Compounds for delivery of granulin across blood brain barrier

Abstract

To provide compounds for the delivery of progranulin, a progranulin fragment, and / or at least one granulin protein subunit across the blood brain barrier.SOLUTION: The present invention provides compounds for the delivery of progranulin, a progranulin fragment, and / or at least one granulin protein subunit across the blood brain barrier, the compounds including a progranulin domain, a TfR1 binding domain, and optionally an albumin binding domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (Reference to sequence listing) This application has been submitted with a Sequence Listing in ST.26 XML format. The Sequence Listing is provided as file entitled "30330," created on June 19, 2023, and is 66 kilobytes in size. The Sequence Listing information in ST.26 XML format is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to novel compounds for delivering progranulin or a fragment thereof across the blood-brain barrier. The present invention also relates to novel compounds comprising a progranulin domain, a transferrin receptor 1 (TfR1) binding domain, and an albumin binding domain. [Background technology]

[0003] Progranulin, a 593 amino acid protein, is a precursor protein encoded by the GRN gene in humans. Individual granulin proteins are cleaved from progranulin. Naturally occurring progranulin is represented by paragranulin (p) and contains a proprotein bound to the 7 granulin protein structure GFBACDE, where each granulin protein is represented by a capital letter. Overall, naturally occurring progranulin has the structure pGFBACDE.

[0004] Various neurodegenerative conditions, including neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), among others, have been linked to a deficiency of progranulin in the brain or cerebrospinal fluid (CSF). Progranulin deficiency accounts for approximately 25 percent of all inherited forms of frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. Progranulin acts protectively in several disease models with increased progranulin levels, accelerating behavioral recovery from ischemia (Tao, J et al., (2012) Brain Res 1436, 130-136; Egashira, Y. et al., (2013). J Neuroinflammation 10, 105), suppressing motor deficits in Parkinson's disease models (Van Kampen, JM et al. (2014). PLoS One 9, e97032), attenuating pathology in models of amyotrophic lateral sclerosis (Laird, A. S. et al., (2010). PLoS One 5, e13368), and arthritis (Tang, W. et al., (2011). Science 332, 478-484), and preventing memory impairment in Alzheimer's disease models (Minami, S. S. et al. al.,(2014).Nat Med 20,1157-1164).

[0005] Therefore, there is a need to develop therapies that can address disorders caused by loss of progranulin function or reduced progranulin levels. Unfortunately, it can be difficult to deliver progranulin across the blood-brain barrier (BBB) ​​and / or provide progranulin with a half-life long enough to persist in the brain and / or CSF of patients with progranulin deficiency. Therefore, there is a need for compounds that can provide effective delivery of progranulin or at least one granulin protein across the BBB. Furthermore, there is a need for compounds that can provide progranulin with a half-life long enough to persist in the brain and / or CSF.

[0006] Compounds designed to deliver Progranulin variants across the BBB are known, such as those disclosed in U.S. Patent Application No. 2022 / 0213155, and include (1) Progranulin variants designed to interact with Sortilin rather than the naturally occurring sequence, and (2) Fc dimers as transferrin receptor 1 (TfR1) binding domains, each of which is described herein. Summary of the Invention

[0007] Provided herein are compounds for delivering native progranulin or fragments of native progranulin across the BBB with a half-life long enough to provide therapeutic benefit to patients with progranulin deficiency.

[0008] In a previous attempt to deliver Progranulin across the BBB, Progranulin has been modified by substituting residues 574-576 to reduce C-terminal clipping of the Progranulin protein and allow it to specifically bind to Sortilin (SEQ ID NO: 4).

[0009] However, it was unexpectedly discovered that clipping (the unintended separation of portions of the Progranulin peptide) could be further reduced by instead attaching an albumin-binding domain to the C-terminus of wild-type Progranulin. Furthermore, it was unexpectedly discovered that modifying Progranulin to induce a secondary interaction with sorritin did not improve transport across the BBB, as shown in Figure 3. As shown in Figure 4, the use of the albumin-binding domain resulted in an increased half-life of the Progranulin domain and / or an increased amount of the Progranulin variant delivered across the BBB.

[0010] Furthermore, in previous attempts to deliver progranulin across the BBB, an Fc dimer has been used as the TfR1-binding domain. However, as shown in Figure 3, it was unexpectedly found that a TfR1-binding domain containing a Fab region with higher binding affinity to the human TfR1 receptor resulted in increased delivery across the BBB.

[0011] Generally, disclosed herein are compounds comprising a progranulin domain and a transferrin receptor 1 (TfR1) binding domain that can improve delivery of progranulin or progranulin fragments across the BBB.

[0012] Also provided herein are Progranulin domains, TfR1-binding domains, and albumin-binding domains that can improve delivery of Progranulin or at least one Granulin protein across the BBB and have improved half-lives.

[0013] Also provided herein are a progranulin domain and a TfR1-binding domain, wherein the TfR1-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, The amino acid sequence of HCDR1 is SEQ ID NO: 45 The amino acid sequence of HCDR2 is SEQ ID NO: 46 The amino acid sequence of HCDR3 is SEQ ID NO: 47 The amino acid sequence of LCDR1 is SEQ ID NO: 48; The amino acid sequence of LCDR2 is SEQ ID NO: 49; The amino acid sequence of LCDR3 is SEQ ID NO:50.

[0014] Also provided herein is a compound comprising a Progranulin fragment, wherein the amino acid sequence of the Progranulin fragment is SEQ ID NO: 2. The Progranulin fragment may be 100 to 500 residues in length.

[0015] Also provided herein are compounds comprising: (a) a progranulin domain X; (b) a TfR1-binding domain Y linked to X by a first linker L1; and (c) an albumin-binding domain Z linked to X or Y by a second linker L2.

[0016] Also provided herein are compounds comprising: (a) a progranulin domain; (b) a TfR1-binding domain linked to the progranulin domain by a first linker; and (c) an albumin-binding domain linked to the progranulin domain or the TfR1-binding domain by a second linker.

[0017] Also provided herein are compounds comprising an amino acid sequence having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:25, the heavy chain of H09 Fab-PGRN-C90.43.

[0018] Also provided herein are compounds comprising (a) a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:25, and (b) a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:19.

[0019] Also provided herein is a compound comprising (a) a heavy chain having the amino acid sequence given by SEQ ID NO:25, and (b) a light chain having the amino acid sequence given by SEQ ID NO:19.

[0020] Also provided herein are compounds comprising an amino acid sequence having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:26, the heavy chain of H09 Fab-PGRNΔpGF-C90.43.

[0021] Also provided herein are compounds comprising (a) a heavy chain having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:26, and (b) a light chain having at least 90% or at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:19.

[0022] Also provided herein is a compound comprising (a) a heavy chain having the amino acid sequence given by SEQ ID NO:26, and (b) a light chain having the amino acid sequence given by SEQ ID NO:19.

[0023] Also provided herein are compounds comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises SEQ ID NO: 45, HCDR2 comprises SEQ ID NO: 46, HCDR3 comprises SEQ ID NO: 47, LCDR1 comprises SEQ ID NO: 48, LCDR2 comprises SEQ ID NO: 49, and LCDR3 comprises SEQ ID NO: 50.

[0024] Also, the present specification provides a antibody comprising: (a) a Progranulin domain, the amino acid sequence of which is SEQ ID NO: 1 or SEQ ID NO: 2; and (b) a transferrin receptor 1 (TfR1) binding domain linked to the Progranulin domain by a first linker, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3. R3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50; and (c) an albumin binding domain linked to the progranulin domain or the TfR1 binding domain by a second linker.

