individual prior to administering the anti-sortilin antibody. In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure to a subject in accordance with the methods provided herein at a dose of about 600mg results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 40% or more as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure to a subject in accordance with the methods provided herein at a dose of about 600mg results in an increase in the level of granulin precursor in the cerebrospinal fluid of the subject of at least about 35% or more as compared to the level of granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, subcutaneously administering an anti-sortilin antibody of the disclosure to a subject in accordance with the methods provided herein at a dose of about 600mg results in an increase in the level of granulin precursor in the cerebrospinal fluid of the subject of at least about 20% or more as compared to the level of granulin precursor in the cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 1 day, about 2 days, about 3 days, about 6 days, about 8 days, about 13 days, about 18 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days, about 34 days, about 35 days, about 36 days, about 37 days, about 39 days, about 41 days, about 42 days, or longer after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 21 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs within about 1 day after administration of the anti-sortilin antibody to about 42 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 7 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 14 days or more after administration of the anti-sortilin antibody. In some embodiments, the increase in the level of granulin precursor protein in the cerebrospinal fluid of the subject occurs about 27 days or more after administration of the anti-sortilin antibody.

In some embodiments, the level of granulin precursor protein in the plasma or cerebrospinal fluid of the subject is measured in a sample obtained from the subject. In some embodiments, the level of granulin precursor protein in the plasma of the individual is measured in a blood sample obtained from the individual. In some embodiments, the level of granulin precursor in the cerebrospinal fluid of the subject is measured in a cerebrospinal fluid sample obtained from the subject. In some embodiments, the level of granulin precursor in the plasma or cerebrospinal fluid of the subject is determined using any method known in the art for quantifying protein. Non-limiting examples of methods that can be used to quantify the granulin precursor protein include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA). In certain embodiments, the level of granulin precursor protein in the plasma or cerebrospinal fluid of an individual is determined using an ELISA assay.

Sortilin protein

In some embodiments, the methods of treating or slowing the progression of a disease or disorder provided herein further comprise measuring the level of sortilin in leukocytes. In some embodiments, the level of sortilin in leukocytes is measured in a blood sample obtained from an individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure.

In some embodiments, administration of an anti-sortilin antibody of the disclosure to an individual according to the methods provided herein results in a decrease in the level of sortilin in the white blood cells of the individual as compared to the level of sortilin in the white blood cells of the individual prior to administration of the anti-sortilin antibody. In some embodiments, after administration of an anti-sortilin antibody, the level of sortilin in leukocytes of the individual is reduced by at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% as compared to the level of sortilin in leukocytes of the individual prior to administration of the anti-sortilin antibody. In some embodiments, the decrease in sortilin level in leukocytes of the individual occurs at any of about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, or more after administration of the anti-sortilin antibody. In some embodiments, the reduction in sortilin level in leukocytes of the individual occurs at any time of about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or more after administration of the anti-sortilin antibody. In some embodiments, the decrease in sortilin level in leukocytes of the individual is at any of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 34 days, about 35 days after administration of the anti-sortilin antibody. About 36 days, about 37 days, about 38 days, about 39 days, about 40 days, about 41 days, about 42 days, about 43 days, about 44 days, about 45 days, about 46 days, about 47 days, about 48 days, about 49 days, about 50 days, about 51 days, about 52 days, about 53 days, about 54 days, about 55 days, about 56 days, about 57 days, about 58 days, about 59 days, about 60 days, about 61 days, about 62 days, about 63 days, about 64 days, about 65 days, about 66 days, about 67 days, about 68 days, about 69 days, about about 70 days, about 71 days, about 72 days, about 75 days, about 76 days, about 77 days, about 78 days, about 79 days, about 80 days, about 81 days, about 82 days, about 83 days, about 84 days, about 85 days, about 86 days, about 87 days, about 88 days, about 89 days, about 90 days, about 91 days, about 92 days, about 93 days, about 94 days, about 95 days, about 96 days, about 97 days, about 98 days, about 99 days, about 100 days, about 101 days, about 102 days, about 103 days, about 104 days, about about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 169, about 170, about 171, any time in about 172, about 173, about 174, about 175, about 179, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 200, about 201, or more days.

In some embodiments, the level of sortilin in the leukocytes of an individual is determined using any method of quantifying protein known in the art. Non-limiting examples of methods that can be used to quantify sortilin include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blots, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent (ELISA) assays. In certain embodiments, the level of sortilin in leukocytes is determined using an ELISA assay.

Disease and neuroinflammation biomarkers

In some embodiments, the methods of treating or slowing the progression of a disease or disorder provided herein further comprise measuring the level of neurofilament light chain (NF-L). In some embodiments, the level of NF- κl is measured in a blood (e.g., plasma) or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. Non-limiting examples of methods that can be used to measure NF-L levels in samples obtained from individuals include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blots, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, the methods of treating or slowing the progression of a disease or disorder provided herein further comprise measuring the level of Tau. In some embodiments, the level of Tau in a blood or cerebrospinal fluid sample obtained from an individual is measured before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. Non-limiting examples of methods that can be used to measure Tau levels in samples obtained from individuals include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, the methods of treating or slowing the progression of a disease or disorder provided herein further comprise measuring the level of one or more biomarkers of neuroinflammation. In some embodiments, the level of one or more biomarkers of neuroinflammation is measured in a blood or cerebrospinal fluid sample obtained from an individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. Biomarkers of neuroinflammation include, but are not limited to, IL-6, SPP1, IFI2712A, CHIT1, YKL-40, GFAP, YWHAE, CSF1, AIF1, LY86, CD86, and TOP2A. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

Method for monitoring treatment

Also provided herein is a method of monitoring treatment of an individual administered an anti-sortilin antibody of the disclosure.

In some embodiments, the method of monitoring treatment comprises measuring the level of one or more biomarkers selected from the group consisting of granulin precursor protein, GCase protein, neurofilament light chain (NF-L), tau, one or more markers of neuroinflammation, or alpha-synuclein in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. In some embodiments, the method further comprises the step of assessing the activity of the anti-sortilin antibody in the individual based on the level of one or more biomarkers in the sample. In some embodiments, the sample is from cerebrospinal fluid of the subject or blood of the subject, e.g., plasma. In some embodiments, the sample is from cerebrospinal fluid of the subject. In some embodiments, the sample is from the blood of an individual, such as plasma. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, the method of monitoring treatment comprises measuring one or more biomarkers of the cerebrospinal fluid protein group in a cerebrospinal fluid sample obtained from an individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. In some embodiments, the method further comprises the step of assessing the activity of the anti-sortilin antibody in the individual based on the level of one or more biomarkers in the sample. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), mass spectrometry, western blotting, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

In some embodiments, the method of monitoring treatment comprises measuring the level of one or more biomarkers of lysosomal function in a sample obtained from an individual before and after the individual has received one or more doses of an anti-sortilin antibody of the disclosure. In some embodiments, the one or more biomarkers of lysosomal function are selected from the group consisting of GCase protein, GCase activity, lyso-Gb1, or glucosylceramide. In some embodiments, the method further comprises the step of assessing the activity of the anti-sortilin antibody in the individual based on the level of one or more biomarkers in the sample. In some embodiments, the sample is from cerebrospinal fluid of the subject or blood of the subject, e.g., plasma. In some embodiments, the sample is from cerebrospinal fluid of the subject. In some embodiments, the sample is from the blood of an individual, such as plasma. Non-limiting examples of methods that can be used to measure the level of one or more biomarkers in a sample obtained from an individual include somcan assays (see, e.g., candia et al, (2017) Sci Rep 7,14248), western blotting, mass spectrometry, flow cytometry, and enzyme-linked immunosorbent assays (ELISA).

Pharmaceutical composition

Provided herein are pharmaceutical compositions and/or pharmaceutical formulations comprising an anti-sortilin antibody of the present disclosure and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutically acceptable carrier is preferably non-toxic to the recipient at the dosage and concentration used. The antibodies described herein may be formulated in solid, semi-solid, liquid, or gaseous form. Examples of such formulations include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Depending on the desired formulation, the pharmaceutically acceptable carrier may include a pharmaceutically acceptable non-toxic carrier of diluents, which is a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. In certain embodiments, the pharmaceutical composition may comprise a formulation material for altering, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeation of the composition.

In certain embodiments, pharmaceutically acceptable carriers include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); an antimicrobial agent; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., borates, bicarbonates, tris-HCl, citrates, phosphates, or other organic acids); fillers (e.g., mannitol or glycine); chelating agents (e.g., ethylenediamine tetraacetic acid); complexing agents (e.g. caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); a filler; a monosaccharide; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrins); proteins (e.g., serum albumin, gelatin, or immunoglobulins); coloring agents, flavoring agents, and diluents; an emulsifying agent; hydrophilic polymers (e.g., polyvinylpyrrolidone); a low molecular weight polypeptide; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); a suspending agent; surfactants or wetting agents (e.g., pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapol); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancing agents (e.g., alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); conveying the carrier; a diluent; excipients and/or pharmaceutical adjuvants. Other examples of formulations suitable for various types of administration can be found in Remington, the Science and Practice of Pharmacy, pharmaceutical Press, 22 nd edition, (2013). For a brief review of drug delivery methods, see Langer, science 249:1527-1533 (1990).

