Humanized and stabilized FC5 variants for enhancement of blood brain barrier transport

Abstract

To provide humanized and stabilized FC5 variants for enhancement of blood brain barrier transport.SOLUTION: Featured herein are compositions comprising humanized and engineered variants of an antibody variable domain (e.g., FC5), chimeric molecules comprising the antibody variable domain, compositions comprising the chimeric molecules, and uses thereof. This disclosure relates generally to compositions for delivering a desired agent (e.g., a therapeutic or diagnostic agent) across the blood brain barrier, and methods of using the same for treatment and / or prevention of neurological disorders. This application relates to molecules comprising an antibody variable domain (e.g., an FC5 variant) that can be used to transport a therapeutic agent across the blood brain barrier.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 751,962, filed October 29, 2018, the contents of which are incorporated by reference in their entirety.

[0002] The present disclosure relates generally to compositions for delivering a desired agent (e.g., therapeutic agent, diagnostic agent) across the blood-brain barrier and methods of using the same for the treatment and / or prevention of neurological disorders. [Background technology]

[0003] Delivery of drugs to the central nervous system (CNS) has been a challenge in the treatment of neurological diseases (such as Alzheimer's and Parkinson's diseases). For drugs to reach the nervous system, they must first penetrate the blood-brain barrier (BBB), a major challenge due to the selectivity of the BBB. The BBB acts as a semipermeable membrane, preventing most molecules from entering the nervous system from the blood, allowing only low molecular weight (less than 400 Da) and lipophilic compounds to pass through. Most small molecules and macromolecules (such as monoclonal antibodies and antisense oligonucleotides) cannot pass through this barrier. Due to this difficult process of drug penetration across the BBB, only a small percentage of therapeutic agents for neurological diseases make it to clinical trials.

[0004] There is a need in the art for improved compositions and methods for delivering therapeutic agents to the CNS. Summary of the Invention [Means for solving the problem]

[0005] The present application relates to molecules comprising antibody variable domains (e.g., FC5 variants) that can be used to transport therapeutic agents across the blood-brain barrier.

[0006] In one aspect, the disclosure features an antibody variable domain that translocates across the blood-brain barrier. The antibody variable domain comprises an amino acid sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:1. The amino acid sequence comprises (i) an amino acid substitution at one or more of positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1, compared to SEQ ID NO:1; (ii) a complementarity determining region (CDR)1 comprising the sequence set forth in SEQ ID NO:47 or 57, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51.

[0007] In some instances, the amino acid sequence comprises amino acid substitutions, as compared to SEQ ID NO:1, at at least four of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. In other instances, the amino acid sequence comprises amino acid substitutions, as compared to SEQ ID NO:1, at at least five of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. In yet other instances, the amino acid sequence comprises amino acid substitutions, as compared to SEQ ID NO:1, at at least six of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. In one instance, the amino acid sequence comprises amino acid substitutions, as compared to SEQ ID NO:1, at each of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1.

[0008] In certain instances, CDR1 comprises the sequence set forth in SEQ ID NO: 47. In other instances, CDR1 comprises the sequence set forth in SEQ ID NO: 57.

[0009] In some cases, the amino acid sequence includes one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; or a leucine at a position corresponding to position 117 of SEQ ID NO:1.

[0010] In certain instances, the amino acid sequence includes a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; and a leucine at a position corresponding to position 117 of SEQ ID NO:1.

[0011] In some instances, the amino acid sequence comprises substitutions at one or more (i.e., 1, 2, 3, 4, 5, or 6) of positions corresponding to positions 1, 37, 44, 45, 47, or 79 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some embodiments, the amino acid sequence comprises one or more (i.e., 1, 2, 3, 4, 5, or 6) of glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; leucine at a position corresponding to position 45 of SEQ ID NO:1; tryptophan at a position corresponding to position 47 of SEQ ID NO:1; or leucine at a position corresponding to position 79 of SEQ ID NO:1. In some instances, the amino acid sequence comprises substitutions at positions corresponding to positions 44 and 45 of SEQ ID NO:1, as compared to SEQ ID NO:1. In certain embodiments, the amino acid sequence comprises a glycine at a position corresponding to position 44 of SEQ ID NO:1; and a leucine at a position corresponding to position 45 of SEQ ID NO:1. In other cases, the amino acid sequence comprises substitutions at positions corresponding to positions 1, 44, and 45 of SEQ ID NO:1, as compared to SEQ ID NO:1. In certain embodiments, the amino acid sequence comprises a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; and a leucine at a position corresponding to position 45 of SEQ ID NO:1. In still other cases, the amino acid sequence comprises substitutions at positions corresponding to positions 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some embodiments, the amino acid sequence comprises a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1. In some cases, the amino acid sequence comprises substitutions at positions corresponding to positions 1, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In certain embodiments, the amino acid sequence comprises a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1.In certain instances, the amino acid sequence comprises substitutions at positions corresponding to positions 37, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some embodiments, the amino acid sequence comprises a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1. In other instances, the amino acid sequence comprises substitutions at positions corresponding to positions 1, 37, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In certain embodiments, the amino acid sequence comprises a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1. In some instances, the amino acid sequence comprises substitutions at positions corresponding to positions 37, 44, 45, 47, and 79 of SEQ ID NO:1, as compared to SEQ ID NO:1. In certain embodiments, the amino acid sequence comprises a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; a tryptophan at a position corresponding to position 47 of SEQ ID NO:1; and a leucine at a position corresponding to position 79 of SEQ ID NO:1.

[0012] In certain instances, the amino acid sequence is a sequence set forth in any one of SEQ ID NOs:10-17.

[0013] In another aspect, the disclosure relates to an antibody variable domain that translocates across the blood-brain barrier, wherein the antibody variable domain comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:1. The amino acid sequence comprises complementarity determining region (CDR) 1, CDR2, and CDR3, wherein CDR2 comprises the sequence set forth in SEQ ID NO:50. In one embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:47, CDR3 comprises the sequence set forth in SEQ ID NO:51, and the amino acid sequence comprises cysteines at positions corresponding to positions 49 and 70 of SEQ ID NO:1. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:48, CDR3 comprises the sequence set forth in SEQ ID NO:51, and the amino acid sequence comprises a cysteine ​​at a position corresponding to position 79 of SEQ ID NO:1. In yet another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:49, and CDR3 comprises the sequence set forth in SEQ ID NO:52. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:52. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:53. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:53. In yet another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:54. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:54. In another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:55. In yet another embodiment, CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:55.

[0014] In some instances, the amino acid sequence includes a substitution at one or more of the positions corresponding to positions 1, 5, 6, 14, 37, 44, 45, 47, 75, 87, 88, 93, 114, or 117 of SEQ ID NO:1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) compared to SEQ ID NO:1. In some embodiments, the amino acid sequence comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) of a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; a tryptophan at a position corresponding to position 47 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; or a leucine at a position corresponding to position 117 of SEQ ID NO:1.