[0025] Also provided herein are methods of treating a disorder, comprising administering any of the disclosed compounds to a patient in need thereof, preferably wherein the disorder is neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

[0026] Also provided herein are compounds for use in treating type 11 neuronal ceroid lipofuscinosis, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

[0027] Also provided herein are pharmaceutical compositions comprising any of the disclosed compounds for use in treating neurological ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

[0028] Also provided herein is the use of any of the disclosed compounds in the manufacture of a medicament for the treatment of type 11 neuronal ceroid lipofuscinosis, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

[0029] Also provided herein are compositions comprising any one of the disclosed compounds and a pharmaceutically acceptable carrier. [Brief explanation of the drawings]

[0030] [Figure 1] 1 shows that constructs with Progranulin fragments had extended durability compared to constructs with the full-length Progranulin peptide. [Figure 2] 1 shows the effect of progranulin length on tau turnover. [Figure 3] Figure 1 shows the impact of the architecture of the TfR shuttle on PGRN delivery across the BBB. [Figure 4] 1 shows the effect of TfR shuttle affinity on the delivery of constructs across the BBB. [Figure 5] We show that the TfR-binding domain may be a suitable Progranulin shuttle, delivering Progranulin to neurons in place of the Sortilin-binding domain. [Figure 6] 1 shows CNS exposure of sandwich example 2 in hTfR KI mice. [Figure 7] 1 shows the effects of BBB-crossing PGRN on lipofuscin pathology, neuronal injury, neuroinflammation, and lysosomal function in Grn KO mice. DETAILED DESCRIPTION OF THE INVENTION

[0031] compound The disclosed compounds deliver Progranulin, a fragment of Progranulin, and / or at least one granulin protein across the BBB with a half-life long enough to persist in the brain and / or CSF. The disclosed compounds comprise a Progranulin domain, a TfR1-binding domain, and optionally an albumin-binding domain. The disclosed compounds also comprise a Progranulin domain X, a TfR1-binding domain Y linked to X by a first linker L1, and an albumin domain Z linked to X or Y by a second linker L2, and are as shown in Formula I, II, or III. Y-L1-X-L2-Z Formula I.

[0032] In some embodiments, the compound can be represented by Formula I. The TfR1-binding domain Y can be linked to the Progranulin domain X with a first linker L1. The TfR1-binding domain can be linked to the Progranulin domain at the N-terminus or C-terminus of the Progranulin domain. The albumin-binding domain Z can be linked to the Progranulin domain with a second linker L2 at the N-terminus or C-terminus of the Progranulin domain. The TfR1-binding domain and the albumin-binding domain can be linked at opposite ends of the Progranulin domain. For example, the TfR1-binding domain can be linked at the N-terminus of the Progranulin domain or within 5 amino acids thereof, while the albumin-binding domain can be linked at the C-terminus of the Progranulin domain or within 5 amino acids thereof, or the TfR1-binding domain can be linked at the C-terminus of the Progranulin domain or within 5 amino acids thereof, while the albumin-binding domain can be linked at the N-terminus of the Progranulin domain or within 5 amino acids thereof. Z-L2-Y-L1-X Formula II. Compound

[0033] In some embodiments, the compound can be represented by Formula II. The progranulin domain X can be linked to the TfR1-binding domain with a first linker L1. The progranulin domain can be linked to the TfR1-binding domain at the N-terminus or C-terminus of the TfR1-binding domain. The albumin-binding domain Z can be linked to the TfR1-binding domain with a second linker L2 at the N-terminus or C-terminus of the progranulin domain. The progranulin domain and the albumin-binding domain can be linked at opposite ends of the TfR1-binding domain. For example, the progranulin domain can be linked at the N-terminus of the TfR1-binding domain or within 5 amino acids thereof, while the albumin-binding domain can be linked at the C-terminus of the TfR1-binding domain or within 5 amino acids thereof, or the progranulin domain can be linked at the C-terminus of the TfR1-binding domain or within 5 amino acids thereof, while the albumin-binding domain can be linked at the N-terminus of the TfR1-binding domain or within 5 amino acids thereof.

[0034] In some embodiments, the progranulin domain and the albumin binding domain can be linked at either the same end or opposite ends of the TfR1 binding domain.

[0035] In some embodiments, the Progranulin domain X can be linked to the TfR1-binding domain with a first linker L1. The Progranulin domain can be linked to the TfR1-binding domain at the N-terminus or C-terminus of the TfR1-binding domain. The Albumin-binding domain Z can be linked to the TfR1-binding domain at the N-terminus or C-terminus of the Progranulin domain using a second linker L2. The Progranulin domain and the Albumin-binding domain can be linked at the same end of the TfR1-binding domain. For example, the Progranulin domain and the Albumin-binding domain can be linked at the N-terminus or C-terminus of the TfR1-binding domain, or within 5 amino acids thereof.

[0036] In some embodiments, the Progranulin domain can be linked to the heavy or light chain of the TfR1 binding domain.

[0037] In some embodiments, the compound is made up of one or more polypeptide and / or protein sequences. Thus, in some embodiments, the compound can be a fusion protein. In some embodiments, the compound is a polypeptide, protein, and / or fusion protein.

[0038] In some embodiments, any two of the Progranulin domain, the TfR1-binding domain, and / or the albumin-binding domain do not form a dimer. For example, in some embodiments, even if the TfR1-binding domain and the albumin-binding domain are at the same end of the Progranulin domain, they do not interact to form a TfR1-binding domain-albumin-binding domain dimer. The dimer may be a heterodimer, such as an Fc heterodimer.

[0039] In some embodiments, the compound comprises a heavy chain comprising SEQ ID NO: 25, SEQ ID NO: 26, and / or SEQ ID NO: 29. In some embodiments, the compound comprises a light chain comprising SEQ ID NO: 19, SEQ ID NO: 32, and / or SEQ ID NO: 33.

[0040] In some embodiments, the compound comprises a heavy chain comprising SEQ ID NO: 25. In some embodiments, the compound comprises a light chain comprising SEQ ID NO: 19.

[0041] In some embodiments, the compound comprises a heavy chain comprising SEQ ID NO: 26. In some embodiments, the compound comprises a light chain comprising SEQ ID NO: 19.

[0042] In some embodiments, the compound comprises a heavy chain comprising SEQ ID NO: 29. In some embodiments, the compound comprises a light chain comprising SEQ ID NO: 32.

[0043] In some embodiments, the compound comprises a heavy chain comprising SEQ ID NO: 29. In some embodiments, the compound comprises a light chain comprising SEQ ID NO: 33.

[0044] In some embodiments, the compound comprises a heavy chain having an amino acid sequence having at least 90%, 95%, and / or 99% sequence identity to the amino acid sequence given by SEQ ID NO:25, SEQ ID NO:26, and / or SEQ ID NO:29.

[0045] In some embodiments, the compound comprises a light chain having an amino acid sequence having at least 90%, 95%, and / or 99% sequence identity to the amino acid sequence provided by SEQ ID NO:19, SEQ ID NO:32, and / or SEQ ID NO:33.

[0046] Progranulin Domain The disclosed compounds contain a progranulin domain. The progranulin domain is a portion of the disclosed compounds that contains an amino acid sequence representing at least one unmodified, naturally occurring, and / or wild-type granulin protein. Progranulin is a precursor protein of the granulin protein that is cleaved from the full-length progranulin precursor. Progranulin contains a proprotein (p) followed by the seven granulin protein sequences: GFBACDE.

[0047] The progranulin domain can comprise at least one, at least two, at least three, at least four, at least five, at least six, or all seven unmodified granulin proteins.

[0048] The Progranulin domain can include the full-length unmodified Progranulin sequence, including the proprotein and all seven granulin proteins: pGFBACDE, which is represented by SEQ ID NO:1.

[0049] A Progranulin domain can comprise a fragment of the full-length sequence of Progranulin. For example, a Progranulin domain can comprise the Progranulin fragment known as BACDE or PGRNΔpGF, which is set forth in SEQ ID NO: 2. A Progranulin domain can also comprise other fragments of Progranulin (e.g., pGF, GFBACDE, etc., among others).

[0050] A Progranulin domain can comprise an unmodified, wild-type, and / or naturally occurring Progranulin sequence or a fragment thereof, hi some embodiments, a Progranulin domain does not include and / or is free of non-naturally occurring modified Granulin protein sequences.

[0051] It was unexpectedly found that when the Progranulin domain comprises a fragment of Progranulin, i.e., the Progranulin fragment set forth in SEQ ID NO: 2 or PGRNΔpGF, the compound remains stable longer than when the Progranulin domain comprises the full-length Progranulin sequence. Without being bound by theory, it is believed that PGRNΔpGF, a fragment of full-length Progranulin, has longer stability due to the reduced clipping present in the smaller Progranulin domain.