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickening agents, stabilizers and preservatives.

Formulations can be optimized to remain and stabilize in the brain or central nervous system. When the agent is administered into the cranial cavity, the agent desirably remains in the cavity and does not diffuse or otherwise cross the blood brain barrier. Stabilization techniques include crosslinking, multimerization, or attachment to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, and the like, to achieve an increase in molecular weight.

Other strategies to increase retention include capturing antibodies in biodegradable or bioerodible implants, such as the anti-sortilin antibodies of the disclosure. The release rate of the antibody is controlled by the transport rate through the polymer matrix and the biodegradation of the implant. The implant may be a particle, sheet, patch, plate, fiber, microcapsule, etc., and may be of any size or shape compatible with the selected insertion site. Biodegradable polymer compositions that can be used can be organic esters or ethers that, when degraded, produce physiologically acceptable degradation products, including monomers. Anhydrides, amides, orthoesters, and the like, alone or in combination with other monomers. The polymer may be a polycondensate. The polymer may be crosslinked or uncrosslinked. Of particular interest are polymers, homopolymers or copolymers of hydroxy aliphatic carboxylic acids and polysaccharides. Polyesters of interest include polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. Polysaccharides of interest include calcium alginate and functionalized carboxymethyl cellulose, particularly carboxymethyl cellulose esters and the like characterized by being insoluble in water and having a molecular weight of about 5kD to 500 kD. Biodegradable hydrogels may also be used in the implants of the present disclosure. Hydrogels are typically copolymer materials characterized by their ability to absorb liquids.

In some embodiments, the pharmaceutical compositions or formulations provided herein comprise an anti-sortilin antibody of the disclosure and are suitable for administration to human subjects by intravenous infusion and/or subcutaneous injection.

Kit/article of manufacture

Provided herein are articles of manufacture (e.g., kits) comprising the anti-sortilin antibodies of the disclosure. The article of manufacture may comprise one or more containers comprising an antibody as described herein. The container may be any suitable package including, but not limited to, a vial, bottle, can, flexible package (e.g., sealed mylar or plastic bag), and the like. The container may be a unit dose, a bulk package (e.g., a multi-dose package) or a subunit dose.

In some embodiments, the kit may further comprise a second dose. In some embodiments, the second agent is a pharmaceutically acceptable buffer or diluent, including but not limited to bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. In some embodiments, the second agent is a pharmaceutically active agent.

In some embodiments of any article, the article further comprises instructions for use according to the methods of the present disclosure. The instructions generally include information regarding the dosage, dosing regimen, and route of administration of the intended treatment. In some embodiments, these instructions include instructions for administering an antibody of the present disclosure (e.g., an anti-sortilin antibody described herein) according to any method of the present disclosure to prevent, reduce the risk of, or treat a disease, disorder, or injury selected from the group consisting of: frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis or osteoarthritis. In some embodiments, the disease, disorder, or injury is frontotemporal dementia. In some embodiments, the disease, disorder, or injury is alzheimer's disease. In some embodiments, the disease, disorder, or injury is parkinson's disease. In some embodiments, the instructions comprise instructions for using the anti-sortilin antibody and a second agent (e.g., a second pharmaceutically active agent).

The present disclosure will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present disclosure. All citations in this disclosure are expressly incorporated herein by reference.

Examples

Example 1: phase I studies assessing the safety, tolerability, pharmacokinetics, pharmacodynamics and bioavailability of intravenous and subcutaneous anti-sortilin antibodies ALX in healthy volunteers.

This example describes phase I studies assessing the safety, tolerability, pharmacokinetics, pharmacodynamics and bioavailability of an anti-sortilin antibody ALX administered intravenously or subcutaneously in healthy volunteers.

The anti-sortilin antibody ALX comprises a polypeptide comprising SEQ ID NO:31 or 32 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30, and a light chain of the amino acid sequence of seq id no. In this study, the expression of one or more sequences encoding a polypeptide comprising SEQ ID NO:31 and a heavy chain comprising the amino acid sequence of SEQ ID NO:30 to produce an anti-sortilin antibody ALX.

I. Purpose of investigation

The main objective of this study was to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD) and bioavailability of antibody ALX in healthy Human Volunteers (HV) by Intravenous (IV) infusion or as single Subcutaneous (SC) administration at progressively higher single doses. The study was also designed to evaluate the administration of multiple doses of antibody ALX by IV infusion or administration as SC in HV.

A. Measurement of primary results

The main outcome measure of this study was to evaluate the safety and tolerability of antibody ALX, based on the number of subjects who developed Adverse Events (AEs) and Dose Limiting Adverse Events (DLAEs).

B. Secondary outcome measure

Secondary outcome measures for this study include:

the Pharmacokinetic (PK) of antibody ALX was assessed based on the plasma and cerebrospinal fluid (CSF) concentrations of antibody ALX.

Maximum concentration of antibody ALX in serum or plasma and CSF (C _max )。

Area Under Curve (AUC) of antibody ALX based on plasma and CSF concentrations of antibody ALX.

Study participants

The study included healthy male and female volunteers.

A. Criteria for inclusion

The study included healthy human volunteers meeting the following criteria.

·Screening was performed in men or women 18-65 years old (both ends included).

·Body mass index 18.0-35.0kg/m ² (with two ends). />

·The weight is 45-120kg (including two ends).

·There was no clinically significant finding based on medical history, physical examination, laboratory examination, 12-lead Electrocardiogram (ECG) and vital signs, and the body was healthy.

B. Exclusion criteria

Subjects meeting any of the following criteria are not included in the study.

A history of severe allergy, allergy or other hypersensitivity reactions to chimeric, human or humanized antibodies or fusion proteins is known.

Past history of seizure except for febrile seizure in children.

·ScreeningSerious infection with oral or intravenous antibiotics was required for the first 30 days.

·Clinically significant systemic immune compromised conditions are due to the sustained action of immunosuppressive drugs.

A history of major depression, schizophrenia, schizoaffective disorder or bipolar disorder.

·A history of cancer is not actively treated with anti-cancer therapy or radiation therapy, except where it is considered likely to be curative, and is unlikely to require treatment in the next 3 years, and is considered to have a low probability of recurrence.

Hepatitis B surface antigen, anti-hepatitis C virus antibodies or anti-Human Immunodeficiency Virus (HIV) -1 and-2 antibodies or antigens, or a history of spirochete infections of the central nervous system (e.g., syphilis, lyme disease or borreliosis).

·Calculated using the Cockcroft-Gault formula<Screening creatinine Clearance (CL) of 30mL/min indicates the presence of chronic kidney disease, which if retested remains at 30mL/min.

·Impaired liver function is indicated by screening for aspartic acid Aminotransferase (AST) or alanine Aminotransferase (ALT) at > 2 x above the upper limit of normal or total bilirubin at > 1.5 x above the upper limit of normal, and if retested due to slightly elevated initial results or abnormalities in clinically significant synthetic function tests, remain above these limits.

·Over the past 2 years, patients with unstable or clinically significant cardiovascular disease (e.g., myocardial infarctionHeart failure of the heart association class II or above of the heart of new york, angina pectoris).

·Uncontrolled hypertension (i.e. systolic blood pressure)>140mmHg or diastolic pressure>Sustained resting pressure of 90 mmHg).

·A history or presence of clinically significant abnormal electrocardiography, including complete left bundle branch block, secondary or tertiary heart block, or evidence of past myocardial infarction.

QT interval corrected using the friedricia formula: male participants were >450ms and female participants were >470ms (average of three screening measurements).

A history of ventricular arrhythmias or risk factors for ventricular arrhythmias, such as structural heart disease (e.g., severe left ventricular contractile dysfunction or left ventricular hypertrophy), coronary heart disease (symptomatic or ischemic as evidenced by diagnostic testing), clinically significant electrolyte abnormalities (e.g., hypokalemia, hypomagnesemia, or hypocalcemia), or a family history of sudden death or long QT syndrome.

III, study design

The present study was the first human phase I study designed to investigate the safety, tolerability, PK, PD and bioavailability of a single dose of anti-sortilin antibody ALX administered via the IV and SC routes. An overview of the study design is provided in figure 1. The study also evaluated the administration of multiple doses of antibody ALX via IV and SC routes.

The study included intravenous administration of a Single Ascending Dose (SAD) of antibody ALX in about 3 cohorts of 11 HV participants. The dosage levels for the first 3 cohorts were 6mg/kg, 15mg/kg and 30mg/kg. In some embodiments, an additional IV SAD queue of 60mg/kg is included.