[0015] In certain instances, the amino acid sequence comprises a substitution at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, or 117 of SEQ ID NO:1, as compared to SEQ ID NO:1. In one embodiment, the amino acid sequence comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; or a leucine at a position corresponding to position 117 of SEQ ID NO:1.

[0016] In other instances, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 44 and 45 of SEQ ID NO:1. In some embodiments, the amino acid sequence includes a glycine at the position corresponding to position 44 of SEQ ID NO:1; and a leucine at the position corresponding to position 45 of SEQ ID NO:1.

[0017] In yet another example, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 1, 44, and 45 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; and a leucine at a position corresponding to position 45 of SEQ ID NO:1.

[0018] In some instances, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 44, 45, and 47 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1.

[0019] In another example, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 1, 44, 45, and 47 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1, a glycine at a position corresponding to position 44 of SEQ ID NO:1, a leucine at a position corresponding to position 45 of SEQ ID NO:1, and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1.

[0020] In certain instances, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 37, 44, 45, and 47 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a valine at a position corresponding to position 37 of SEQ ID NO:1, a glycine at a position corresponding to position 44 of SEQ ID NO:1, a leucine at a position corresponding to position 45 of SEQ ID NO:1, and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1.

[0021] In some instances, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 1, 37, 44, 45, and 47 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1, a valine at a position corresponding to position 37 of SEQ ID NO:1, a glycine at a position corresponding to position 44 of SEQ ID NO:1, a leucine at a position corresponding to position 45 of SEQ ID NO:1, and a tryptophan at a position corresponding to position 47 of SEQ ID NO:1.

[0022] In another example, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 37, 44, 45, 47, and 79 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a valine at a position corresponding to position 37 of SEQ ID NO:1, a glycine at a position corresponding to position 44 of SEQ ID NO:1, a leucine at a position corresponding to position 45 of SEQ ID NO:1, a tryptophan at a position corresponding to position 47 of SEQ ID NO:1, and a leucine at a position corresponding to position 79 of SEQ ID NO:1.

[0023] In certain instances, the amino acid sequence includes substitutions compared to SEQ ID NO:1 at positions corresponding to positions 1, 37, 44, 45, 47, and 79 of SEQ ID NO:1. In one embodiment, the amino acid sequence includes a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1, a valine at a position corresponding to position 37 of SEQ ID NO:1, a glycine at a position corresponding to position 44 of SEQ ID NO:1, a leucine at a position corresponding to position 45 of SEQ ID NO:1, a tryptophan at a position corresponding to position 47 of SEQ ID NO:1, and a leucine at a position corresponding to position 79 of SEQ ID NO:1.

[0024] In one embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:20. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:21. In yet another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:22. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:23. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:24. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:25. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:26. In another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:27. In yet another embodiment, the amino acid sequence is the sequence set forth in SEQ ID NO:28.

[0025] In another aspect, the disclosure features a chimeric molecule comprising any of the antibody variable domains described above.

[0026] In certain instances, the chimeric molecule comprises an antibody Fc region. In some instances, the chimeric molecule comprises an antibody, an antigen-binding fragment of an antibody (e.g., Fab), a single-chain antibody, a diabody, a nanobody, an enzyme, a nucleic acid (e.g., an antisense oligonucleotide), a small molecule drug, or a liposome or lipid nanoparticle that encapsulates a nucleic acid (e.g., an antisense oligonucleotide), a small molecule drug, or a peptide. In some instances, the chimeric molecule comprises a display scaffold (such as ankyrin repeats (Darpin), fibronectin (Adnectin), protein A (Affibody), or stefin A (Affimer)).

[0027] In some instances, in the chimeric molecule, the antibody variable domain is linked, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody hinge region. The C-terminus of the hinge region is fused to an Fc portion. In certain embodiments, the chimeric molecule is bivalent with respect to the antibody variable domain (i.e., it comprises two antibody variable domains, each linked, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody hinge region, and the C-terminus of the hinge region is fused to an Fc portion).

[0028] In certain cases, the hinge region comprises the sequence AEPKSCD (SEQ ID NO:56), AEPKSSD (SEQ ID NO:59), or KTHTCPPCP (SEQ ID NO:19).

[0029] In some cases, the intervening amino acid sequence comprises an antibody heavy chain variable region; an antibody light chain variable region; an enzyme; a peptide, or a linker comprising the amino acid sequence 3x(G4S) (SEQ ID NO:60), G4S (SEQ ID NO:5), GG, or G.

[0030] In some cases, the Fc portion is hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P)Fc, hIgG4(S228P) / G1 agly Fc (in this format which minimizes effector function, the CH1 and CH2 domains are IgG4 with a "fixed" hinge (S228P) and are not glycosylated). The CH3 domain is hIgG1, or hIgG4(S228P)agly Fc.

[0031] In certain cases, the Fc portion is linked, either directly or via a second intervening amino acid sequence, to a second antibody variable domain (e.g., the variable domain(s) of a therapeutic antibody); a Fab; an scFv; a single domain antibody; a nanobody, a diabody, a peptide; a polypeptide, including an enzyme; or a nucleic acid (e.g., an antisense oligonucleotide).

[0032] In some cases, in the chimeric molecule, the hinge region is linked to the nucleic acid. In some cases, in the chimeric molecule, the hinge region is chemically conjugated to the nucleic acid. In certain embodiments, the nucleic acid is an antisense oligonucleotide.

[0033] In another aspect, the disclosure provides chimeric molecules comprising an antibody variable domain described herein linked, either directly or via an intervening amino acid sequence, to a liposome, polymeric nanocarrier, or lipid nanoparticle.

[0034] In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate small molecules or nucleic acids. In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate peptides. In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate enzymes. In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate antibodies or antibody fragments, including Fab, Fab'2, scFv. In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate nanobodies. In some instances, the liposomes, polymeric nanocarriers, or lipid nanoparticles encapsulate antisense oligonucleotides. In some embodiments, the antisense oligonucleotides are gapmers. In other embodiments, the antisense oligonucleotides are splice-switching antisense oligonucleotides.

[0035] In another aspect, the disclosure provides chimeric molecules comprising an antibody variable domain described herein, where the antibody variable domain is linked to the whole antibody directly or via an intervening amino acid sequence. In some embodiments, the whole antibody is an antibody that binds to human LINGO-1, human LINGO-2, human LINGO-3, human LINGO-4, human NOGO receptor, human TREM2, a dipeptide repeat (DPR) of poly-glycine-alanine (GA) having at least six repeats (GA)6 translated from the human chromosome 9 open reading frame 72 (C9orf72) gene [human C9orf72 DPR], human TWEAK, human TWEAK receptor, human beta amyloid, human tau, human alpha-synuclein, human TDP-43, human DR6, human SOD1, DR6, JC virus (or other infectious disease target), or human ErbB2, VEGF, CD20, Her2 (or other human brain tumor target). In some instances, the whole antibody is bevacizumab, rituximab, trastuzumab, an anti-PD1 antibody, or an anti-PDL-L1 antibody.