[0052] The progranulin fragment can have a length of less than 550 residues, less than 500 residues, 100 to 500 residues, 350 to 450 residues, 400 to 425 residues, or 414 residues.

[0053] TfR1 binding domain The disclosed compounds can also include a transferrin receptor 1 (TfR1) binding domain. The TfR1 binding domain is a portion of a compound that specifically binds to the TfR1 receptor and / or the human TfR1 receptor. The TfR1 receptor is found on neurons and glial cells; therefore, without being bound by theory, it is believed that linking a TfR1 binding domain, which has high binding affinity for the TfR1 receptor, to a progranulin domain enhances penetration across the blood-brain barrier (BBB) ​​compared to a progranulin sequence that is not linked to the TfR1 binding domain. Furthermore, it is believed that enhanced delivery of the progranulin domain across the BBB may enable peripheral administration to patients with progranulin deficiency.

[0054] The TfR1-binding domain can be a peptide, a protein, an antibody, an antibody fragment, an Fc region, a Fab region, a single-domain antibody, or a combination thereof. The TfR1-binding domain can include a Fab region. The TfR1-binding domain also can not include and / or be free of an Fc region.

[0055] The TfR1-binding domain can also be described by its affinity for the TfR1 receptor. The TfR1-binding domain can have an affinity for the human TfR1 receptor of about 1 nM to about 100 nM, about 1 nM to about 50 nM, less than 100 nM, greater than 1 nM to less than 20 nM, about 5 nM to less than 20 nM, about 5 nM to about 20 nM, about 10 nM, or 10 nM. Preferably, the affinity of the TfR1-binding domain can be about 10 nM or 10 nM. Typically, transport across the BBB is expected to be maximized as the affinity concentration of the TfR1-binding domain decreases. Unexpectedly, it has been found that transport across the BBB is maximized when the affinity is less than 20 nM but greater than 1 nM or about 10 nM.

[0056] In some embodiments, the TfR1 binding domain can have a heavy chain comprising SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and / or SEQ ID NO: 18. In some embodiments, the TfR1 binding domain can have a light chain comprising SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and / or SEQ ID NO: 22.

[0057] In some embodiments, the TfR1 binding domain comprises a heavy chain comprising SEQ ID NO:15 and a light chain comprising SEQ ID NO:19.

[0058] In some embodiments, the TfR1 binding domain comprises a heavy chain comprising SEQ ID NO:16 and a light chain comprising SEQ ID NO:20.

[0059] In some embodiments, the TfR1 binding domain comprises a heavy chain comprising SEQ ID NO:17 and a light chain comprising SEQ ID NO:21.

[0060] In some embodiments, the TfR1 binding domain comprises a heavy chain comprising SEQ ID NO:18 and a light chain comprising SEQ ID NO:22.

[0061] In some embodiments, the TfR1 binding domain can comprise an amino acid sequence having at least 90%, 95%, and / or 99% sequence identity to the amino acid sequence provided by SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and / or SEQ ID NO:22.

[0062] In some embodiments, a suitable TfR1-binding domain can be defined by its complementary determining regions (CDRs), which are regions of the domain that are thought to interact with the TfR1 receptor. Suitable CDRs for the heavy chain of a TfR1-binding domain can include SEQ ID NO: 45, SEQ ID NO: 46, and / or SEQ ID NO: 47. Suitable CDRs for the light chain of a TfR1-binding domain can include SEQ ID NO: 48, SEQ ID NO: 49, and / or SEQ ID NO: 50.

[0063] In some embodiments, the TfR1 binding domain comprises one or more of SEQ ID NOs: 45-50.

[0064] In some embodiments, the TfR1 binding domain comprises SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50.

[0065] In some embodiments, a suitable TfR1 binding domain can comprise the CDRs set forth in Table A.

[0066] [Table 1] The CDRs in Table A are defined as described in Kabat. (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)), and with the exception of HCDR2, which is defined as described in the North numbering system (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0067] [Table 2] The CDRs in Table A-1 may be identified by sequences similar to those described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international The term "nucleic acid" is defined according to methods well known to those skilled in the art, such as those described in the ImMunoGeneTics database; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0068] Albumin-binding domain The disclosed compounds can also include an albumin-binding domain. The albumin-binding domain is a portion of the compound that specifically binds to albumin and / or human serum albumin. In some embodiments, the albumin-binding domain binds to human serum albumin, but also to serum albumin from other species, such as, but not limited to, mouse, rat, and cynomolgus monkey.

[0069] It has been unexpectedly found that attaching an albumin-binding domain to the C-terminus of a Progranulin domain improves the half-life of the Progranulin domain and / or improves the transport of the Progranulin domain across the BBB. Without being bound by theory, it is believed that the attachment of an albumin-binding domain at the C-terminus of the Progranulin domain prevents C-terminal clipping of the Progranulin domain and / or allows Progranulin to be recycled through the albumin turnover process, thereby improving the half-life of the Progranulin domain.

[0070] In some embodiments, the albumin binding domain is attached to the C-terminus of the progranulin domain via a linker such as L1 or L2.

[0071] The albumin binding domain may be a peptide, a protein, an antibody, a fragment of an antibody, an Fc region, a Fab region, a single domain antibody, or a combination thereof. Preferably, the albumin binding domain is V H It may be a single domain antibody such as H.

[0072] In some embodiments, the albumin binding domain can be represented by SEQ ID NO:23 or SEQ ID NO:24.

[0073] In some embodiments, the albumin binding domain can comprise an amino acid sequence having at least 90%, 95%, and / or 99% sequence identity to the amino acid sequence given by SEQ ID NO:23 or SEQ ID NO:24.

[0074] In some embodiments, a suitable albumin binding domain may be defined by its complementarity determining regions (CDRs), which are the regions of the domain that are thought to interact with albumin. Suitable CDRs for albumin binding domains may include SEQ ID NO:51, SEQ ID NO:52, and / or SEQ ID NO:53.

[0075] In some embodiments, the TfR1 binding domain comprises SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0076] In some embodiments, a suitable albumin binding domain may comprise the CDRs set forth in Table B.

[0077] [Table 3] The CDRs are defined as described in Kabat. (Kabat, et al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242 (1991)), and with the exception of CDR2, which is defined as described in the North numbering system (North et al., A New Clustering of Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011)).

[0078] Linker The disclosed compounds include one or more linkers L1, L2, L3, etc. The linkers are used to connect the progranulin domain, the TfR1-binding domain, and the albumin-binding domain to one another. In some embodiments, the disclosed compounds have one linker, two linkers, three linkers, or one to three linkers.

[0079] In some embodiments, a first linker, L1, connects the TfR1-binding domain Y to the progranulin domain X. In some embodiments, a second linker, L2, connects the albumin-binding domain Z to the progranulin domain X or the TfR1-binding domain Y. In some embodiments, L1 and L2 are the same. In some embodiments, L1 and L2 are not the same.

[0080] The linkers can independently comprise a covalent bond, a peptide linker, a PEG linker, a disulfide bond, a thioacetal bond, or a thioester bond. The linkers can independently be selected from a covalent bond, a peptide linker, a PEG linker, a disulfide bond, a thioacetal bond, and a thioester bond.

[0081] In some embodiments, the linker is a peptide linker. Suitable peptide linkers include peptides of 2 to 50 amino acids in length. Other suitable peptide linkers include (G4U) n where U is any suitable amino acid and n is an integer from 1 to 10. Suitable amino acids that may be represented by U include glutamine (Q), serine (S), asparagine (N), and alanine (A), among others. Preferably, the linker is (GQ) n or (G4S) n where n is 3 to 5.

[0082] In some embodiments, the linker may be represented by one or more of SEQ ID NOs: 5-14.

[0083] In some embodiments, the first linker and the second linker can be represented by SEQ ID NO:7.

[0084] In some embodiments, the linker is a PEG linker. Suitable PEG linkers include those represented by Formula III, where n is an integer from 1 to 10.