In addition to the SAD IV cohort, a Single Dose (SD) cohort evaluates the bioavailability and tolerability of SC administration of antibody ALX. The SD SC cohort included 9 HV participants who administered antibody ALX at a fixed dose of 600 mg. If dosing flexibility is required, another SDSC queue is entered. The following queues were also evaluated: as described below, single dose SC 150mg cohort, multi-dose (MD) SC cohort, and multi-dose IV cohort.

All cohorts required cerebrospinal fluid (CSF) samples to be taken at both pre-dose (baseline) and post-dose time points to assess PK and PD in the brain. The subgroups in each cohort had CSFs sampled at different time points post-dose to allow more time points in the cohort to sample CSF (three total time points post-dose). The CSF subset of IV and SC queues is described below.

All participants in this study were followed up for 12 weeks after single dose administration of antibody ALX to assess safety, PK, PD and bioavailability. In some embodiments, the participants follow-up for 16 weeks after their single dose of antibody ALX.

A. Single dose escalation IV cohort

In each SADIV cohort, 11 HV participants received either antibody ALX or Placebo (PBO) at random, with a ratio of 8:3 (antibody ALX: PBO) in up to 3 dose cohorts (cohorts 1, 2, and 3). A total of about 33 HV participants were included. The first SAD IV cohort was dosed with 6mg/kg antibody ALX. The dose levels for the subsequent SAD IV cohorts were 15mg/kg and 30mg/kg antibody ALX. An additional SAD IV dose of 60mg/kg antibody ALX (not shown in FIG. 1) may be added. In some embodiments, an additional SAD queue of 60mg/kg is included. An overview of the SAD IV queue is provided in Table 3.

Table 3 single dose escalation IV cohort.

CSF samples were collected at baseline, day 25, and day 43 for the first 6 participants of each SAD IV cohort. CSF samples were collected at baseline, day 43 and day 57 for the remaining 5 participants in each SAD IV cohort. The overall randomization of 11 participants in each SAD IV cohort was 8:3 (antibody ALX: PBO). In each SAD IV cohort, 4 participants dosed with antibody ALX on days 25 and 57 and 8 participants dosed with antibody ALX on baseline and 43 were sampled for CSF. If the emerging PK and PD data allow, the CSF time point after administration is altered.

In another embodiment, CSF is sampled at baseline and 2 time points on days 25, 43 or 57 for a 6mg/kg IV cohort (FIG. 15). In another embodiment, CSF is sampled at baseline and 2 points on days 25, 43 or 57 for a 15mg/kg IV cohort (FIG. 15). In another embodiment, CSF is sampled at baseline and 2 time points on days 25, 43, 57 or 85 for a 30mg/kg IV cohort (FIG. 15). In another embodiment, CSF is sampled at baseline and 2 time points on day 43, 57 or 85 for a 60mg/kg IV cohort (FIG. 15). In another embodiment, CSF is sampled at baseline and 2 time points on days 25, 43, 57 or 85 for placebo SDIV cohorts (fig. 15).

Based on the emerging safety, tolerability, PK and PD data from the previous queues, dose levels may be adjusted, one or more queues may be omitted or extended, or intermediate dose queues may be added. The enlarged cohort or cohort of intermediate doses is openly labeled and includes up to 9 participants to whom antibody ALX was administered.

B. Single dose SC cohort-600 mg

The SD SC 600mg queue is the 9HV open label queue. If clinical safety data (including day 13 access) was accessed according to SAD IV cohort 2 (15 mg/kg) day 13, antibody ALX at a dose of 15mg/kg was generally safe and tolerable, then the cohort was entered. A single dose of 600mg antibody ALX was administered to the SDSC cohort, which corresponds to a maximum dose level of 13.3mg/kg of the lowest body weight (45 kg) allowed in this study. SD SC queue has no PBO group.

CSF samples were collected at baseline, day 25 and day 43 for the first 3 participants. The next 3 participants collected CSF samples at baseline, day 25 and day 57. CSF samples were collected at baseline, day 43 and day 57 for the remaining 3 participants. CSF samples of 6 participants were included at each post-dose CSF time point (day 25, day 43 and day 57). If the emerging PK and PD data allow, the CSF time point after administration is altered.

In another embodiment, the first participant in the SD SC600 mg cohort collects CSF samples at baseline, day 25, and day 43. The second participant collected CSF samples at baseline, day 13 and day 25. Participants 3 collect CSF samples at baseline, day 18 and day 25. The next 3 participants collected CSF samples at baseline, day 8 and day 13. The next 3 participants collected CSF samples at baseline, day 8 and day 18. In one embodiment, CSF samples are collected at the time points shown in table 4 below.

Table 4. CSF sample collection time points for single dose SC600 mg cohorts.

C. Single dose SC cohort-150 mg

The single dose SC150 mg cohort is a cohort of 6 HV participants, and a single dose of 150mg antibody ALX was administered on day 1. CSF samples (LP, lumbar puncture) were collected at baseline, day 6 and day 13 (fig. 12).

D. Multi-dose SC cohort

A multi-dose (MD) SC cohort seven doses of 300mg antibody ALX were administered in a cohort of 10 HV participants (fig. 13). A 300mg antibody ALX dose was administered to the subject on day 1, day 15, day 29, day 43, day 57, day 71, and day 85. CSF samples (LP) were collected at baseline, day 92 and day 97.

E. Multiple dose IV cohort

In the MD IV cohort, 10 HV participants received either antibody ALX or Placebo (PBO) at a ratio of 8:2 (antibody ALX: PBO) (FIG. 14). 30mg/kg antibody ALX or placebo was administered to MD IV cohorts on day 1, day 29, day 57 and day 85. CSF samples (LP) were collected at baseline and day 97 and day 113.

F. Drug for testing, dosage and administration route

For IV cohorts, antibody ALX was administered as a single peripheral IV infusion using an infusion pump for about 60 minutes.

For SC cohorts, antibody ALX was administered by slow injection over 15 minutes. The total volume of antibody ALX administered at a dose of 600mg was 12mL.

G. Placebo control, dose and route of administration

For the IV cohort, saline (0.9% NaCl) was administered as PBO as a single peripheral IV infusion using an infusion pump for about 60 minutes.

For the SC queues, there is no PBO group.

H. Duration of the study

The total study duration for each participant was approximately 4 months. In some embodiments, the total study duration for each participant is about 3-6 months. This includes a screening period of up to 28 days prior to study drug administration, a single IV infusion or SC administration of antibody ALX or PBO (IV-only cohort) on day 1, or multiple doses of IV or SC antibody ALX or PBO (IV-only cohort), each according to the dosing regimen of each cohort, and follow-up according to the follow-up period specified for each cohort to day 57 (8 weeks after study drug administration), day 85 (12 weeks after study drug administration), day 113 (16 weeks after study drug administration), or day 141 (20 weeks after study drug administration).

Study procedure

A. Pre-dosing procedure and assessment

Prior to administration of antibody ALX or PBO, the following evaluations were performed:

physical examination or limited, symptom-driven examination.

Neurological examination.

Height and body weight.

Vital signs, including Blood Pressure (BP), pulse, body temperature, and respiratory rate.

Triplicate 12 lead ECG.

Blood and urine samples for chemical, hematology, coagulation, serology and urine analysis.

Blood and urine samples for anti-drug antibody (ADA) and PK analysis; plasma samples for granulin precursors and exploratory biomarkers; and whole blood samples for leukocyte analysis.

Whole blood samples for exploratory biomarkers.

Whole Genome Sequencing (WGS).

CSF samples obtained by lumbar puncture.

Concomitant medication review.

Record Serious Adverse Events (SAE) and AE.

B. Administration of drugs

Antibodies ALX or PBO were administered to participants as described above. No fasting was applicable on the day of administration unless clinical laboratory testing was performed.

C. After administration of the drug

Following administration of antibody ALX or PBO, the following evaluations were performed:

serum samples for PK analysis; plasma samples for granulin precursors and biomarkers; and whole blood samples for leukocyte analysis.

Triplicate 12 lead ECG.

Vital signs, including BP, pulse, body temperature, and respiratory rate.

Concomitant medication review.

Record SAE and AE.

V. study evaluation

A. Clinical assessment

Any past drug received within 30 days prior to screening was recorded. All concomitant medications and concomitant treatments were recorded from screening to follow-up.

All serious adverse events, as well as any adverse events associated with the protocol procedure, were recorded upon informed consent from the first administration of study drug. Any serious adverse events and adverse events, including worsening or alteration of medical history, were recorded from the first administration of study drug to the follow-up.

Demographic information (year of birth, sex, race) was recorded.

All relevant medical history, including medical history or current disease, other relevant medical history and information about underlying disease, was recorded at the time of screening prior to study drug administration.

Complete Physical Examination (PE) was performed at the completion of screening and study or Early Termination (ET). Complete PE includes assessment of head, eyes, ears, nose and throat, assessment of cardiovascular system, skin system, musculoskeletal system, respiratory system and gastrointestinal system. Abnormalities observed at baseline were recorded. Height (in cm) and weight (in kg) were measured at screening and BMI was calculated.