[0036] In another aspect, the present disclosure provides a chimeric molecule comprising an antibody variable domain described herein and human serum albumin or a binding portion of human serum albumin. In some instances, the chimeric molecule further comprises a therapeutic agent. In one embodiment, the therapeutic agent is an antibody variable domain (e.g., a variable domain(s) of a therapeutic antibody); a Fab; an scFv; a single domain antibody; a nanobody, a diabody, a peptide; an enzyme; a nucleic acid (e.g., an antisense oligonucleotide).

[0037] In another embodiment, the present disclosure provides chimeric molecules encompassing any of the molecules depicted in Figure 49. In some cases, the Fc region of the depicted molecules is replaced by human serum albumin, and these chimeric molecules are also part of the present disclosure. In some instances, the cargo is linked using chemical conjugation such as sulfhydryls (maleimide, iodo, vinylsulfone).

[0038] In yet another aspect, the disclosure provides chimeric molecules comprising an antibody variable domain as described herein linked, either directly or via an intervening amino acid sequence, to a viral capsid (e.g., AAV) containing a therapeutic vector. See, e.g., Naso et al., BioDrugs. 2017;31(4):317-334.

[0039] In another aspect, the disclosure features a composition that includes: (1) a chimeric protein comprising (i) an antibody variable domain described herein and (ii) a first protein comprising a first hinge region and a first Fc portion of an antibody, where the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the first hinge region; (2) a second protein comprising a second hinge region and a second Fc portion of an antibody, where the C-terminus of the second hinge region is linked to the N-terminus of the second Fc portion, where the second protein is paired with the first protein; and (3) a therapeutic agent. In some instances, a linker connects the first antibody variable region to the first protein; and / or the chimeric protein to the therapeutic agent. In some cases, the linker is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G.

[0040] In another aspect, the disclosure provides a first chimeric protein comprising: (1)(i) a first antibody variable domain of any one of the antibody variable domains disclosed herein; and (ii) a first protein comprising an antibody first hinge region and a first Fc portion, wherein the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the first antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the first hinge region; (ii) a second protein comprising a second antibody variable domain of any one of the antibody variable domains of the first protein; (iii) a second protein comprising a second hinge region of an antibody and a second Fc portion, wherein the C-terminus of the second hinge region is linked to the N-terminus of the second Fc portion and the C-terminus of the second antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the second hinge region, such that the second protein is paired with the first protein; and (iv) a therapeutic agent. In some instances, a linker connects the first antibody variable region to the first protein; and / or the second antibody variable region to the second protein; and / or the chimeric protein to the therapeutic agent. In some instances, the linker is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G.

[0041] In another aspect, the disclosure provides a first chimeric protein comprising: (1) (i) a first antibody variable domain of any one of the antibody variable domains disclosed herein; and (ii) a first protein comprising an antibody first hinge region and a first Fc portion, wherein the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the first antibody variable domain is fused to the N-terminus of the first hinge region via a first intervening amino acid sequence; and (2) (i) an antibody variable domain disclosed herein; and (ii) a second protein comprising a second hinge region of an antibody and a second Fc portion, the C-terminus of the second hinge region being linked to the N-terminus of the second Fc portion and the C-terminus of the second antibody variable domain being fused to the N-terminus of the second hinge region via a second intervening amino acid sequence, the second protein being paired with the first protein, the first and second intervening amino acid sequences comprising a Fab that is a therapeutic agent. In some instances, a linker connects the first antibody variable region to the first protein; and / or the second antibody variable region to the second protein; and / or the chimeric protein to a therapeutic agent. In some instances, the linker is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G.

[0042] In some instances, the hinge region comprises the sequence AEPKSCD (SEQ ID NO:56) or AEPKSSD (SEQ ID NO:59). In other instances, the hinge region comprises the sequence KTHTCPPCP (SEQ ID NO:19).

[0043] In certain instances, the first and second Fc moieties have identical amino acid sequences, hi other instances, the first and second Fc moieties have different amino acid sequences.

[0044] In some instances, the second and / or first Fc moieties are aglycosylated.

[0045] In certain instances, the first and second Fc moieties are hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P)Fc, hIgG4(S228P) / G1 agly Fc, and hIgG4(S228P)agly Fc.

[0046] In some instances, the compositions described above further comprise a half-life extending moiety. In some cases, the first and / or second Fc moiety is a mutant variant of a wild-type Fc that acts to extend half-life. The Fc variants (such as YTE and LS) have resulted in improved antibody half-life in humans. In some instances, the variant Fc region differs from the parent Fc region in that the variant Fc region comprises amino acid substitutions at positions 428 and 434, the amino acid substitutions being M428L and N434S. See, e.g., U.S. Patent Nos. 8,546,543 and 8,624,007; US20140056879A1; Brian J. Booth, et al. (2018) Extending human IgG half-life using structure-guided design, mAbs, DOI:10.1080 / 19420862.2018.1490119; and Shen Y, et al. (2017) Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates. PLoS ONE 12(8):e0183326.doi.org / 10.1371 / journal.pone.0183326. In other instances, the composition further comprises a half-life extending HSA mutation. See, e.g., U.S. Patent No. 8,748,380; and WO2014179657A1. In some instances, the HSA variant is mutated at one or more residues selected from the group consisting of Y411, V415, V418, T422, L423, V426, L430, L453, L457, L460, V473, R485, F488, L491, F502, F507, F509, K525, A528, L529, L532, V547, M548, F551, K573, L575, V576, S579, and L583. In still other instances, the composition further comprises XTEN.See, e.g., WO2013130683A2 and Podust et al., Journal of Controlled Release, Volume 240, 28 October 2016, Pages 52-66.

[0047] In some instances, the first and second hinge regions have identical amino acid sequences.

[0048] In certain instances, the therapeutic agent is a binding molecule comprising an antibody variable domain. In one embodiment, the binding molecule binds to human LINGO-1, human LINGO-2, human LINGO-3, human LINGO-4, human TREM2, human C9orf72 DPR, human TWEAK, human TWEAK receptor, human β-amyloid, human tau, human α-synuclein, or human TDP-43. In some instances, the binding molecule is a Fab, and the N-terminus of the VH or VL domain of the Fab is linked to the C-terminus of the first Fc portion.

[0049] In some instances, the therapeutic agent is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a gapmer. In another embodiment, the antisense oligonucleotide is a splice-switching antisense oligonucleotide. In some instances, the antisense oligonucleotide is linked to the first and / or second hinge region.