[0085] [ka]

[0086] Other suitable peptide linkers include DKT(G4U) n where U is any suitable amino acid and n is an integer from 1 to 10. Suitable amino acids that may be represented by U include glutamine (Q), serine (S), asparagine (N), alanine (A), among others. In some embodiments, the linker is DKT(G4Q). n or DKT(G4S) n where n is 3 to 5.

[0087] Compositions and Routes of Administration The compounds of the present invention can be used as pharmaceuticals in human medicine, administered by various routes. Most preferably, such compositions are for parenteral administration. Such pharmaceutical compositions can be prepared by methods well known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 19th ed. (1995), A. Gennaro et al., Mack Publishing Co.), and comprise the compounds disclosed herein and pharmaceutically acceptable carriers, diluents or excipients.

[0088] Treatment method The compounds of the present disclosure can be used to aid in the treatment of patients, particularly CNS diseases including neurodegenerative diseases such as neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), etc. Treatment includes the administration of the compounds of the present disclosure for the treatment of CNS diseases or conditions in humans that would benefit from delivery of a progranulin domain across the blood-brain barrier to alleviate the effects of low levels of progranulin in the patient.

[0089] definition As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibody embodiments include monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific or multispecific antibodies, or conjugated antibodies. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0090] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG)-type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino-terminal portion of each of the four polypeptide chains contains a variable region of about 100 to 125 amino acids or more that is primarily responsible for antigen recognition. The carboxy-terminal portion of each of the four polypeptide chains contains a constant region that is primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (VL) and a light chain constant region. IgG isotypes can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0091] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are critical regions of the antibody for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods of Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics This can be done according to well-known schemes, including those described in the Imgt database, available at www.imgt.org (see Lefranc et al., Nucleic Acids Res. 1999;27:209-212).

[0092] Embodiments of the present disclosure also include antibody fragments or antigen-binding fragments, as used herein, that comprise at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fv (sdFv), Fd fragments, etc.

[0093] As used herein, the terms "bind" and "binds," unless otherwise indicated, refer to the ability of a binding domain of a compound to form a chemical bond or attractive interaction with another protein or molecule, resulting in close proximity of the two proteins or molecules as determined by common methods known in the art.

[0094] The term "Progranulin domain" refers to a portion of a compound or polypeptide sequence of the present disclosure that includes an amino acid sequence representing at least one unmodified or wild-type granulin protein. In some embodiments, a Progranulin domain can include amino acid sequences representing up to seven granulin proteins and a proprotein naturally contained in Progranulin. In some embodiments, a Progranulin domain can include amino acid sequences representing two or more but fewer than seven granulin proteins and a proprotein naturally contained in Progranulin, i.e., a Progranulin fragment. In one non-limiting example, a Progranulin domain is represented by an amino acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO: 2, and / or SEQ ID NO: 3. In another non-limiting example, a Progranulin domain does not include SEQ ID NO: 4.

[0095] The term "TfR1 binding domain" refers to an antibody, portion of an antibody, portion of a compound, or polypeptide sequence that binds to the transferrin receptor, or TfR1.

[0096] The term "albumin binding domain" refers to an antibody, portion of an antibody, portion of a compound, or polypeptide sequence that binds to albumin. In one non-limiting example, an albumin binding domain refers to a portion of a compound of the present disclosure that binds to human serum albumin.

[0097] As used interchangeably herein, the terms "patient," "subject," and "individual" refer to a human. In certain embodiments, the patient is further characterized by a CNS disease, disorder, or condition (e.g., a CNS neurodegenerative disease). In some embodiments, the patient may be further characterized as being at risk for developing a CNS disorder, disease, or condition.

[0098] As used herein, "Fab" refers to a fragment antigen-binding region of an antibody. Further, as used herein, the Fab region comprises a heavy chain linked to another portion of the disclosed compounds, such as a linker, a progranulin domain, a TfR1-binding domain, and / or an albumin-binding domain. The Fab region also comprises a light chain covalently linked to a heavy chain.

[0099] As used herein, a "single domain antibody" is an antibody fragment consisting of a single monomeric variable antibody chain. A single domain antibody comprises an antigen-binding region.

[0100] As used herein, "V H "H fragment" is a single domain antibody engineered from a heavy chain antibody found in camelids.

[0101] As used herein, the terms "peptide" or "peptide chain" generally refer to a polymer comprising two or more amino acids and / or amino acid derivatives linked via peptide bonds. Peptide embodiments may include modifications or amino acid derivatives, including post-translational modifications such as phosphorylation, hydroxylation, sulfonation, palmitoylation, glycosylation, and disulfide formation.

[0102] As used herein, the term "linked to" or "linked with" refers to a first nucleotide (or polynucleotide) or peptide being associated, bound, connected, or otherwise joined to a second nucleotide (or polynucleotide) or peptide. For example, a first polynucleotide can be linked to a second polynucleotide sequence such that, when the sequence is translated, it forms a fusion peptide or protein. Similarly, a first peptide sequence can be linked to a second peptide sequence via covalent or non-covalent interactions to form a multimeric peptide. Alternatively, "linked to" or "linked with" refers to a nucleotide (or polynucleotide sequence) or peptide that is associated, connected, or joined to a non-nucleotide or non-peptide moiety. For example, a peptide can be linked (i.e., acylated) to a fatty acid moiety to form a conjugated peptide.

[0103] The term "conjugate group," as used herein, refers to a group that is bound or linked to a peptide. The conjugate group can include a conjugate moiety and a conjugate linker for binding or linking the conjugate moiety to the peptide.

[0104] As used herein, the term "conjugate linker" refers to an atom, group of atoms, molecule, or compound (such as an amino acid or group of amino acids) that comprises at least one bond that attaches or connects a conjugate moiety to a peptide herein.

[0105] The term "conjugate moiety" as used herein means a molecule or compound, particularly a non-peptide molecule or compound, that is bound or linked to a peptide herein, either directly or via a conjugate linker. [Example]

[0106] Example 1 - Expression of the Disclosed Compounds The compounds of the present invention can be expressed essentially as follows: Suitable host cells, such as HEK293 or CHO, can be either transiently or stably transfected with an expression system for antibody secretion using an optimal predetermined HC:LC vector ratio (such as 1:3, 1:2, or 1:1), or with a single vector system encoding both the HC and LC.

[0107] Kappa Design Refinement To evaluate the purification characteristics of the CI pool, 1 L of sCHO supernatant containing CI was purified using a three-column process. The supernatant was applied to a pre-packed, approximately 60 mL CaptoL resin column pre-equilibrated with 20 mM Tris, pH 7.0. The supernatant-loaded column was subsequently washed with 5 column volumes of 20 mM Tris, pH 7.0. The mAb was eluted from the CaptoL resin by applying 5 column volumes of mixed acid elution buffer (20 mM acetic acid, 5 mM citric acid). The CaptoL elution pool was neutralized to pH 5 with 0.5 M Tris base, centrifuged, and applied to a 0.22 micron sterile filter. This CaptoL-purified material was further purified by PrismA resin affinity purification. The CaptoL pool was adjusted to pH 7 using 1 M Tris pH 7.5 and applied to a 58 mL PrismA pre-packed column pre-equilibrated in 50 mM Tris (pH 8.0). It was washed with 5 column volumes of equilibration buffer and eluted with 5 column volumes of mixed acid elution buffer (20 mM acetic acid, 5 mM citrate). The PrismA pool was neutralized to pH 5 using 0.5 M Tris base. The pool was further purified using cation exchange chromatography (Cation Exchange, CEX). The CaptoL pool was loaded onto a 70 mL pre-packed Poros HS50 CEX column equilibrated with 20 mM acetic acid, pH 5, and eluted with a 5-60% linear gradient over 15 column volumes using the same buffer supplemented with 1 M NaCl. Subsequently, the elution fractions containing the monomer were pooled, sterile filtered, and dialyzed overnight at 4 °C into PBS. After dialysis, the protein was sterile filtered, and the protein concentration / yield was determined at A280 using the calculated extinction coefficient. This sCHO material was used to analyze all potential development and HCP proteins.