Complete neurological examination includes assessment of consciousness, direction, cranial nerves, motor and sensory systems, coordination and gait, and reflex. Changes in baseline abnormalities were recorded in each subsequent neurological examination. If it is considered clinically significant, the new or worsening abnormality is recorded as AE.

Vital signs were assessed and abnormalities observed at baseline were recorded. In subsequent visits, a new or worsening abnormality is recorded as AE if it is deemed clinically significant. All ECGs were analyzed on a clinical safety basis (no intensive QT analysis). After review of the ECG reports associated with the participant's medical history, PE and concomitant medication, the clinical significance of the ECG changes was determined.

Blood and urine samples were collected for clinical safety laboratory tests (chemistry, hematology, urinalysis, serology, drug and alcohol screening and pregnancy tests). If clinical significance is confirmed and considered, or if participants are required to stop the study or receive treatment, out-of-range values are recorded as AE after the study drug is initiated.

Serum samples were collected for determination of anti-drug antibodies (ADA). Additional ADA samples were collected in participants with signs and symptoms of infusion-related reactions. Corresponding additional PK samples were obtained at the same time points.

B. Security assessment

Adverse Events (AEs) were recorded and rated according to the World Health Organization (WHO) toxicity rating scale. If AE is not specified in the WHO toxicity rating scale, the AE is rated as follows: grade 1 (mild: transient or mild discomfort; unrestricted activity; no need for medical intervention or treatment; participants may know signs or symptoms but are reasonably well tolerated); grade 2 (moderate: mild to moderate restricted activity; no or minimal medical intervention/treatment); grade 3 (severe: significant limitation of activity; need for medical intervention/treatment, e.g. hospitalization); class 4 (life threatening: risk of death due to occurrence of adverse events); or grade 5 (death).

If an AE is considered clear, it is likely or likely to be associated with a study treatment, e.g., there is clear evidence that the event is associated with the use of a study drug, or that the event cannot be explained by a participant's medical condition, concomitant with treatment or other cause, and that there is a reasonable temporal relationship between the event and the administration of the study drug, then the AE is associated with the study drug (i.e., antibody ALX). If the correlation with the study drug is deemed unlikely or definitely unrelated to the study treatment, the AE is not related to the study drug, e.g., the event may be readily explained by the potential medical condition of the participant, concomitant therapy or other reasons, or more likely an event that is alternative to explanation (e.g., concomitant medication therapy or ongoing medical condition) or a temporal relationship with study drug administration and/or exposure indicates a causal relationship is unlikely.

Serious Adverse Events (SAE) are any AEs that occur at any dose that results in death, life threatening AEs, hospitalization, prolonged, sustained or significant disability/disability or congenital abnormalities/birth defects of the existing hospitalization. Other important medical events are also considered SAE when they endanger the participants or require intervention to prevent one of the listed results.

Dose Limiting AE (DLAE) refers to an AE that was evaluated as being related to the study drug, confirming that the participants received the study drug, and is: serious adverse events occurring during infusion or within 24 hours after completion of infusion (no other clear attributable to reasons other than study drug), AE grade 3 or higher (no other clear attributable to reasons other than study drug), or infusion-related toxicity grade 2 or higher (e.g., allergic/hypersensitivity reactions, drug fever, urticaria, dyspnea, flushing, bronchospasm, wheezing, hypoxia or infusion site pain), but could not be promptly addressed by supportive care and/or infusion rate reduction.

C. Pharmacokinetic assessment

Serum samples were collected for assessment of serum concentration of antibody ALX.

Samples of cerebrospinal fluid were also assessed for antibody ALX concentration.

D. Pharmacodynamic evaluation

Plasma samples were collected for evaluation of the levels of the granulin precursors. Whole blood samples were collected for evaluation of sortilin expression in leukocytes and for evaluation of other analytes.

The level of granulin precursors in the cerebrospinal fluid samples is evaluated.

E. Biomarker assessment

Plasma samples were collected for evaluation of biomarkers, including neurofilament light chain (NF-L), tau, levels of neuroinflammatory markers, and other analytes related to disease biology and response to antibody ALX.

Biomarkers for cerebrospinal fluid samples are evaluated, including NF-L, tau, markers of neuroinflammation, and levels of other analytes related to disease biology and response to antibody ALX.

Blood samples are collected at the time of screening DNA extraction to enable analysis by whole genome sequencing to identify common and rare genetic variants that predict responses to antibody ALX, correlate with progression to more severe disease states, correlate with susceptibility to progression to AE, or may increase awareness and understanding of disease biology.

VI study endpoint and statistics

Summary statistics (e.g., number of non-missing values, mean, median, standard deviation, minimum and maximum values of continuous variables, number and percentage of classified variables) of all measurements are provided, including demographics and baseline values, safety endpoints, PK, PD, and PK/PD correlations. No formal statistical inference is made of the security parameters. Missing data did not use interpolation.

A. Safety analysis and endpoint

The safety population includes all enrolled subjects receiving any number of study drugs (antibodies ALX or PBO). The safety population is described and summarized by treatment dose level/cohort.

Safety endpoints for this study included:

Incidence, nature and severity of AE.

Incidence of Dose Limiting AE (DLAE).

AE resulted in the incidence of treatment disruption.

Average change from baseline in clinical laboratory examination results; abnormal laboratory values for treatment occurrence and incidence of abnormal laboratory values reported as AE.

Average change in ECG assessment from baseline and incidence of abnormal ECG assessment.

Average change and incidence of abnormalities and heart rate.

Average change of vital signs from baseline and incidence of abnormal vital sign measurements.

Physical and neurological examination abnormalities.

Incidence of anti-drug antibodies (ADA) during the study (relative to ADA prevalence at baseline).

All adverse events of treatment incident occurring at or after administration are summarized in Medical Dictionary for Regulatory Activities (MedDRA) in terms of code, appropriate thesaurus levels and severity. In addition, all Serious Adverse Events (SAE), including death and events leading to discontinuation, are listed and summarized separately. DLAE are listed and summarized in terms of therapeutic dose level/cohort. All AEs were encoded using the latest version of MedDRA and classified by the MedDRA system broad class and preferred terminology.

B. Pharmacokinetic analysis and endpoint

The PK population includes all participants in the safety population with adequate assessment for determining PK parameters.

PK endpoints for this study included:

ALX antibody and cerebrospinal fluid concentration.

Antibody ALX plasma concentration or PK parameters versus safety endpoint. PK parameters include: c (C) _max Time to maximum observed concentration (T _max ) AUC from time zero to time t, AUC from time zero to infinity, percentage of AUC extrapolated to infinity, AUC from time zero to tau, elimination rate constant, terminal half-life (t _1/2 ) Distribution volume during the final phase (V _s. ) And Clearance (CL).

Relationship between blood concentration, CSF concentration or PK parameters of antibody ALX and activity or PD endpoint.

Bioavailability of SC-administered antibody ALX.

Individual and mean plasma antibody ALX concentration-time data are tabulated and plotted as therapeutic dose levels/cohorts. Serum PK of antibody ALX, if applicable, is summarized by estimating total exposure based on results obtained after single or multiple dosing, e.g. AUC, C _max Total serum CL, V _z And t _1/2 As shown. The estimated values of PK parameters are tabulated and summarized by descriptive statistics (mean, standard deviation, minimum, maximum, geometric mean and coefficient of variation); using C to be logarithmically converted _max And regression (power) models of specified AUC parameters with log-transformed dose. Individual and average antibody ALXCSF concentration-time data are tabulated as therapeutic dose levels/cohorts and plotted over time. Bioavailability of SC-administered antibody ALX was estimated. Potential correlations of relevant PK parameters with dose, demographics, safety (including QT variation) and PD measurements were investigated. Additional modeling was performed to characterize these correlations, including population PK analysis.

C. Pharmacodynamic analysis and endpoint

The PD cohort includes all participants in the safety cohort who had a baseline PD assessment and at least one post-dose PD assessment.

The PD endpoint of this study included changes from baseline in plasma and CSF levels of granulin precursors following administration.

PD endpoints were described and summarized in terms of therapeutic dose levels/cohorts.

D. Biomarker analysis and endpoint

The biomarker population includes all participants in the safety population that have the necessary baseline and post-dose measurements to provide interpretable results for the particular parameter of interest.

The biomarker endpoints of this study included:

changes in the level of markers of neuroinflammation and other analytes in CSF from baseline following administration.

Relationship between baseline biomarker and safety, PK, activity, immunogenicity, or other biomarker endpoints.

Biomarkers evaluated in blood and CSF samples include NF-L, as well as other blood/CSF markers that are involved in frontotemporal dementia (FTD) or other neurodegenerative disorders. In addition, biomarkers include rare genetic variants identified by whole genome sequencing of DNA extracted from blood.

Example 2: the results of phase I studies of the intravenous and subcutaneous anti-sortilin antibodies ALX were evaluated in healthy volunteers for safety, tolerability, pharmacokinetics, pharmacodynamics and bioavailability.