[0050] In certain instances, the therapeutic agent is a small molecule drug, a peptide, an enzyme, a nanobody, or a nucleic acid (e.g., an antisense oligonucleotide) that is encapsulated in a liposome, a polymeric nanocarrier, or a lipid nanoparticle.

[0051] In another aspect, the disclosure provides a composition comprising: (1) a chimeric protein comprising (i) any one of the antibody variable domains described herein and (ii) an antibody light chain, wherein the C-terminus of the antibody variable domain is fused directly, or via an intervening amino acid sequence, to the N-terminus of the antibody light chain; and (2) an antibody heavy chain that pairs with the antibody light chain.

[0052] In another aspect, the disclosure features a composition including: (1) a chimeric protein comprising (i) any one of the antibody variable domains described herein and (ii) an antibody heavy chain, where the C-terminus of the antibody variable domain is fused directly, or via an intervening amino acid sequence, to the N-terminus of the antibody heavy chain; and (2) an antibody light chain that pairs with the antibody heavy chain.

[0053] In another aspect, the present disclosure provides a composition comprising: (1) a first chimeric protein comprising (i) a first antibody variable domain of any one of the antibody variable domains described herein and (ii) an antibody light chain, where the C-terminus of the first antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody light chain; and (2) a second chimeric protein comprising (i) a second antibody variable domain of any one of the antibody variable domains described herein and (ii) an antibody heavy chain, where the C-terminus of the second antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody heavy chain.

[0054] In some instances, the intervening amino acid sequence is 3x(G4S) (SEQ ID NO:60), G4S (SEQ ID NO:5), GG, or G.

[0055] In certain instances, the antibody binds to human LINGO-1, human LINGO-2, human LINGO-3, human LINGO-4, human TREM2, human C9orf72 DPR, human TWEAK, human TWEAK receptor, human beta amyloid, human tau, human alpha-synuclein, or human TDP-43. In some embodiments, the antibody comprises six CDRs of an antibody selected from the group consisting of Li81, Li113, huP2D10, 12F6A, 12F4, 21D11, 6C5, 40E8, BMS-986168, 15O7, 5J10, 41D1, 14W3, and 51C1. In other embodiments, the antibody comprises the heavy chain variable region (VH) and light chain variable region (VL) of an antibody selected from the group consisting of Li81, Li113, huP2D10, 12F6A, 12F4, 21D11, 6C5, 40E8, BMS-986168, 15O7, 5J10, 41D1, 14W3, and 51C1. In yet other embodiments, the antibody comprises the heavy chain and light chain of an antibody selected from the group consisting of Li81, Li113, huP2D10, 12F6A, 12F4, 21D11, 6C5, 40E8, BMS-986168, 15O7, 5J10, 41D1, 14W3, and 51C1.

[0056] In another aspect, the disclosure features a composition comprising a chimeric protein comprising: (i) an antibody variable domain of any one of the antibody variable domains described herein; (ii) a protein comprising an antibody hinge region and an Fc portion, wherein the C-terminus of the antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the hinge region of the protein; and (iii) a therapeutic agent. In some cases, the composition comprises a dimer of the chimeric protein. In some cases, the hinge region comprises the sequence AEPKSCD (SEQ ID NO:56) or AEPKSSD (SEQ ID NO:59). In other cases, the hinge region comprises the sequence KTHTCPPCP (SEQ ID NO:19). In certain cases, the Fc portion is not glycosylated. In some cases, the Fc portion is a hIgG1 The antibody variable domain is selected from the group consisting of: hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P) / G1 agly Fc, hIgG4(S228P)Fc, and hIgG4(S228P)agly Fc. In some cases, the C-terminus of the antibody variable domain is directly fused to the N-terminus of the hinge region of the protein. In certain cases, the therapeutic agent is a binding molecule comprising a second antibody variable domain. In some embodiments, the binding molecule binds to human LINGO-1, human LINGO-2, human LINGO-3, human LINGO-4, human TREM2, human C9orf72 DPR, human TWEAK, human TWEAK receptor, human beta amyloid, human tau, human alpha-synuclein, or human TDP-43. In some instances, the binding molecule is a Fab, and the N-terminus of the VH or VL domain of the Fab is linked to the C-terminus of the Fc portion. In certain instances, the therapeutic agent is an antisense oligonucleotide. In one embodiment, the antisense oligonucleotide is a splice-switching oligonucleotide. In another embodiment, the antisense oligonucleotide is a gapmer. In certain instances, the antisense oligonucleotide is linked to a hinge region. In some instances, the therapeutic agent is a small molecule, peptide, enzyme, single chain antibody, nanobody, or nucleic acid (e.g., ASO) encapsulated in a liposome, polymeric nanocarrier, or lipid nanoparticle.

[0057] In another aspect, the disclosure provides a pharmaceutical composition comprising any of the antibody variable domains described herein, the chimeric molecules described herein, or the compositions described herein, and a pharma- ceutically acceptable carrier.

[0058] In another aspect, the disclosure provides a nucleic acid(s) encoding an antibody variable domain, a chimeric molecule, or a composition described herein.

[0059] In another aspect, the disclosure provides a vector(s) comprising such a nucleic acid.

[0060] In yet another aspect, the disclosure features a host cell containing the above-described vector(s).

[0061] In another aspect, the present disclosure provides a method for producing an antibody variable domain, a chimeric molecule, or a composition. The method includes culturing a host cell as described above in a cell culture medium under conditions that result in expression of the antibody variable domain, the chimeric molecule, or the composition, and isolating the antibody variable domain, the chimeric molecule, or the composition from the cell culture medium. In some instances, the host cell is a CHO cell, a COS cell, or a HEK293 cell. In other instances, the host cell is a bacterial cell. In still other instances, the host cell is a yeast cell (e.g., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe). In certain instances, the host cell is a fungal cell (e.g., Aspergillus).

[0062] In another aspect, the disclosure provides a method of producing a fusion polypeptide comprising an antibody variable domain described herein, the method comprising culturing a host cell containing a vector(s) encoding the fusion polypeptide in a cell culture under conditions resulting in expression of the fusion polypeptide, and isolating the fusion polypeptide from the cell culture.

[0063] In another aspect, the disclosure provides a method of treating Alzheimer's disease in a human subject in need thereof. The method comprises administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human β-amyloid. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 12F6A (see U.S. Patent No. 8,906,367). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of 12F6A.

[0064] In another aspect, the disclosure provides a method of treating progressive supranuclear palsy (PSP) disease in a human subject in need thereof. The method comprises administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human β-amyloid. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 12F6A (see U.S. Patent No. 8,906,367). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of 12F6A.