[0108] [Table 4]

[0109] Refining the sandwich design To evaluate the purification characteristics of the CI pool, 3 L of sCHO supernatant containing CI was purified using a three-column process. The supernatant was applied to a pre-packed PrismA resin column of approximately 138 mL pre-equilibrated with 50 mM Tris (pH 8.0). The column loaded with the supernatant was then washed with 5 column volumes of 50 mM Tris (pH 8.0). The mAb was eluted from the PrismA resin by applying 5 column volumes of mixed acid elution buffer (20 mM acetic acid, 5 mM citric acid). The PrismA elution pool was neutralized to pH 5 with 0.5 M Tris base, centrifuged, and applied to a 0.22 micron sterile filter. This PrismA-purified material was further purified by CaptoL resin affinity purification. The PrismA pool was adjusted to pH 7 using 1 M Tris pH 7.5 and applied to a 120 mL CaptoL pre-packed column pre-equilibrated in 20 mM Tris (pH 7). It was washed with 5 column volumes of equilibration buffer and eluted with 5 column volumes of mixed acid elution buffer (20 mM acetic acid, 5 mM citric acid). The CaptoL pool was neutralized to pH 5 using 0.5 M Tris base. The pool was further purified using cation exchange chromatography (CEX). The CaptoL pool was loaded onto a 70 mL pre-packed Poros HS50 CEX column equilibrated with 20 mM acetic acid, pH 5, and eluted with a 5-60% linear gradient over 15 column volumes using the same buffer supplemented with 1 M NaCl. Subsequently, the elution fractions containing the monomer were pooled, sterile filtered, and dialyzed overnight at 4 °C into PBS. After dialysis, the protein was sterile filtered, and the protein concentration / yield was determined at A280 using the calculated extinction coefficient. This sCHO material was used to analyze all potential development and HCP proteins.

[0110] [Table 5] 1 A linker used to connect the TfR1-binding domain / progranulin domain and the progranulin domain / albumin-binding domain

[0111] Expression and purification of Comparative Example 1 Comparative Example 1 was expressed and purified as described in WO2019246071(A1) (SEQ ID NOs: 227 and 291 in WO2019246071(A1)).

[0112] [Table 6]

[0113] Example 2 - Transferrin Receptor 1 (TfR1) Binding of the H09 Binding Domain To determine the binding kinetics and affinity of H09-PGRN and BACDE to human TfR1 (hTfR1), human TfR2 (hTfR2), and cynomolgus monkey TfR1, the binding affinity and kinetics of the antibodies to the antigen were determined using a BIACORE® T200 instrument. The binding kinetics and affinity of the antibodies H09-PGRN and BACDE to the human TfR1 ECD were determined using a surface plasmon resonance biosensor such as the BIAcore® T200 (GE Healthcare, Piscataway, NJ). Briefly, a BIAcore® T200 instrument was used to measure the binding kinetics of sandwich example 1 (H09-PGRN-C90.43) and sandwich example 2 (H09-PGRNΔpGF-C90.43) to hTfR1, hTfR2, and cyno Tfr1 by surface plasmon resonance (SPR) at 25° C. Samples were dissolved in 1× HBS-EP+ running buffer (Teknova catalog number H802), and the His-tagged ECDs of hTfR1, hTfR2, and cyno Tfr1 were amine-coupled to Cytiva CM3 Series S sensor chips.

[0114] Human TfR1 ECD protein with a hexahistidine tag was generated, expressed in 293 cells, and purified by nickel-charged IMAC followed by size-exclusion chromatography. hTfR2 and cynomolgus TfR ECD were purchased from SYNGENE.

[0115] Binding is assessed using multiple analytical cycles. Each cycle is performed at 25°C with a flow rate of 50 μl / min for ligand binding and dissociation. Each kinetic cycle consists of the following steps: Sandwich Example 1 (H09-PGRN-C90.43) and Sandwich Example 2 (H09-PGRNΔpGF-C90.43) are injected across all four flow cells for 240 seconds (starting at 125 nM and using a two-fold serial dilution for each cycle), followed by a dissociation phase for 900 seconds and regeneration with 3M MgCl2 hydrochloride for a 30 second contact time. The respective binding rates (i.e., k on ) and dissociation rate (i.e., k off ) is evaluated using standard double referencing and fitted to a "1:1 (Langmuir) binding" model in the BiaEvaluation software in batch mode. Affinity (KD) is calculated from the binding kinetics according to the relationship K) = Koff / Kon.

[0116] In experiments performed as described, the protein constructs of the present invention exhibited the following affinities for TfR1 ligands as shown in Table 4. As shown in Table 4, both Sandwich Example 1 and Sandwich Example 2 exhibited 1:1 binding.

[0117] [Table 7]

[0118] Example 3 - PD durability in Grn KO mice Bis(monoacylglycero)phosphate (BMP) is a structural lipid important for lysosomal protein membrane docking, and its function has been proposed as an important biomarker of lysosomal dysfunction. PGRN is critically involved in maintaining BMP levels in lysosomes (Logan et al., Cell, 2021; Boland et al., Nat Comm, 2022). To evaluate the effect of PGRN mutants on rescuing BMP deficiency, 3- to 4-month-old Grn KO mice were treated intravenously (iv) with 10E10 kappa PGRN (kappa Ex. 5, HC: SEQ ID NO: 34 and LC: SEQ ID NO: 35) or 10E10 kappa PGRNΔpGF (kappa Ex. 6, HC: SEQ ID NO: 34 and LC: SEQ ID NO: 36) at 1.5, 5, and 15 mg / kg. Untreated wild-type littermates and Grn KO mice served as positive and negative controls, respectively. Brain BMP 22:6 levels were assessed at 1, 4, 7, 14, 21, 35 and 63 days after treatment.

[0119] [Table 8]

[0120] Table 5 and Figure 1 show that a construct containing a progranulin fragment (PGRNΔpGF, i.e., the progranulin fragment does not include p-proprotein, G granulin, and F granulin) has a prolonged PD effect and prolonged durability in inducing a pharmacological response in Grn KO mice compared to a construct containing the full-length progranulin peptide. Without being bound by theory, it is believed that a construct containing PGRNΔpGF has prolonged durability compared to a construct containing PGRN.

[0121] Example 4 - Tau turnover To assess the effect of BBB-crossing PGRN on tau turnover, wild-type mice were treated with 5 mg / kg intravenous administration of kappa H09 PGRN (kappa Example 3) and kappa H09 PGRNΔpGF (kappa Example 4). Soluble tau protein levels were tested 7 days after treatment using ELISA. Statistical significance was determined using one-way ANOVA with correction for multiple comparisons.

[0122] Table 7 and Figure 2 show that both constructs (full-length PGRN and PGRNΔpGF) reduced tau turnover, a biomarker that indicates that both constructs are delivering progranulin across the BBB in wild-type mice.

[0123] [Table 9]

[0124] Example 5 - TfR shuttle delivers PGRN across the BBB To investigate whether BBB-crossing PGRN can rescue lipofuscin accumulation in Grn KO mice, 8-month-old Grn KO mice were treated with Kapp PGRN or kappa PGRNΔpGF at various doses and administration intervals. Brain tissue and CSF were collected 12 weeks after the first administration. To avoid any immunogenicity to the test article, CD4 T cells were depleted using the GK1.5 anti-CD4 monoclonal antibody. The effects of BBB-crossing PGRN on lipofuscin accumulation, microglial and astrocyte activation, and CSF neurofilament light chain (NfL) levels were assessed at 1, 6, 10, 24, 48, 72, and 168 hours after treatment. To estimate PGRN exposure in the interstitial fluid and intracellular compartments of the brain, phosphate-buffered saline (PBS) buffer-soluble proteins and RIPA buffer-soluble proteins (a buffer used for lysis of cells and tissues to facilitate the isolation of cytoplasmic, membrane, nuclear, and mitochondrial proteins) were sequentially extracted. PGRN levels in PBS, the RIPA fraction of brain tissue, and cerebrospinal fluid (CSF) were measured using enzyme-linked immunoassay (ELISA). PTV11 (Comparative Example 1) was also evaluated as a reference compound.