This example provides the results of single dose escalation (SAD) Intravenous (IV) cohorts 1 and 2 of the phase I study described in example 1.

As detailed in example 1, healthy Volunteers (HV) in SAD IV cohort 1 were administered a single intravenous dose of 6mg/kg of antibody ALX, while HV in SAD IV cohort 2 were administered a single intravenous dose of 15mg/kg of antibody ALX.

Table 5 provides an overview of the allocation of participants at different time points in the determination of plasma levels of the granulin precursors.

Table 5. Plasma granulin precursor level assessment in healthy volunteer participants allocation and cohorts 1 and 2.

Figures 2A-2B show the plasma granulin precursor levels of each subject in cohort 1 at the indicated time on day 1 of the study. Fig. 2A provides plasma granulin precursor levels for subjects administered antibody ALX (n=8), and fig. 2B provides plasma granulin precursor levels for subjects administered placebo (n=3). Figure 2C provides the average level of granulin precursor in the plasma of subjects administered placebo or antibody ALX in cohort 1 from day specified to day 85 of the study. These results indicate that administration of antibody ALX at a dose of 6mg/kg resulted in an increase in plasma granulin precursor levels by about 2-fold and about 3-fold from baseline, and that plasma granulin precursor levels returned to baseline levels about 42 days after administration of antibody ALX.

Figures 3A-3B show the plasma granulin precursor levels of each subject in cohort 2 at the indicated time on day 1 of the study. Fig. 3A provides plasma granulin precursor levels for subjects administered antibody ALX (n=6), and fig. 3B provides plasma granulin precursor levels for subjects administered placebo (n=2). Figure 3C provides the average level of granulin precursor in the plasma of subjects administered placebo or antibody ALX in cohort 2 from day specified to day 57 of the study. Taken together, these results show that antibody ALX administered at a dose of 15mg/kg resulted in an increase in plasma granulin precursor levels by about 2 and about 3 fold compared to baseline, and that plasma granulin precursor levels returned to baseline levels at about day 57 of the study.

Fig. 4 provides a comparison of the plasma levels of granulin precursors in subjects administered antibody ALX or placebo on days specified to 57 (cohort 2) or 85 (cohort 1) of the study, cohorts 1 and 2.

Figure 5A shows the plasma levels of granulin precursors in subjects administered placebo cohorts 1 and 2 on the indicated days after administration of placebo. Figures 5B-5C show the levels of granulin precursors in the plasma of subjects administered antibody ALX in cohorts 1 (figure 5B) and 2 (figure 5C) on the indicated days after administration of antibody ALX. Figure 5D provides a comparison of granulin precursor levels in plasma of subjects administered antibody ALX or placebo in cohorts 1 and 2 on a specified day after administration of placebo or antibody ALX. In fig. 5D, subjects administered placebo are pooled in cohorts 1 and 2. Taken together, these results demonstrate that administration of antibody ALX results in an increase in plasma granulin precursor levels from about 2 to about 3 fold over baseline, and that plasma granulin precursor levels return to baseline levels about 42 days (cohort 1) or about 56 days (cohort 2) after administration of antibody ALX.

Figure 6 shows the average percent change in granulin precursor levels in CSF of subjects administered antibody ALX or placebo in cohorts 1 and 2 from baseline on the day specified to day 57 of the study. On about day 25 of the study, the CSF granulin precursor level of the cohort 1 subjects increased by about 18% (6 mg/kg) from baseline, and the CSF granulin precursor level of the cohort 2 subjects increased by about 80% (15 mg/kg) from baseline. Subjects who were administered antibody ALX in cohorts 1 and 2 had elevated CSF granule protein precursor levels until day 57 of the study.

The safety results currently available have demonstrated that antibody ALX shows good tolerability at single doses up to 30 mg/kg.

Taken together, the results described in these examples demonstrate that administration of a single IV dose of antibody ALX results in a significant increase in the levels of granulin precursors in plasma and CSF, and that this increase persists for at least 6 weeks.

Example 3: phase IIa studies evaluating the safety, tolerability, pharmacokinetics and pharmacodynamics of the anti-sortilin antibody ALX in patients with Parkinson's disease.

This example describes a randomized, placebo-controlled, dose-escalated phase IIa study of the anti-sortilin antibody ALX that evaluates the safety, tolerability, pharmacokinetic and pharmacodynamic responses of selected biomarkers in Parkinson's Disease (PD) patients.

I. Purpose of investigation

The objective of this study was to evaluate the safety, tolerability and effect on key biomarkers in sporadic (idiopathic) PD patients as well as PD patients with at least one pathogenic mutation in the GBA1 gene for the 6 month low dose (15 mg/kg) and high dose (30 mg/kg) regimen of antibody ALX. Key biomarkers for analysis include granulin precursors, GCase and alpha-synuclein.

A. Measurement of primary results

The primary outcome measures of this study included Adverse Events (AEs), severe Adverse Events (SAE), and the number of AEs leading to discontinuation in each treatment group during the study. Safety and tolerability are based on additional safety assessments (e.g., clinical safety laboratory tests, vital signs, body weight, electrocardiogram [ ECG ] parameters, and physical examination). Other major outcome measures of this study include changes in cerebrospinal fluid (CSF) and blood-based biomarkers (e.g., granulin precursors, GCase and alpha-synuclein). Changes in CSF and blood-based biomarkers from baseline to end of treatment (EOT) were assessed.

B. Secondary outcome measure

Secondary outcome measures for this study included changes in movement examinations, unified parkinsonism rating scale (UPDRS) third part (MDS-UPDRS third part) and UPDRS total amount table assessment by the institute of Movement (MDS) sponsored revisions. The change in overall cognitive function assessed by a montreal cognitive assessment (MoCA) score may also be measured. Secondary outcome measures are assessed from baseline to end of treatment (EOT).

Study participants

The study included 48 subjects with sporadic PD or PD patients with at least one pathogenic mutation in the GBA1 gene.

A. Criteria for inclusion

Subjects meeting the following criteria were included in the study:

·adult males and females, age greater than or equal to 40 years and less than or equal to 80 years.

·Diagnosis of sporadic PD or PD with at least one pathogenic GBA1 variant is demonstrated, as well as Hoehn and Yahr criteria with a confirmation phase between I-III (including I and III).

Subjects taking PD drugs, such as glutamate antagonists, anticholinergic agents, dopamine agonists, levodopa (L-DOPA and decarboxylase DDC inhibitors), monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, beta blockers, selective serotonin uptake inhibitors (SSRI), tricyclic antidepressants (TCA) or indomethacin, prior to the start of the study may continue to take the drugs during the study.

B. Exclusion criteria

The study did not include subjects meeting the following criteria:

an ongoing anticoagulant (e.g., warfarin) that may prevent safe completion of lumbar puncture

And (3) treating.

A medical history of known sensitivity to antibody ALX or excipients thereof.

·Kidney function is impaired.

·Active cancer diagnosis, except for squamous cell carcinoma of the skin.

·Moderate/severe liver function impairment.

·Significant cardiovascular events (e.g., myocardial infarction, acute coronary syndrome, decompensated congestive heart failure, pulmonary embolism, or coronary revascularization) occurred within six months after study screening.

III, study design

The present study is a randomized, placebo-controlled, sequential study of two dose levels of antibody ALX (15 mg/kg and 30 mg/kg) administered monthly by Intravenous (IV) administration. Subjects were stratified by PD performance (sporadic or GBA1 gene mutation).

Study subjects were randomized into 3 study groups: antibody ALX dose is 15mg/kg, n=16; antibody ALX at a dose of 30mg/kg, n=16; and placebo, n=16. In each treatment group, the randomization ratio of antibody ALX to placebo was 2:1. Study treatment was administered by intravenous infusion. Antibody ALX or placebo was administered once a month (i.e., once every 28 days) for a total of six administrations. Dosage levels and frequencies may be modified to remove or increase dosage levels, e.g., 60mg/kg, and/or to alter the frequency of intravenous administration (e.g., every six weeks (q 6 w) or every eight weeks (q 8 w)), and/or to include administration by subcutaneous administration, as described herein. Placebo is saline with appropriate procedures to ensure blindness.

Biomarker and target engagement

As described above, the primary outcome measures of this study include changes in CSF and blood-based biomarkers (e.g., granulin precursors, GCase, and alpha-synuclein).

Plasma and CSF samples were collected for biomarker analysis. Changes in CSF and blood-based biomarkers from baseline to end of treatment (EOT) were assessed.

Plasma and CSF granulin precursor levels after antibody ALX treatment were measured as markers for target engagement.

To monitor the lysosomal pharmacodynamic effects of antibody ALX, changes in the GCase protein, GCase activity and its substrates lyso-Gb1 and glucosylceramides in CSF and plasma were analyzed.

The α -synuclein and CSF protein groups are used to assess changes in disease states.

All experiments were tightly controlled for quality and confirmed.

Example 4: study of anti-sortilin antibody ALX in cynomolgus monkeys.

This example describes the results of four-week toxicology studies of anti-sortilin antibody ALX in cynomolgus monkeys.