[0065] In another aspect, the disclosure features a method of treating a synucleinopathy (e.g., Parkinson's disease) in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human alpha-synuclein. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 12F4 (see U.S. Pat. No. 8,940,276) or 21D11 (see U.S. Pat. No. 9,580,493). In some instances, the antibody or antigen-binding fragment comprises the VH and VL of 12F4 or 21D11.

[0066] In another aspect, the disclosure features a method of treating a tauopathy (e.g., Alzheimer's disease) in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human tau. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 6C5, 40E8 (see U.S. Patent No. 9,598,484), 4E4 (see U.S. Patent No. 9,605,059), or BMS-986168 / hIPN002 (see U.S. Patent No. 9,447,180 and U.S. Application Publication No. US2017 / 0174755A1). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of 6C5, 40E8, 4E4, or BMS-986168 / hIPN002.

[0067] In another aspect, the disclosure features a method of treating progressive supranuclear palsy in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human tau. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 6C5, 40E8 (see U.S. Patent No. 9,598,484), 4E4 (see U.S. Patent No. 9,605,059), or BMS-986168 / hIPN002 (see U.S. Patent No. 9,447,180 and U.S. Application Publication No. US2017 / 0174755A1). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of 6C5, 40E8, 4E4, or BMS-986168 / hIPN002.

[0068] In another aspect, the disclosure features a method of treating frontotemporal dementia in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked to an antibody or antigen-binding fragment thereof that specifically binds human TDP-43, either directly or via an intervening amino acid sequence. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of 41D1, 14W3, 51C1 (see U.S. Pat. No. 9,587,014), or C9orf72 DPR. In some cases, the antibody or antigen-binding fragment comprises the VH and VL of 41D1, 14W3, 51C1, or an anti-C9orf72 DPR antibody (such as those disclosed herein).

[0069] In another aspect, the disclosure features a method of treating amyotrophic lateral sclerosis (ALS) in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human SOD1, DR6, or C9orf72 DPR. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises the hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises six CDRs of an antibody described in any one of U.S. Patent Nos. 9,283,271; 8,759,029; or Broering et al., doi.org / 10.1371 / journal.pone.0061210.

[0070] In another aspect, the disclosure provides a method of treating multiple sclerosis in a human subject in need thereof. The method comprises administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human LINGO-1. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of Li81 (see U.S. Pat. No. 8,128,926) or Li113 (see U.S. Pat. No. 8,058,406). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of Li81 or Li113.

[0071] In another aspect, the disclosure provides a method of treating optic neuritis (e.g., AON) in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human LINGO-1. In certain instances, the intervening amino acid sequence is a 3×(G4S) linker (SEQ ID NO:60), a G4S linker (SEQ ID NO:5), a GG linker, or a G linker. In other instances, the intervening amino acid sequence comprises a hinge region of an antibody fused to an Fc portion. In some instances, the antigen-binding fragment is a Fab. In some cases, the antibody or antigen-binding fragment comprises the six CDRs of Li81 (see U.S. Pat. No. 8,128,926) or Li113 (see U.S. Pat. No. 8,058,406). In some cases, the antibody or antigen-binding fragment comprises the VH and VL of Li81 or Li113.

[0072] In yet another aspect, the disclosure features a method of treating spinal muscular atrophy in a human subject in need thereof. The method includes administering to the subject a chimeric molecule comprising an antibody variable domain described herein linked to nusinersen (CAS Registry Number 1258984-36-9; UNII:5Z9SP3X666) directly or via an intervening amino acid sequence. In some cases, nusinersen is encapsulated in a liposome, a polymeric nanocarrier, or a lipid nanoparticle. In other cases, the intervening amino acid sequence includes a hinge region fused to an Fc portion, and nusinersen is linked to the hinge region, an engineered cysteine ​​(e.g., S442C) in the chimeric molecule (e.g., hinge region), or a non-targeting site (e.g., using amine chemistry).

[0073] In another aspect, the disclosure features a method of assessing instability of an antibody variable domain. The method includes providing an antibody variable domain. The antibody variable domain is added to a serum sample to generate a mixture. The mixture is incubated. The antibody variable domain is purified and peptide mapping is performed.

[0074] In another aspect, the disclosure features a method of screening for stabilized forms of FC5. The method includes screening for stabilized forms of FC5 using one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65) amino acid sequence that differs from SEQ ID NO:1 or is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:1. The antibody variable domain is added to a serum sample to produce a mixture. The mixture is incubated. The antibody variable domain is purified and peptide mapping is performed. The antibody variable domain that shows increased peptide recovery is selected as a stabilized form of FC5.