[0125] Table 8 shows the levels of Progranulin delivered across the BBB to the brain and / or CSF up to 168 hours after treatment with Sandwich Example 1. Table 9 shows the levels of Progranulin delivered across the BBB to the brain and / or CSF up to 168 hours after treatment with Kappa Example 3. Sandwich Example 1 and Kappa Example 3 have the same components, i.e., H09 Fab and C90.43 VHH linked to full-length Progranulin with a (G4Q)3 peptide linker. However, in Sandwich Example 1, the heavy chain of the H09 Fab antibody portion is linked to the N-terminus of the Progranulin sequence, as shown in SEQ ID NO:25, and the C90.43 VHH is linked to the N-terminus of the Progranulin sequence. HIn Kappa Example 3, the heavy chain of the H09 Fab antibody portion is linked to the C90.43 V VHH, and the light chain of the H09 Fab antibody portion is linked to the N-terminus of the Progranulin sequence. As shown in Table 8 and Figure 3, administration of Sandwich Example 1 resulted in increased PGRN levels in the brain and CSF compared to Kappa Example 3. This result was consistent throughout the entire post-treatment period up to 168 hours post-treatment.

[0126] Table 10 shows the levels of progranulin delivered across the BBB to the brain and / or CSF up to 168 hours after treatment with Comparative Example 1. Comparative Example 1 is a literature compound disclosed in U.S. Patent Application Publication No. 2021 / 0284702. As shown in Table 10 and FIG. 3, administration of both constructs of the present invention, i.e., sandwich Example 1 and kappa Example 3, resulted in higher levels of progranulin in the brain and CSF compared to administration with Comparative Example 1. This result was consistent throughout the entire post-treatment period up to 168 hours after treatment. Therefore, the constructs disclosed herein are believed to improve delivery of PGRN across the BBB to the brain and CSF.

[0127] [Table 10]

[0128] [Table 11]

[0129] [Table 12]

[0130] [Table 13]

[0131] FIG. 6 shows CNS exposure of sandwich example 2 in hTfR KI mice, and FIG. 7 shows the effect of BBB-crossing PGRN on lipofuscin pathology, neuronal injury, neuroinflammation, and lysosomal function in Grn KO mice.

[0132] Example 6 - Evaluation of TfR1 shuttle affinity for PGRN CNS delivery To better understand the affinity requirements of the TfR1 shuttle in delivering PGRN across the BBB, Grn- / - mice were treated with three 10E10-PGRN mutants with TfR binding of 10 nM (kappa Example 6), 300 nM (kappa Example 7), and 1000 nM (kappa Example 8) at 5 mg / kg. Brain BMP 22:6 levels were assessed 7 days after treatment. Untreated wild-type littermates and Grn- / - mice were used as positive and negative controls, respectively. His-tagged PGRN (SYNGENE, SEQ ID NO: 55) was also assessed as a control.

[0133] Table 11 shows that wild-type mice had brain BMP 22:6 levels of 0.74 ± 0.07, while Grn − / − mice had brain BMP 22:6 levels of 0.39 ± 0.02. Peripheral doses of His-tagged PGRN (His-PGRN) did not improve brain BMP 22:6 levels in Grn − / − mice. A construct containing a TfR-binding domain with 1000 nM TfR1 affinity (kappa Example 8) resulted in a brain BMP 22:6 level of 0.65 ± 0.09, indicating that this construct improved brain PGRN levels better than PGRN alone or no treatment, but the improvement did not approach the amount of PGRN seen in wild-type mice. Unexpectedly, a construct containing a TfR1-binding domain with stronger affinity (300 nM) (kappa Example 7) did not improve the amount of PGRN seen in Grn − / − mice. Instead, the construct with 300 nM shuttle (kappa example 7) resulted in levels of PGRN in Grn− / − mice that were comparable to those seen in untreated Grn− / − mice or in Grn− / − mice treated with PGRN that did not contain the TfR1-binding domain.

[0134] Unexpectedly, a construct containing a TfR1-binding domain with an affinity of 10 nM for TfR1 (kappa Example 6) improved the amount of PGRN seen in Grn− / − mice. Indeed, the levels of PGRN seen in Grn− / − mice treated with a construct containing a 10 nM TfR1-binding domain were similar to those seen in positive control (wild-type mice). The data in Table 11 and Figure 4 demonstrate that a construct containing a TfR1-binding domain with an affinity of approximately 10 nM results in transport of the bound progranulin domain across the BBB, improving the deficiency in PGRN levels seen in Grn− / − mice.

[0135] [Table 14]

[0136] Example 7 - TfR mediated PGRN internalization in the absence of sortilin binding and promoted cathepsin D metabolism in vitro It has been reported that PGRN deficiency results in cathepsin D dysregulation in PGRN FTD (Gotzl et al., Acta Neuropathol, 2014). Therefore, HEK293 cells were treated with 100 ng / mL of PGRN-TfR fusion protein, which either binds or does not bind to human TfR or sortilin receptor, for 24 hours. Mature cathepsin D levels in cell lysates were assessed by ELISA.

[0137] As shown in Table 12 and Figure 5, TfR mediated PGRN internalization and promoted cathepsin D metabolism in vitro in the absence of sortilin binding. As shown in Figure 5, Comparative Example 2 was able to shuttle PGRN into HEK293 cells. Comparative Example 2 can bind to sortilin but not to TfR. However, 8D3-PGRN-C90.43 was unable to shuttle PGRN into HEK293 cells because it was unable to bind to sortilin or TfR. Unexpectedly, it was found that PGRN could be effectively shuttled into HEK293 cells when the 8D3 Fab portion was replaced with H3.03. One difference is that H3.03 can bind to TfR but not to sortilin, as shown in Figure 5. Without wishing to be bound by theory, the data shown in Table 12 and Figure 5 are believed to demonstrate that TfR binding may be an effective alternative to sortilin binding for transporting PGRN across the BBB.

[0138] [Table 15]

[0139] Example 8 - Effect of linker on protein quality To evaluate the purification characteristics of the Progranulin linker mutants, 100 mL of tCHO supernatant containing the linker mutants was purified using a one-column process. The supernatant was applied to a 5 mL pre-packed HiTrap PrismA resin column pre-equilibrated with 3 column volumes of 3 M Tris (pH 8.0). The column loaded with the supernatant was subsequently washed with 2.5 column volumes of 20 mM Tris (pH 7), 2.5 column volumes of 20 mM Tris, 1 M NaCl (pH 7), and 2.5 column volumes of 20 mM Tris (pH 7). The fusion protein was eluted from the PrismA resin by applying 5 column volumes of mixed acid elution buffer (20 mM acetic acid, 5 mM citric acid). The PrismA elution pool was neutralized to pH 5 with 1 M Tris (pH 8), centrifuged, and applied to a 0.22 micron sterile filter. Protein concentration / yield was determined by A280 using the calculated extinction coefficient. Purity was assessed by analytical size exclusion chromatography on a TSKgel UP-SW3000 column using isocratic elution in 50 mM phosphate, 300 mM NaCl (pH 7) buffer.

[0140] As shown in Table 13, when the linker length was increased from (G4Q)1 to (G4Q)2 to (G4Q)3, the protein quality and purity increased in the case of both constructs.