The cynomolgus monkey is administered the antibody ALX intravenously at a dose of 20, 60 or 200mg/kg once a week for four weeks for a total of five doses. The study also included a 6 week recovery period.

Figures 7A-7B show the average concentration of antibody ALX in serum at the indicated times (hours) after administration of a dose of antibody ALX on days 1, 8 and 15 (figure 7A) or 22 and 29 (figure 7B) of the study. By maximum concentration (C) _max ) And area under the curve (AUC) from time 0 to 168 hours _0-168 ) The estimated exposure increases with increasing dose level from 20 mg/kg/dose to 200 mg/kg/dose and is typically dose proportional. Day 22, 20mg/kg dose combined sex combined mean C _max And AUC _0-168 855 μg/mL and 70600 μg h/mL, respectively; the dose group of 60mg/kg was 2770 μg/mL and 248000 μg h/mL, respectively; the mg/kg dose groups were 7960 μg/mL and 744000 μg h/mL, respectively. Accumulation of antibody ALX was observed after multiple dosing in monkeys. Average C of gender combinations _max And AUC _0-168 The ratio (day 22/day 1) ranges from 1.40 to 1.44 and from 1.59 to 1.84 in each dose.

[ sortilin expression was consistently down-regulated in leukocytes (WBC) after weekly administration of antibody ALX, no significant difference in sortilin down-regulation was observed at dose levels of 20, 60 and 200mg/kg (8A). As shown in fig. 8B, the peripheral serum levels of granulin precursors increased with the down-regulation of sortilin and remained raised more than 3-fold above baseline throughout the study. At all time points sampled during the dosing phase, CSF granulin precursor levels were 2-3 times higher than baseline (fig. 8C). At the end of the 6-week recovery period, serum and CSF granule protein precursor levels remained significantly elevated in 3 out of 4 animals in the 200mg/kg group, indicating a durable pharmacodynamic effect (fig. 8B-8C).

The results of this study also show that antibody ALX administered weekly in cynomolgus monkeys at doses up to 200mg/kg is well tolerated. Pharmacokinetic analysis showed that antibody ALX-treated animals remained exposed during the treatment period and no antibody ALX-related adverse findings were observed during the study period. The level of adverse reaction (NOAEL) not observed in cynomolgus monkeys was 200mg/kg.

Other toxicology studies by rccchan: WIST rats administered antibody ALX at a dose of up to 300mg/kg for 4 weeks showed that antibody ALX was well tolerated. Pharmacokinetic analysis showed that antibody ALX treated animals remained exposed during the treatment period and no adverse findings associated with antibody ALX. The unobserved side effect level (NOAEL) was 300mg/kg in the rats.

Example 5: the results of phase I studies of the intravenous and subcutaneous anti-sortilin antibodies ALX were evaluated in healthy volunteers for safety, tolerability, pharmacokinetics, pharmacodynamics and bioavailability.

This example provides additional results from the single increment dose (SAD) Intravenous (IV) cohorts 1-4 and single dose 600mg Subcutaneous (SC) cohorts of the phase I study described in example 1.

As detailed in example 1, HV in an additional Single Dose (SD) subcutaneous cohort was administered antibody ALX at a fixed dose of 600 mg. This dose corresponds to a maximum dose level of 13.3mg/kg of the lowest body weight (45 kg) allowed in the study. After the safety review board determines that the 15mg/kgIV dose level is generally safe and tolerable, the cohorts are entered.

Figure 9 provides an overview of the participant treatments of the group entry population at various time points during the study. Of the total 55 participants in the group study, 54 participants (92.8%) completed the medication and 43 participants (78.2%) completed the study. As shown in table 6, the baseline demographics and characteristics of each group were similar. In all groups, most participants were white or black, and most were not spanish or latin.

Table 6: baseline demographics and characteristics (safety population)

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As shown in table 7, most Adverse Events (AEs) were mild to moderate in severity, with the most common AEs being headache (25.6%), anemia (9.3%) and surgical pain (9.3%). 1 subject developed 1 severe and severe infusion response event (60 mg/kg IV group) considered relevant to study treatment, thus stopping study medication shortly after infusion, but recovering within the same day. Another subject showed 1 severe and severe influenza a adverse event (placebo group) and was considered unrelated to study treatment. The last subject (30 mg/kg group) showed both a severe decrease in glomerular filtration rate and an increase in creatinine, but not severe, and was considered unrelated to study treatment.

Table 7: TEAE occurring in two or more subjects in all groups (safety populations)

Figures 10A-10B show the mean serum and cerebrospinal fluid (CSF) concentrations of ALX in each group after administration of SAD IV doses or single subcutaneous doses of ALX. Fig. 10A shows that the mean serum concentration of ALX increases in a dose-dependent manner after administration of a dose of SADIV of ALX, and was detected for up to 30 days after 600mg ALX of a single SC dose. Fig. 10B shows that CSF concentration increases in a dose-dependent manner after administration of a SAD IV dose of ALX and detected up to 17 days after 600mg ALX of a single SC dose. The mean serum and CSF concentrations of ALX were detected up to 84 days after a single IV dose of 60mg/kg (FIGS. 10A-10B).

Table 8 provides a summary of the mean serum Pharmacokinetic (PK) parameters for ALX for cohorts 1-4 and SD SC cohorts. As shown in Table 8, the average serum partition coefficient was 0.0015 to 0.0007.

Table 8: summary of ALX (PK population) mean (standard deviation) serum PK parameters

AUC _inf Extrapolation from time 0 to the area under the infinite concentration-time curve; CL, total clearance; c (C) _max Maximum observed concentration; CSF, cerebrospinal fluid; IV, intravenous; NE, not evaluable; PC, assigning coefficients; PK, pharmacokinetics; SC, subcutaneous; t is t _1/2 Terminal elimination half-life; t is t _max Time of maximum observed concentration. ^a PC cannot be calculated because CSF ALX samples were not collected. ^b PC cannot be calculated because CSF samples were below the limit of quantitation.

Figures 11A-11B show the percent change from baseline in plasma and CSF concentrations of granulin precursors for each group on the day indicated for the study. The SAD IV dose of ALX causes an increase in the levels of granulin Precursor (PGRN) in the periphery (FIG. 11A) and brain (FIG. 11B), with a dose-dependent effect on the duration of the increase. Fig. 11A shows that an increase in ALX-induced plasma PGRN levels prolonged with increasing IV dose levels. SC administration of 600mg ALX caused a strong increase in plasma PGRN, lasting until 29 days post-administration. Figure 11B shows a parallel increase in CSF PGRN lasting up to 24 days post-dose.

Taken together, the results described in these examples demonstrate that ALX was found to be safe and well tolerated for single dose IV or SC administration. These results indicate that ALX exposure increases in a dose-proportional manner and is distributed into the central nervous system as demonstrated by CSF ALX concentration. These results demonstrate that ALX is a potent modulator of PGRN levels in CSF, with PK/Pharmacodynamic (PD) characteristics that support the development of IV and SC ALX in chronic conditions.

Table 9: sequence.

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Claims (97)

1. A method of treating or delaying progression of a disease, disorder, or injury in an individual, the method comprising administering to the individual an anti-sortilin antibody at a dose of at least about 6mg/kg, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

2. A method of increasing the level of a granulin precursor in an individual suffering from a disease, disorder or injury, comprising administering to said individual an anti-sortilin antibody at a dose of at least about 6mg/kg, wherein said antibody comprises a light chain variable domain and a heavy chain variable domain, wherein said heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

3. The method of claim 2, wherein the increase in the level of the granulin precursor comprises an increase in the level of the granulin precursor in cerebrospinal fluid of the subject, plasma of the subject, or both.

4. The method of any one of claims 1-3, comprising administering the anti-sortilin antibody by intravenous infusion or by subcutaneous injection.

5. The method of any one of claims 1-4, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of at least about 6mg/kg, at least about 15mg/kg, at least about 30mg/kg, or at least about 60 mg/kg.

6. The method of any one of claims 1-5, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of between about 6mg/kg and about 30 mg/kg.

7. The method of any one of claims 1-5, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 6mg/kg, about 15mg/kg, about 30mg/kg, or about 60 mg/kg.

8. The method according to any one of claims 1-4, comprising:

(a) Administering the anti-sortilin antibody by intravenous infusion at an initial dose of about 60mg/kg, followed by one or more lower doses of the anti-sortilin antibody of between about 6mg/kg and about 59mg/kg, or between about 6mg/kg and about 30mg/kg, or between about 6mg/kg and about 15 mg/kg;

(b) Administering the anti-sortilin antibody by intravenous infusion at an initial dose of about 30mg/kg, followed by one or more lower doses of the anti-sortilin antibody of between about 6mg/kg and about 29mg/kg, or between about 6mg/kg and about 15 mg/kg;

(c) Administering the anti-sortilin antibody at an initial dose of about 15mg/kg by intravenous infusion followed by one or more lower doses of the anti-sortilin antibody of between about 6mg/kg and about 14 mg/kg;

(d) Administering the anti-sortilin antibody by intravenous infusion at an initial dose of about 6mg/kg, followed by one or more higher doses of the anti-sortilin antibody of between about 7mg/kg and about 30mg/kg, between about 15mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg;

(e) Administering the anti-sortilin antibody by intravenous infusion at an initial dose of about 15mg/kg, followed by one or more higher doses of the anti-sortilin antibody of between about 16mg/kg and about 30mg/kg, or between about 30mg/kg and about 60 mg/kg; or (b)

(f) The anti-sortilin antibody is administered by intravenous infusion at an initial dose of about 30mg/kg followed by one or more higher doses of the anti-sortilin antibody of between about 31mg/kg and about 60 mg/kg.

9. The method of any one of claims 5-8, comprising administering the anti-sortilin antibody about once every four weeks or less frequently.

10. The method of claim 9, comprising administering the anti-sortilin antibody about once every four weeks, about once every five weeks, about once every six weeks, about once every seven weeks, about once every eight weeks, about once every nine weeks, or about once every ten weeks.

11. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 15mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks.

12. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 15mg/kg about once every six weeks or about once every eight weeks.

13. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 30mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks.

14. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 30mg/kg about once every six weeks or about once every eight weeks.

15. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 60mg/kg about once every four weeks, about once every six weeks, or about once every eight weeks.

16. The method of claim 1 or claim 2, comprising administering the anti-sortilin antibody by intravenous infusion at a dose of about 60mg/kg about once every six weeks or about once every eight weeks.

17. The method of any one of claims 1-4, comprising administering the anti-sortilin antibody at a dose of at least about 270mg by subcutaneous injection.

18. The method of any one of claims 1-4, comprising administering the anti-sortilin antibody at a dose of between about 150mg and about 600mg by subcutaneous injection.

19. The method of claim 18, comprising administering the anti-sortilin antibody by subcutaneous injection at any of about 150mg, about 270mg, about 300mg, or about 600 mg.

20. The method of claim 18, comprising administering the anti-sortilin antibody at a dose of about 150mg by subcutaneous injection.

21. The method of claim 18, comprising administering the anti-sortilin antibody at a dose of about 270mg by subcutaneous injection.

22. The method of claim 18, comprising administering the anti-sortilin antibody at a dose of about 300mg by subcutaneous injection.

23. The method of claim 18, comprising administering the anti-sortilin antibody at a dose of about 600mg by subcutaneous injection.

24. The method of any one of claims 17-23, comprising administering the anti-sortilin antibody by subcutaneous injection at any frequency of about every two weeks, about every four weeks, about every six weeks, or about every eight weeks.

25. The method of any one of claims 17-23, comprising administering the anti-sortilin antibody about once every four weeks by subcutaneous injection.

26. The method of any one of claims 1-25, wherein the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:20 and a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable domain of an amino acid sequence of seq id no.

27. The method of any one of claims 1-26, wherein the anti-sortilin antibody has a human IgG1 isotype.

28. The method of claim 27, wherein the anti-sortilin antibody comprises an Fc region comprising amino acid substitutions L234A, L235A and P331S, wherein numbering of residue positions is according to EU numbering.

29. The method of any one of claims 1-28, wherein the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:31 or 32.

30. The method of any one of claims 1-29, wherein the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO:32, and a heavy chain of an amino acid sequence of seq id no.

31. The method of any one of claims 1-29, wherein the anti-sortilin antibody comprises a polypeptide comprising SEQ ID NO:30 and a light chain comprising the amino acid sequence of SEQ ID NO: 31.

32. The method of any one of claims 1-31, further comprising measuring the level of granulin precursors in a blood or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

33. The method of any one of claims 1-32, further comprising measuring sortilin levels in leukocytes of a blood sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

34. The method of any one of claims 1-33, further comprising measuring the level of neurofilament light chain (NF-L), tau, one or more biomarkers of neuroinflammation, one or more inflammatory biomarkers, one or more biomarkers of complement function, and/or one or more biomarkers of microglial activity in a blood or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

35. The method according to claim 34, wherein:

(a) The one or more biomarkers of neuroinflammation are selected from IL-6, SPP1, IFI2712A, CHIT1, YKL-40, GFAP, YWHAE, CSF1, AIF1, LY86, CD86, and TOP2A;

(b) The one or more inflammatory biomarkers are selected from osteopontin (SPP 1), YWHAE (14-3-3 protein epsilon), allograft inflammatory factor 1 (AIF 1), colony stimulating factor 1 (CSF 1), chitinase 1 (CHIT 1), lymphocyte antigen 86 (LY 86), and CD86;

(c) The one or more biomarkers of complement function are selected from the group consisting of C1qb and C1qc; and/or

(d) The biomarker of one or more microglial activities is selected from the group consisting of YKL-40, GFAP, and interleukin-6.

36. The method of any one of claims 1-7 and 26-35, wherein administering a dose of 6mg/kg or 15mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the subject's plasma level of granulin precursor of at least about 1.4-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.6-fold, at least about 2.8-fold, or at least about 3-fold as compared to the subject's plasma level of granulin precursor prior to administration of the anti-sortilin antibody.

37. The method of claim 36, wherein the increase in the level of granulin precursor in the plasma of the individual occurs within about 1 day after administration of the anti-sortilin antibody.

38. The method of claim 36 or claim 37, wherein the dose of the anti-sortilin antibody is 6mg/kg, and wherein an increase in the level of progranulin in the plasma of the individual is present about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 17 days, about 24 days, about 29 days, or about 42 days after administration of the anti-sortilin antibody.

39. The method of claim 36 or claim 37, wherein the dose of the anti-sortilin antibody is 15mg/kg, and wherein an increase in the level of progranulin in the plasma of the individual occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 17 days, about 24 days, about 29 days, about 42 days, or about 56 days after administration of the anti-sortilin antibody.

40. The method of any one of claims 1-7 and 26-35, wherein administering a dose of 6mg/kg, 15mg/kg, 30mg/kg, or 60mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% compared to the level of granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody.

41. The method of claim 40, wherein the dose of the anti-sortilin antibody is 6mg/kg, and wherein an increase in the level of progranulin in the plasma of the individual occurs about 1 day, about 7 days, about 14 days, about 21 days, or about 28 days after administration of the anti-sortilin antibody.

42. The method of claim 40, wherein the dose of the anti-sortilin antibody is 15mg/kg, and wherein an increase in the level of progranulin in the plasma of the individual occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, or about 42 days after administration of the anti-sortilin antibody.

43. The method of claim 40, wherein the dose of the anti-sortilin antibody is 30mg/kg, and wherein an increase in the level of progranulin in the plasma of the individual occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody.

44. The method of claim 40, wherein the dose of the anti-sortilin antibody is 60mg/kg, and wherein an increase in the level of progranulin in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days after administration of the anti-sortilin antibody.

45. The method of any one of claims 1-4, 18-20, and 26-35, wherein administering a dose of about 150mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody.

46. The method of any one of claims 1-4, 17-19, 22, and 26-35, wherein administering a dose of about 300mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody.

47. The method of any one of claims 1-4, 17-19, 23, and 26-35, wherein administering a dose of about 600mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in the plasma of the subject of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, or at least about 200% as compared to the level of granulin precursor in the plasma of the subject prior to administration of the anti-sortilin antibody.

48. The method of any one of claims 45-47, wherein an increase in the level of granulin precursor in the plasma of the subject occurs about 1 day, about 7 days, about 14 days, about 21 days, or about 28 days after administration of the anti-sortilin antibody.

49. The method of any one of claims 1-7, 26-38, and 40-41, wherein administering a dose of 6mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, or at least about 20% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

50. The method of any one of claims 1-7, 26-37, 39-40, and 42, wherein administering a dose of 15mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

51. The method of any one of claims 1-7, 26-35, 40 and 43, wherein administering a dose of 30mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

52. The method of any one of claims 1-5, 7, 26-35, 40, and 44, wherein administering a dose of 60mg/kg of the anti-sortilin antibody to the subject by intravenous infusion results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

53. The method of any one of claims 49-51, wherein an increase in the level of granulin precursor in the cerebrospinal fluid of the individual occurs about 1 day, about 2 days, about 5 days, about 7 days, about 12 days, about 14 days, about 17 days, about 21 days, about 24 days, about 28 days, about 29 days, about 35 days, about 42 days, about 49 days, or about 56 days after administration of the anti-sortilin antibody.

54. The method of claim 52, wherein an increase in the level of granulin precursor in the cerebrospinal fluid of the individual occurs about 1 day, about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days after administration of the anti-sortilin antibody.

55. The method of any one of claims 1-4, 18-20, 26-35, 45, and 48, wherein administering a dose of about 150mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

56. The method of any one of claims 1-4, 17-19, 22, 26-35, 46, and 48, wherein administering a dose of about 300mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

57. The method of any one of claims 1-4, 17-19, 23, 26-35, 47, and 48, wherein administering a dose of about 600mg of the anti-sortilin antibody to the subject by subcutaneous injection results in an increase in the level of granulin precursor in cerebrospinal fluid of the subject of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% as compared to the level of granulin precursor in cerebrospinal fluid of the subject prior to administration of the anti-sortilin antibody.