[0141] [Table 16]

[0142] Sequence Listing Progranulin sequence SEQ ID NO:1 - Human Progranulin TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

[0143] SEQ ID NO:2 - Human Progranulin ΔPGF TPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

[0144] SEQ ID NO:3-PGRNdesQLL TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLQDPALR

[0145] SEQ ID NO: 4 - Human progranulin with QLL changed to PIL at amino acid residues 574-576 TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRPIL

[0146] Peptide Linker SEQ ID NO:5-(G4Q) 1 GGGGQ SEQ ID NO: 6-(G4Q) 2 GGGGQGGGGQ SEQ ID NO: 7-(G4Q) 3 GGGGQGGGGQGGGGQ SEQ ID NO:8-(G4Q) 4 GGGGQGGGGQGGGGQGGGGQ SEQ ID NO: 9-(G4Q) 5 GGGGQGGGGQGGGGQGGGGQGGGGQ SEQ ID NO: 10-(G4S) 1 GGGGS SEQ ID NO: 11-(G4S) 2 GGGGSGGGGS SEQ ID NO: 12-(G4S) 3 GGGGSGGGGGSGGGGGS SEQ ID NO: 13-(G4S) 4 GGGGSGGGGGSGGGGGSGGGGGS SEQ ID NO: 14-(G4S) 5 GGGGSGGGGGSGGGGGSGGGGGSGGGGGS

[0147] Fab heavy chain as a TfR1-binding domain SEQ ID NO: 15-H09 Fab heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO: 16-H3.03 Fab heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISRSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIHGYSNSDAFDKWGQGTLVTVSSASTKGPCVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO: 17-8d3 Fab heavy chain EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSASTKGPCVFPLASSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPSCDKTHT SEQ ID NO: 18-10E10 Fab heavy chain QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWVRQAPGKGLEWIGCIYSTSGGRTYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDLWGPGTLVTVSSASTKGPCVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP

[0148] Fab light chain as a TfR1-binding domain SEQ ID NO: 19-H09 Fab light chain DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPKGVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20-8D3 Fab light chain DIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGGGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 21-H3.03 Fab light chain DIQMTQSPSAMSASVGDRVTITCRASQGISNYLAWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 22-10E10 Fab light chain ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC V as an albumin-binding domain H H Sequence number 23-C90.43 V H H EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP Sequence number 24-C80.43 VH H EVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0149] Sandwich PGRN payload SEQ ID NO: 25-H09 Fab-PGRN-C90.43 heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLLGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 26-H09 Fab-PGRNΔpGF-C90.43 heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTGGGGQGGGGQGGGGQTPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLLGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 27-8D3-PGRN-C90.43 heavy chain EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKGLEWIAMIYYDSSKMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLLGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO:28-H3.03-PGRN-C90.43 heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISRSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIHGYSNSDAFDKWGQGTLVTVSSASTKGPCVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLLGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP

[0150] Kappa PGRN Payload SEQ ID NO: 29-H09-C90.43 heavy chain EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCGARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 30-H09 PGRNdesQLL light chain DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLQDPALR SEQ ID NO: 31-H09 PGRNΔpGF desQLL light chain DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQTPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALR SEQ ID NO: 32-H09 PGRN light chain DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLQDPALRQLL SEQ ID NO: 33-H09 PGRNΔpGF light chain DIQMTQSPSAMSASVGDRVTITCRASQGISHYLVWFQQKPGKVPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQTPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL SEQ ID NO: 34-10E10-C80.43 heavy chain QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWICWVRQAPGKGLEWIGCIYSTSGGRTYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDLWGPGTLVTVSSASTKGPCVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPGGGGQGGGGQGGGGQGGGGQGGGGQEVQLLESGGGLVQPGGSLRLSCAASGRYIDETAVAWFRQAPGKGREFVAGIGGGVDITYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAARPGRPLITSKVADLYPYWGQGTLVTVSSPP SEQ ID NO: 35-10E10 PGRN light chain ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGQGGGGQGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL SEQ ID NO: 36-10E10 PGRNΔpGF light chain ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGQGGGGQGGGGQGGGGQGGGGQTPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL SEQ ID NO: 37-10E10 R32W (300 nM) PGRN light chain ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNWLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGQGGGGQGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL SEQ ID NO: 38-10E10 T52G PGRN light chain (1000 nM) AIDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYGASTLASGVPSRFKGSGSGTQFTLTISGVQCDDSATYYCQGTYFSSGWSWAFGGGTEVVVKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNECGGGGQGGGGQGGGGQGGGGQGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

[0151] Knob and hole SEQ ID NO: 39 - scFv 8D3-hIgG1-LALA knob EVQLVESGGGLVQPGNSLTLSCVASGFTFSNYGMHWIRQAPKKCLEWIAMIYYDSSKMNYADTVKGRFTISRDNSKNTLYLEMNSLRSEDTAMYYCAVPTSHYVVDVWGQGVSVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQSPASLSASLEEIVTITCQASQDIGNWLAWYQQKPGKSPQLLIYGATSLADGVPSRFSGSRSGTQFSLKISRVQVEDIGIYYCLQAYNTPWTFGCGTKLELKGGGGQGGGGQGGGGQDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40 - hIgG1 LALA Whole-PGRN DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL SEQ ID NO: 41-3F5 light chain AEVVMTQTPSSVSAAVGGTVTIKCQASQNINSWLSWYQQKPGQRPKLLIYSASTLASGVPSRFEGSGSGTEYTLTISDLECDDAATYYCQSSYGSSYDFGGGTEVVVKGDLVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC SEQ ID NO: 42-3F5 hIgG1 LALA knob QVQLVQSGAEVKKPGSSVKVSCTASGFSFSSGYWICWVRQAPGQGLEWMGCIHSVRSHMTYYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARDASGVWNYFTLWGQGTLVTVSSASTKGPCVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 43 - Stump hIgG1 TV LALA Knob TRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLLGGGGSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 44 - Stump hIgG1 LALA knob DKTHTCPCPPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0152] TfR1 binding domain complementarity determining region SEQ ID NO: 45 - Heavy chain VH CDR1-North VASGFTFSSYSMN SEQ ID NO: 46 - Heavy chain VH CDR2 - Kabat SISSSSSYIYYADSVKG SEQ ID NO: 47 - Heavy chain VH CDR3-North ARRHGYSNSDAFDN SEQ ID NO: 48 - Light chain kappa CDR1-North RASQGISHYLV SEQ ID NO: 49 - Light chain kappa CDR2-North YAASSLQS SEQ ID NO:50 - Light chain kappa CDR3-North LQHNSYPWT

[0153] Albumin-binding domain complementarity-determining region SEQ ID NO:51-C90.43 CDR1 AASGRYIDETAVA SEQ ID NO:52-C90.43 CDR2 GIGGGVDITYYADSVKG SEQ ID NO:53-C90.43 CDR3 GARPGRPLITSKVADLYPY

[0154] TfR1 binding domain variable heavy chain (V H ) SEQ ID NO:54-H09 Fab V H (HCVR) EVQLVESGGGLVKPGGSLRLSCVASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRHGYSNSDAFDNWGQGTLVTVSS

[0155] Other arrays SEQ ID NO: 55 - His-tagged PGRN HHHHHHGGGGQGGGGQGGGGQENLYFQSGGGGQTRCPDGQFCPVACCLDPGGASYSCCRPLLDKWPTTLSRHLGGPCQVDAHCSAGHSCIFTVSGTSSCCPFPEAVACGDGHHCCPRGFHCSADGRSCFQRSGNNSVGAIQCPDSQFECPDFSTCCVMVDGSWGCCPMPQASCCEDRVHCCPHGAFCDLVHTRCITPTGTHPLAKKLPAQRTNRAVALSSSVMCPDARSRCPDGSTCCELPSGKYGCCPMPNATCCSDHLHCCPQDTVCDLIQSKCLSKENATTDLLTKLPAHTVGDVKCDMEVSCPDGYTCCRLQSGAWGCCPFTQAVCCEDHIHCCPAGFTCDTQKGTCEQGPHQVPWMEKAPAHLSLPDPQALKRDVPCDNVSSCPSSDTCCQLTSGEWGCCPIPEAVCCSDHQHCCPQGYTCVAEGQCQRGSEIVAGLEKMPARRASLSHPRDIGCDQHTSCPVGQTCCPSLGGSWACCQLPHAVCCEDRQHCCPAGYTCNVKARSCEKEVVSAQPATFLARSPHVGVKDVECGEGHFCHDNQTCCRDNRQGWACCPYRQGVCCADRRHCCPAGFRCAARGTKCLRREAPRWDAPLRDPALRQLL