58. The method of any one of claims 55-57, wherein an increase in the level of granulin precursor in the cerebrospinal fluid of the individual occurs about 1 day, about 7 days, about 14 days, or about 21 days after administration of the anti-sortilin antibody.

59. The method of any one of claims 1-58, wherein the disease, disorder, or injury is selected from the group consisting of frontotemporal dementia, progressive supranuclear palsy, alzheimer's disease, vascular dementia, seizures, retinal dystrophy, amyotrophic Lateral Sclerosis (ALS), traumatic brain injury, spinal cord injury, dementia, stroke, parkinson's disease, acute disseminated encephalomyelitis, retinal degeneration, age-related macular degeneration, glaucoma, multiple sclerosis, septic shock, bacterial infection, arthritis, and osteoarthritis.

60. The method of claim 59, wherein the individual is at risk of the disease, disorder, or injury.

61. The method of any one of claims 1-60, wherein the disease, disorder, or injury is parkinson's disease.

62. The method of claim 61, wherein the disease, disorder, or injury is sporadic parkinson's disease.

63. The method of claim 61, wherein the individual has at least one pathogenic mutation in the GBA1 gene.

64. The method of claim 63, wherein the individual is homozygous or heterozygous for the at least one pathogenic mutation in the GBA1 gene.

65. The method of claim 63 or claim 64, wherein the at least one pathogenic mutation in the GBA1 gene is selected from the group consisting of c.1226a > G, c.1448t > C, IVS2+1g > a, recNcil, 84insGG, and any combination thereof; or wherein the at least one pathogenic mutation in the GBA1 gene is a mutation in the GBA1 gene that results in an amino acid substitution in the GBA1 gene product selected from the group consisting of N370S, L444P, R35120W, H255Q, D409H, E326K, T369M, R496H and any combination thereof.

66. The method of any one of claims 61-65, wherein the parkinson's disease is classified as between stage I and stage III based on Hoehn and Yahr criteria.

67. The method of any one of claims 61-66, wherein the individual is receiving one or more parkinson's disease treatments prior to administration of the anti-sortilin antibody.

68. The method of claim 67, wherein the subject continues to receive the one or more parkinson's disease treatments after starting treatment with the anti-sortilin antibody.

69. The method of any one of claims 61-68, comprising administering the anti-sortilin antibody in combination with one or more parkinson's disease treatments.

70. The method of any one of claims 67-69, wherein the one or more parkinson's disease treatments are selected from the group consisting of: glutamate antagonists, anticholinergic agents, dopamine agonists, levodopa (L-DOPA and decarboxylase [ DDC ] inhibitors), monoamine oxidase B (MAO-B) inhibitors, catechol-O-methyltransferase (COMT) inhibitors, beta blockers, selective serotonin uptake inhibitors (SSRI), tricyclic antidepressants (TCA) and indomethacin.

71. The method of any one of claims 61-70, wherein the method further comprises measuring the level of granulin precursors, GCase and/or a-synuclein in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

72. The method of any one of claims 61-71, further comprising measuring the level of one or more biomarkers of lysosomal function in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

73. The method of claim 72, wherein the one or more biomarkers of lysosomal function are selected from the group consisting of: GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD).

74. The method of any one of claims 61-73, further comprising assessing cognitive function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

75. The method according to claim 74, wherein cognitive function is assessed using montreal cognitive assessment (MoCA).

76. The method of any one of claims 61-75, further comprising assessing the motor function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

77. The method of claim 76, wherein the motor function is assessed using a motion disorder institute (MDS) sponsored revision Unified Parkinson's Disease Rating Scale (UPDRS) third part (MDS-UPDRS third part) or a UPDRS total amount table.

78. The method of any one of claims 61-77, comprising administering the anti-sortilin antibody to the individual for at least about 6 months or at least about 24 weeks.

79. The method of any one of claims 1-60, wherein the disease, disorder, or injury is frontotemporal dementia (FTD).

80. The method of claim 79, wherein the individual:

(a) Is heterozygous for one or more mutations in the GRN gene, optionally wherein the one or more mutations are loss-of-function mutations;

(b) Is heterozygous for the C9orf72 hexanucleotide repeat amplification;

(c) FTD-symptomatic, FTD-symptomatic or pre-symptomatic FTD; and/or

(d) There is FTD-GRN, or FTD caused by one or more mutations in the GRN gene.

81. The method of claim 80, wherein:

(a) The individual has a pre-symptomatic FTD and: (i) Elevated levels of one or more biomarkers selected from Nfl, SPP1, ywae, AIF1, CSF1, CHIT1, and LY86, and/or (ii) reduced levels of one or more biomarkers selected from NAGK and CTSB; or (b)

(b) The individual is free of FTD symptoms and: (i) Heterozygous for one or more mutations in the GRN gene, and/or (ii) has a reduced PGRN level or function.

82. The method of any one of claims 1-60, wherein the disease, disorder, or injury is alzheimer's disease.

83. The method of any one of claims 1-60, wherein the disease, disorder, or injury is ALS.

84. The method of any one of claims 79-83, further comprising measuring the level of granulin precursors, GCase and/or a-synuclein in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

85. The method of any one of claims 79 to 83, further comprising measuring the level of one or more biomarkers of lysosomal function in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

86. The method of claim 85, wherein the one or more biomarkers of lysosomal function are selected from the group consisting of: GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD).

87. The method of any one of claims 79-83, further comprising assessing cognitive function of the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

88. The method of any one of claims 1-87, wherein the individual is a human.

89. A method of monitoring treatment of an individual administered an anti-sortilin antibody, comprising measuring the level of one or more biomarkers, wherein the one or more biomarkers are selected from the group consisting of a granule protein precursor, GCase, neurofilament light chain (NF-L), tau, one or more markers of neuroinflammation, one or more biomarkers of inflammatory biomarker, one or more biomarkers of complement function, one or more biomarkers of microglial activity, and alpha-synuclein, wherein the level of the one or more biomarkers is measured in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

90. A method of monitoring treatment of an individual administered an anti-sortilin antibody, comprising measuring the level of a biomarker of one or more lysosomal functions in a plasma or cerebrospinal fluid sample obtained from the individual before and after the individual receives one or more doses of the anti-sortilin antibody.

91. The method of claim 90, wherein the one or more biomarkers of lysosomal function are selected from the group consisting of: GCase protein, GCase activity, lyso-Gb1, one or more cathepsins, LAMP1, N-acetyl-D-glucosamine kinase (NAGK), and glucosylceramide; optionally, wherein the one or more cathepsins are selected from the group consisting of cathepsin B (CTSB) and cathepsin D (CTSD).

92. The method of any one of claims 89-91, further comprising assessing the activity of the anti-sortilin antibody in the individual based on the level of the one or more biomarkers in the sample.

93. The method of claim 89 or claim 92, wherein:

(a) The one or more markers of neuroinflammation are selected from the group consisting of IL-6, SPP1, IFI2712A, CHIT1, YKL-40, GFAP, YWHAE, CSF1, AIF1, LY86, CD86, and TOP2A;

(b) The one or more inflammatory biomarkers are selected from osteopontin (SPP 1), YWHAE (14-3-3 protein epsilon), allograft inflammatory factor 1 (AIF 1), colony stimulating factor 1 (CSF 1), chitinase 1 (CHIT 1), lymphocyte antigen 86 (LY 86), and CD86;

(c) The one or more biomarkers of complement function are selected from the group consisting of C1qb and C1qc; and/or

(d) The biomarker of one or more microglial activities is selected from the group consisting of YKL-40, GFAP, and interleukin-6.

94. An anti-sortilin antibody at a dose of at least about 6mg/kg in a method for treating or delaying progression of a disease, disorder, or injury in an individual, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

95. An anti-sortilin antibody at a dose of at least about 6mg/kg for use in a method of increasing the level of a progranulin in an individual with a disease, disorder, or injury, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

96. Use of an anti-sortilin antibody at a dose of at least about 6mg/kg in the manufacture of a medicament for treating or delaying progression of a disease, disorder, or injury in an individual, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.

97. Use of an anti-sortilin antibody at a dose of at least about 6mg/kg for the manufacture of a medicament for increasing the level of a granulin precursor in an individual with a disease, disorder, or injury, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, wherein the heavy chain variable domain comprises a polypeptide comprising SEQ ID NO:6, HVR-H1 comprising the amino acid sequence of SEQ ID NO:7 and an HVR-H2 comprising the amino acid sequence of SEQ ID NO:8, and the light chain variable domain comprises an HVR-H3 comprising the amino acid sequence of SEQ ID NO:9, HVR-L1 comprising the amino acid sequence of SEQ ID NO:10 and an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11, and HVR-L3 of the amino acid sequence of seq id no.