[0156] TfR1 binding domain complementarity determining region SEQ ID NO: 56 - Heavy chain VH CDR2-North SISSSSSYIY SEQ ID NO: 57 - Heavy chain VH CDR1 - Kabat SYSMN SEQ ID NO: 58 - Heavy chain VH CDR3 - Kabat RHGYSNSDAFDN SEQ ID NO:59 - Light chain kappa CDR1 - Kabat DISCLOSURE SEQ ID NO: 60 - Light chain kappa CDR2 - Kabat AASSLQS SEQ ID NO: 61 - Light chain kappa CDR3 - Kabat LQHNSYPWT SEQ ID NO: 62 - Heavy chain VH CDR1-IMGT GFTFSSYS SEQ ID NO: 63 - Heavy chain VH CDR2-IMGT ISSSSSYI SEQ ID NO: 64 - Heavy chain VH CDR3-IMGT ARRHGYSNSDAFDN SEQ ID NO: 65 - Light chain kappa chain CDR1 - IMGT QGISHY SEQ ID NO: 66 - Light chain kappa chain CDR2 - IMGT AAS SEQ ID NO: 67 - Light chain kappa chain CDR3 - IMGT LQHNSYPWT SEQ ID NO: 68 - Heavy chain VH CDR1 - Chothia GFTFSSY SEQ ID NO: 69 - Heavy chain VH CDR2 - Chothia SSSSSY SEQ ID NO: 70 - Heavy chain VH CDR3 - Chothia RHGYSNSDAFDN SEQ ID NO:71 - Light chain kappa CDR1 - Chothia DISCLOSURE SEQ ID NO:72 - Light chain kappa CDR2 - Chothia AASSLQS SEQ ID NO:73 - Light chain kappa CDR3 - Chothia LQHNSYPWT

Claims

1. (a) a progranulin domain; (b) a transferrin receptor 1 (TfR1) binding domain comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the VL comprises light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; and A compound comprising: The amino acid sequence of HCDR1 is SEQ ID NO:45; The amino acid sequence of HCDR2 is SEQ ID NO:46; The amino acid sequence of HCDR3 is SEQ ID NO:47; The amino acid sequence of LCDR1 is SEQ ID NO:48; The amino acid sequence of LCDR2 is SEQ ID NO:49; A compound wherein the amino acid sequence of LCDR3 is SEQ ID NO:

50.

2. the compound further comprises an albumin binding domain, the albumin binding domain comprising complementarity determining regions (CDRs) CDR1, CDR2, and CDR3; the amino acid sequence of CDR1 is SEQ ID NO:51; the amino acid sequence of CDR2 is SEQ ID NO:52; The compound according to claim 1, wherein the amino acid sequence of CDR3 is SEQ ID NO:

53.

3. The compound of claim 2, wherein the TfR1 binding domain is linked to the Progranulin domain using a first linker, and the albumin binding domain is linked to the Progranulin domain or the TfR1 binding domain using a second linker.

4. The compound has the formula: Y-L １ -XL ２ -Z or Z-L ２ -Y-L １ -X compound, wherein X is the progranulin domain; Y is the TfR1-binding domain; Z is the albumin binding domain, L １ is the first linker; L ２ The compound of claim 2 or 3, wherein:

5. The compound has the formula: Y-L １ -XL ２ -Z, The compound of claim 4, wherein the TfR1 binding domain is linked to the Progranulin domain at the N-terminus of the Progranulin domain and the albumin binding domain is linked to the Progranulin domain at the C-terminus of the Progranulin domain.

6. The compound according to any one of claims 2 to 5, wherein the TfR1-binding domain and the albumin-binding domain do not form a dimer.

7. The compound according to any one of claims 3 to 6, wherein the TfR1-binding domain is a Fab region, and the first linker is linked to the heavy chain of the TfR1-binding domain or the light chain of the TfR1-binding domain.

8. 8. The compound of any one of claims 1 to 7, wherein the TfR1 binding domain has an affinity for the human TfR1 receptor of about 1 nM to about 100 nM, about 5 nM to less than 20 nM, or about 10 nM.

9. The TfR1-binding domain is (a) a heavy chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18; and (b) a light chain, wherein the amino acid sequence of the light chain is SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:

22.

10. The compound according to any one of claims 1 to 9, wherein the TfR1 binding domain comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO: 15 and the amino acid sequence of the LC being SEQ ID NO:

19.

11. The albumin binding domain is V Ｈ The compound according to any one of claims 2 to 10, which is an H antibody fragment.

12. The albumin binding domain is ２ The compound according to any one of claims 3 to 11, wherein the C-terminus of the progranulin domain is linked via

13. The compound according to any one of claims 1 to 22, wherein the amino acid sequence of the albumin binding domain is SEQ ID NO:23 or SEQ ID NO:

24.

14. L １ and L ２ is independently selected from a covalent bond, a peptide linker, a PEG linker, a disulfide bond, a thioacetal bond, or a thioester bond.

15. L １ and L ２ The compound according to any one of claims 3 to 14, wherein are identical.

16. L １ and L ２ is SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14, and preferably １ and L ２ The compound according to any one of claims 3 to 15, wherein the amino acid sequence is SEQ ID NO:

7.

17. The compound according to any one of claims 1 to 16, wherein the Progranulin domain is a naturally occurring sequence.

18. The compound according to any one of claims 1 to 17, wherein the Progranulin domain comprises a fragment of Progranulin, and the amino acid sequence of the Progranulin fragment is SEQ ID NO:

2.

19. The compound according to any one of claims 1 to 18, wherein the amino acid sequence of the Progranulin domain is SEQ ID NO:

1.

20. 20. The compound of any one of claims 1 to 19, wherein the compound comprises a heavy chain having an amino acid sequence having at least 90% sequence identity to the amino acid sequence given by SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:

29.

21. 21. The compound of any one of claims 1 to 20, wherein the compound comprises a light chain having an amino acid sequence having at least 90% sequence identity to the amino acid sequence given by SEQ ID NO:19, SEQ ID NO:32, or SEQ ID NO:

33.

22. The compound is (a) a heavy chain, wherein the amino acid sequence of the heavy chain is SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:29; and (b) a light chain, wherein the amino acid sequence of the light chain is SEQ ID NO: 19, SEQ ID NO: 32, or SEQ ID NO:

33.

23. 23. The compound of any one of claims 1 to 22, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:25 and the amino acid sequence of the LC being SEQ ID NO:

19.

24. 24. The compound of any one of claims 1 to 23, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:26 and the amino acid sequence of the LC being SEQ ID NO:

19.

25. 25. The compound of any one of claims 1 to 24, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:29 and the amino acid sequence of the LC being SEQ ID NO:

32.

26. 26. The compound of any one of claims 1 to 25, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:29 and the amino acid sequence of the LC being SEQ ID NO:

33.

27. A compound comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence given by SEQ ID NO:25, preferably comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:

25.

28. 28. The compound of claim 27, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:25 and the amino acid sequence of the LC being SEQ ID NO:

19.

29. A compound comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence given by SEQ ID NO:26, preferably comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence given by SEQ ID NO:

26.

30. 30. The compound of claim 29, wherein the compound comprises a heavy chain (HC) and a light chain (LC), the amino acid sequence of the HC being SEQ ID NO:26 and the amino acid sequence of the LC being SEQ ID NO:

19.

31. A compound comprising a Progranulin fragment, wherein the amino acid sequence of the Progranulin fragment is SEQ ID NO:

2.

32. The compound of claim 31, wherein the progranulin fragment is between 100 and 500 residues in length.

33. 33. A compound according to any one of claims 1 to 32 for use in the treatment of neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

34. A method for treating a disorder, comprising administering a compound according to any one of claims 1 to 32 to a patient in need thereof.

35. 35. The method of claim 34, wherein the disorder is neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

36. A compound according to any one of claims 1 to 32 for use in therapy.

37. 33. A pharmaceutical composition comprising a compound according to any one of claims 1 to 32 for use in the treatment of neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

38. 36. Use of a compound according to any one of claims 1 to 32 in the manufacture of a medicament for the treatment of neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

39. A composition comprising a compound according to any one of claims 1 to 32 and a pharma- ceutically acceptable carrier.

40. 33. Use of a compound according to any one of claims 1 to 32 for the treatment of neuronal ceroid lipofuscinosis type 11, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or a combination thereof.

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