[0075] In some embodiments of the above two aspects, the serum sample is rat serum. In other embodiments, the serum sample is human serum. In certain embodiments, the antibody variable domain is in serum at a concentration of about 0.01-1 mg / mL. In certain embodiments, the antibody variable domain is in serum at a concentration of about 0.05-0.5 mg / mL. In certain embodiments, the antibody variable domain is in serum at a concentration of about 0.05 mg / mL. In certain embodiments, the antibody variable domain is in serum at a concentration of about 0.1 mg / mL. In certain embodiments, the antibody variable domain is in serum at a concentration of about 0.5 mg / mL. In some embodiments, the mixture is incubated at about 25-37° C. for about 10-100 hours. In one embodiment, the mixture is incubated at about 37° C. for about 70 hours. In one embodiment, the antibody variable domain comprises an FC5 variant. In some embodiments, the FC5 variant is humanized FC5. In another example, the FC5 variant comprises a disulfide stabilized FC5. In yet another example, the FC5 variant comprises a humanized disulfide stabilized FC5.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In the practice or testing of the present invention, methods and materials similar or equivalent to those described herein can be used, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present application, including definitions, will control. The materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0077] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0078] [Figure 1] The amino acid sequence of the wild type FC5 single domain antibody (SEQ ID NO:1) is provided. The position of each amino acid in the FC5 amino acid sequence is also provided (numbering according to the primary sequence). The CDRs are at positions 23-35 (CDR1), 50-66 (CDR2) and 97-111 (CDR3). CDRs 1 and 3 are defined according to North et al. (North, B., Lehmann, A. & Dunbrack, RL, Jr, "A New Clustering of Antibody CDR Loop Conformations", J. Mol. Biol., 406:228-256 (2011)) to account for structural variability, and CDR2 is defined by integrating the definitions of Kabat et al. (Kabat, EA, Wu, TT, Perry, HM, Gottesman, KS & Foeller, C. (1991). Sequences of Proteins of Immunological Interest, 5th edit. National Institutes of Health, Bethesda, MD) and North et al. to account for both structural and sequence variability. [Diagram 2] Provided are the amino acid sequences of the Li81 light chain (SEQ ID NO:2), the FC5-3×(G4S)-HC Li81 heavy chain (SEQ ID NO:3), and the FC5-3X(G4S)-LC Li81 light chain (SEQ ID NO:4). These are the sequences of the constructs used to generate constructs 3014, 3015, and 3016. The signal peptide sequence is shown in light font. [Diagram 3]SDS-PAGE characterization of FC5-(G4S)3-Li81 samples. Samples were subjected to SDS-PAGE on 4-20% Tris-glycine gradient gels from Invitrogen. Non-reduced samples were heated at 95°C for 2 min before analysis (left panel). Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated (right panel). Lane 1, FC5 Li81 HC, wt LC (4D#3014); lane 2, Li81 wt HC, FC5 Li81 LC (4D#3015); lane 3, FC5 Li81 HC, FC5 Li81 LC (4D#3016); lane S, high molecular weight marker. [Figure 4] Deconvoluted mass spectra of the light chain (top) and heavy chain (bottom) for a reduced sample of construct 3014 are shown. [Diagram 5] 1 shows the deconvoluted mass spectra of the heavy and light chains for a reduced sample of construct 3015. [Figure 6] 1 shows the deconvoluted mass spectra of the heavy and light chains for a reduced sample of construct 3016. [Figure 7] Illustrated is the SDS-PAGE characterization of FC5-(G4S)1-Li81 samples. Samples were subjected to SDS-PAGE on 4-20% Tris-Glycine gradient gels from Invitrogen. Non-reduced samples were heated at 95°C for 2 min before analysis. Left panel: lane 1, FC5(G4S)1-Li81 (ID#3438); lane 2, FC5-Fc(G4S)1-Li81 long hinge (ID#3440); lane 3, FC5-Fc(G4S)1-Li81 short hinge (ID#3441); lane 4, heterodimer ID#3442; lane 5, heterodimer ID#3443; lane 6, heterodimer ID#3444; lane 7, heterodimer ID#3443 (batch 2); lane S, high molecular weight marker. Right panel, purification of heterodimer ID#3443 by SEC: lane 1, column load; lanes 2-11, successive elution fractions; lane S, high molecular weight marker. [Figure 8] 1 provides the amino acid sequences of the constructs used to generate the FC5-(G4S)1-Li81 construct: Li81 light chain (SEQ ID NO:2), FC5-(G4S)1-Li81 huIgG1 agly heavy chain (SEQ ID NO:7), FC5-Fc-(G4S)1-Li81 huIgG1 agly long hinge light chain (SEQ ID NO:8), and FC5-Fc-(G4S)1-Li81 huIgG1 agly short hinge (SEQ ID NO:9). The signal peptide sequence is shown in light font. [Figure 9] Illustrated are deconvoluted mass spectra of the heavy chain for the FC5-(G4S)1-Li81 constructs. Li81 / FC5 variant 1, FC5-(G4S)1-Li81; Li81 / FC5 variant 3, FC5-Fc-(G4S)1-Li81 long hinge; Li81 / FC5 variant 4, FC5-Fc-(G4S)1-Li81 short hinge; Li81 / FC5 variant 5, dual monovalent FC5-Fc-(G4S)1-Li81 long hinge; Li81 / FC5 variant 6, dual monovalent FC5-Fc-(G4S)1-Li81 short hinge; and Li81 / FC5 variant 7, mono Fab FC5-Fc-(G4S)1-Li81 short hinge sample. [Figure 10] Figure 1 shows the potency assessment of the FC5-(G4S)1-Li81 construct. The apparent affinity of the FC5-(G4S)1-Li81 samples for LINGO-1 was measured by direct binding ELISA. Data is plotted as absorbance at 405 nm vs. concentration. [Figure 11] Figure 2 illustrates the characterization of FC5-Li81 samples in an in vitro BBB cell culture test. The levels of antibodies in the bottom chamber (15, 30, 60 and 90 min) were determined by MRM-ILIS. Values ​​are the mean Papp ± standard deviation. [Figure 12]Illustrates the effect of dalargin (Dal) cross-linked FC5-Li81 fusion on thermal hyperalgesia in the Hargreaves model of inflammatory pain. Top panel: Withdrawal latencies of control intact or inflamed paws to thermal stimulation were measured following a single intravenous (iv) dose of 13 mg / kg test molecule or PBS at 15 min intervals over 4 h. Data are shown as mean values ​​(2 animals / group; error bars represent individual measurements). Bottom panel: Integrated area under the curve (AUC) data from the top panel were expressed as a percentage of maximum possible effect (MPE) of the control paw. [Figure 13A] Figure 1 shows the effect of FC5-Li81 fusion cross-linked with Dal on thermal hyperalgesia in the Hargreaves model of inflammatory pain. Analgesia achieved by single iv injections of increasing doses of Li81 and FC5-Li81. Data are AUC integrated over the duration of the response (4 hours) expressed as percentage MPE of the control paw (shown as mean ± standard deviation of 2 or 5 animals / group). [Figure 13B] Data from FIG. 13A is shown plotted as the analgesic effect of FC5-Li81(3438) and Li81 cross-linked with dalargin for iv doses. [Figure 14-1] Serum and CSF PK assessment of FC5-Li81 is provided. Samples were administered IV via the tail vein at 20, 65, and 200 nmol / kg, respectively, and antibody levels at the indicated time points after injection were determined in serum (top panel) and CSF (middle panel) samples from the cisterna magna. Paired CSF / serum ratios (%) at each time point are plotted in the bottom panel. Results are the average of two separate animals. [Figure 14-2] Same as above. [Figure 15] FIG. 1 illustrates the analysis of PK of FC5-(G4S)1-Li81 in rats. Serum and CSF levels were quantified by mass spectrometry. Data are presented as ratios of CSF and serum levels. [Figure 16]Illustrated is the SDS-PAGE characterization of FC5-(G4S)1-Li81 samples. Samples were subjected to SDS-PAGE on 4-20% Tris-glycine gradient gels from Invitrogen. Non-reduced samples were treated with 5 mM N-ethylmaleimide for 5 min at room temperature, diluted with Laemmli non-reducing sample buffer, and heated at 95°C for 2 min before analysis. Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated. Lane 1. High molecular weight marker, lane 2. FC5-(G4S)1-Li81 (dosing preparation), lane 3. FC5-(G4S)1-Li81 purified from 72-h rat serum, lane 4. FC5-(G4S)1-Li81-HC purified from rat serum spiked samples at 100 μg / mL. [Figure 17] Characterization of FC5-(G4S)1-Li81 sample by size exclusion chromatography. Sample (25 μg in 300 μL column buffer) was subjected to SEC at room temperature on a Superdex 200 HR10 / 30 FPLC column (GE Healthcare) using 20 mM sodium phosphate, 150 mM NaCl, pH 7.2 as mobile phase. The column was run at 0.3 mL / min. The column effluent was monitored by UV detection at 280 nm. The arrow indicates the time of injection. SEC / light scattering was performed on a BioSep-SEC-S3000 column, 300×7.8 mm (Phenomenex) on a Waters Alliance instrument (Waters 2790, MA) in 20 mM sodium phosphate, 150 mM NaCl, pH 7.2 at a flow rate of 0.6 mL / min. Static light scattering was synchronized with SEC and measured online using a Precision PD2100 Detector (Precision Detectors, MA). Molecular weights were calculated using Discovery 32 Light Scattering Analysis Software. [Figure 18]We present the characterization of FC5-(G4S)1-Li81 samples in an in vitro BBB cell culture test. The levels of antibodies in the bottom chamber (15, 30, 60 and 90 min) were determined by MRM-ILIS. Values ​​are the mean values ​​of Papp ± standard deviation. [Figure 19] 1 shows the mass spectrum of the native FC5-(G4S)1-Li81 sample, the deconvoluted mass spectra of the native FC5(G4S)1Li81 dosing solution, the FC5-(G4S)1-Li81 spike, and the FC5-(G4S)1-Li81 72 hour sample. [Figure 20] Mass spectrum of FC5-Li81 HC sample. Deconvoluted mass spectra of native FC5(G4S)1Li81 dosing solution, FC5-(G4S)1-Li81 spike, and FC5-(G4S)1-Li81 72 hour sample. [Figure 21] Mass spectra of FC5-Li81 LC samples. Deconvoluted mass spectra of native FC5(G4S)1Li81 dosing solution, FC5-(G4S)1-Li81 spike, and FC5-(G4S)1-Li81 72 hour sample. [Figure 22-1] Figure 1 shows EndoLysC / tryptic peptide maps of FC5-Li81 samples. UV traces of Lys-C / tryptic disulfide maps (monitored at 214 nm) of FC5(G4S)1Li81 (control), FC5-(G4S)1-Li81-HC spike, and FC5-(G4S)1-Li81-HC 72 h non-reduced and reduced. [Figure 22-2] Same as above. [Diagram 23] Figure 1 shows the recovery of heavy chain peptides in non-reduced and reduced peptide maps. The % recovery (Y-axis) was normalized to the corresponding peptide in the control sample. The characterized HC peptides are shown on the X-axis. Low recovery peptide, HC 215-249; hydrophobic peptides, HC 171-192 and HC 276-338. [Figure 24]Figure 1 illustrates the recovery of light chain peptides in non-reduced and reduced peptide maps. The % recovery (Y-axis) was normalized to the corresponding peptide in the control sample. The characterized LC peptides are shown on the X-axis. [Diagram 25] Illustrating the design of humanized FC5. Sequence alignment and computational structural model comparing FC5 (SEQ ID NO:1) and humanized variants H12 (SEQ ID NO:11) and H62 (SEQ ID NO:15). Light shading of side chains in the amino acid single letter code of the model and alignment indicates camelid origin, dark shading indicates human origin. H12 has a fully humanized patch (dashed outline) that corresponds to the VH / VL boundary in conventional antibodies. This boundary is covered by the light chain in conventional antibodies but exposed in single domain antibodies. The M34T mutation is introduced to avoid methionine oxidation. [Figure 26] Illustrated characterization of FC5-hFc humanized variants by denaturing microfluidic capillary electrophoresis. 1) H11. 2) H12. 3) H31. 4) H32. 5) H61. 6) H62. 7) H71. 8) H72. Left: non-reduced samples. Right: reduced samples. Molecular weight markers are shown in kDa on either side. Using the instrument software, the electropherogram is presented in 2D as a virtual gel using small molecular markers (LM) for alignment. SP = system peak. [Figure 27-1] 1 shows the deconvoluted intact mass spectrum of reduced FC5-hFc humanized variant sample. H11: predicted mass 38553.7 Da, observed mass 38551. H12: predicted mass 38523.6 Da, observed mass 38521 Da. H31: predicted mass 38548.6 Da, observed mass 38544 Da. H32: predicted mass 38518.6 Da, observed mass 38516 Da. H61: predicted mass 38649.7 Da, observed mass 38645 Da. H62: predicted mass 38619.6 Da, observed mass 38617 Da. H71: predicted mass 38677.8 Da, observed mass 38674 Da. H72: predicted mass 38647.7 Da, observed mass 38645 Da. [Figure 27-2] Same as above. [Figure 28] 1 shows the results of analytical size exclusion chromatography of humanized variants of FC5-hFc. Variants H61, H62, and H71 eluted as a uniform peak at approximately 17.3 minutes, while the other variants show delayed elution times in the order listed. Data are normalized to a constant peak height. [Figure 29] Characterization of humanized variants of FC5-hFc in an in vitro rat BBB cell culture transwell assay. The levels of antibody in the bottom chamber (15, 30 and 60 min) were determined by MRM and used to calculate permeability values. Values ​​are the mean ± standard deviation of PAPP. [Diagram 30] Figure 1 shows the effect of dalargin cross-linked FC5-hFc humanized variants on thermal hyperalgesia in the Hargreaves model of inflammatory pain. Left panel: Withdrawal latencies of control intact or inflamed paws to thermal stimulation were measured following a single iv dose of test molecule at approximately 5mg / kg or PBS at 15 min intervals over 4 hours. Data are shown as mean values ​​(sd for 2 animals / group). Right panel: Integrated response (AUC) data from the left panel expressed as percentage MPE of the control paw. [Diagram 31] The engineering of disulfide bonds within FC5 to stabilize the structure is shown. Confirmation of appropriate distances between positions for cysteine ​​mutations and minimal disruption within the protein was achieved through computational modeling. The top panel shows the positions of the indicated cysteines and the bottom panel shows the model with the disulfides formed. [Diagram 32]Illustrates SDS-PAGE characterization of FC5-(G4S)1-Li81 samples. Samples were subjected to SDS-PAGE on 4-20% Tris-Glycine gradient gels from Invitrogen. Non-reduced samples were heated at 95°C for 2 min before analysis. Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated. Lane 1, 4D 4662; lane 2, 4D 4663; lane 3, 4D 4664; lane 4, 4D 4665; lane 5, 4D 4666; lane 6, 4D 4667; lane 7, 4D 4668; lane 8, 4D 4669; lane 9, 4D 4670; lane 10, high molecular weight marker. [Diagram 33] Characterization of disulfide-stabilized FC5-(G4S)1-Li81 samples in an in vitro BBB cell culture transwell assay is presented. The levels of antibody in the bottom chamber (15, 30, 60 and 90 min) were determined by MRM-ILIS. Values ​​are the mean ± standard deviation of PAPP. [Diagram 34] Assessment of susceptibility of FC5-(G4S)1-Li81 samples to proteolysis by SDS-PAGE. Samples were subjected to SDS-PAGE on 4-20% Tris-glycine gradient gels (Invitrogen). Non-reduced samples were heated at 95°C for 2 min before analysis. Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated. Lane 1, H32(T34C / V79C)-G4S-Li81; lane 2, H32(T34C / V79C)-G4S-Li81+pepsin; lane 3, H32(T33C / T103C)-G4S-Li81; lane 4, H32(T33C / T103C)-G4S-Li81+pepsin; lane 5, Li81; lane 6, Li81+pepsin; lane 7, FC5-H32-G4S-Li81; lane 8, FC5-H32-G4S-Li81+pepsin; lane 9, high molecular weight marker. [Diagram 35]Assessment of susceptibility of FC5-(G4S)1-Li81 samples to proteolysis by SDS-PAGE. Samples were subjected to SDS-PAGE on 4-20% Tris-glycine gradient gels (Invitrogen). Non-reduced samples were heated at 95°C for 2 min before analysis. Reduced samples were treated with sample buffer containing 2% 2-mercaptoethanol and heated. Lane 1, FC5-H62(S49C / I70C)-G4S-Li81; lane 2, FC5-H62(S49C / I70C)-G4S-Li81+pepsin; lane 3, FC5-H62(T34C / V79C)-G4S-Li81+pepsin; lane 4, FC5-H62(T33C / S102C)-G4S-Li81+pepsin; lane 5, FC5-H62(T33C / T103C)-G4S-Li81+pepsin; lane 6, FC5-H62(T3 lane 5, FC5-H62(T33C / A104C)-G4S-Li81+pepsin; lane 6, FC5-H62(T33C / T105C)-G4S-Li81+pepsin; lane 8, FC5-H32-Li81-G4S-Li81+pepsin; lane 9, Li81+pepsin; lane 10, H62-FC5-Fc; lane 11, H62-FC5-Fc+pepsin; lane 12, H32-FC5-Fc; lane 13, H32-FC5-Fc+pepsin; lane 14, high molecular weight marker. To facilitate analysis of the samples, the bottom panel shows a magnified view of lanes 1-9 from the 100-150 kDa region of non-reducing SDS-PAGE. [Diagram 36] Deconvoluted mass spectra of the heavy chain for reduced samples 4662, 4665, 4667, and 4668 are presented. [Figure 37] Deconvoluted mass spectra of the light chain for reduced samples 4662, 4665, 4667, and 4668 are presented. [Figure 38] Graphical representation of heavy chain peptide recovery in non-reduced and reduced peptide maps. % recovery (Y-axis) was normalized to the corresponding peptide in the control sample. Characterized HC peptides are shown on the X-axis. Low recovery peptide, HC 215-249; hydrophobic peptides, HC 171-192 and HC 276-338. [Figure 39] FIG. 1 is a graphical representation of the recovery of light chain peptides in non-reduced and reduced peptide maps. The % recovery (Y-axis) was normalized to the corresponding peptide in the control sample. The characterized LC peptides are shown on the X-axis. [Diagram 40] Illustrates serum and CSF PK assessment of FC5-Li81. Samples were administered IV via the tail vein at 65 nmol / kg each, and antibody levels at the indicated time points after injection were determined in serum (top panel) and CSF (middle panel) samples from the cisterna magna. Paired CSF / serum ratios (%) at each time point are plotted in the bottom panel. Results are the average of two separate animals. [Diagram 41] Serum PK assessment of three disulfide-stabilized FC5-Li81 variants showing the greatest improvement in in vitro stability. Samples were administered iv via tail vein at 65 nmol / kg each and antibody levels were determined in serum at the indicated time points after injection. Results are the average of two separate animals. [Figure 42A] A illustrates the assessment of brain levels of the three disulfide-stabilized FC5-Li81 variants that show the greatest improvement in in vitro stability. Samples were administered iv via the tail vein at 65 nmol / kg each, and antibody levels were determined in serum samples at the indicated time points after injection. B illustrates the assessment of CSF levels of the three disulfide-stabilized FC5-Li81 variants that show the greatest improvement in in vitro stability. Samples were administered iv via the tail vein at 65 nmol / kg each, and antibody levels were determined in CSF samples from the cisterna magna at the indicated time points after injection. C illustrates a plot of the paired CSF / serum ratio (%) of the three disulfide-stabilized FC5-Li81 variants at each time point. Results are the average of two separate animals. D illustrates the brain levels of the three disulfide-stabilized FC5-Li81 variants measured at the 24-hour time point. [Figure 42B] Same as above. [Figure 42C] Same as above. [Fig.42D] Same as above. [Diagram 43] A shows serum and CSF PK assessment of FC5-Li81 in rats. Samples were administered iv via the tail vein at 65 nmol / kg each and antibody levels at the indicated time points after injection were determined in serum samples by MRM. Results are the average of 2-3 separate animals. A schematic of the molecular design is shown on the left. B shows serum and CSF PK assessment of FC5-Li81 in rats. Samples were administered iv via the tail vein at 65 nmol / kg each and antibody levels at the indicated time points after injection were determined in CSF samples from the cisterna magna. Results are the average of 2-3 separate animals. A schematic of the molecular design is shown on the left. C shows paired CSF / serum ratios (%) from the plotted data in A and B. Results are the average of 2-3 separate animals. A schematic of the molecular design is shown on the left. [Diagram 44] Serum and CSF PK assessment of FC5-H62(T33C / A104C)-Li81 (solid line) vs. Li81 (dashed line) in cynomolgus monkeys (n=12). Test articles were administered via a single IV bolus injection at 65 nmol / kg, and antibody levels at the indicated post-injection time points were determined by nanoLC-MRM in serum (top panel) and CSF (middle panel) samples. Paired CSF / serum ratios are also plotted (bottom panel). [Figure 45A]A shows a graph of a direct binding ELISA of FC5-H62(T33C / A104C)-12F6A with amyloid-β. Synthetic amyloid-β (amino acids 1-40) peptide was coated onto an ELISA plate and exposed to multiple dilutions of reagent, and antibody binding at the indicated concentrations was detected with an HRP-conjugated secondary anti-hIgG and measured by absorbance at 450 nm. EC50 values ​​calculated from fitting the data to a sigmoidal curve are shown. B shows the results of an SV40-immortalized brain endothelial cell (svARBEC) transwell assay recorded by MRM measurement of bottom well antibody concentration (mean apparent permeability (PAPP) ± standard deviation for n=3 replicates). The control antibody in each well was anti-HEL mouse IgG1. hFC5.2 is FC5-H62(T33C / A104C). C shows human iPSC-derived brain endothelial cell transwell assay results recorded by MRM measurement of bottom well antibody concentration (mean apparent permeability (PAPP) ± standard deviation for n=3 replicates). The control antibody in each well was anti-HEL mouse IgG1. hFC5.2 is FC5-H62(T33C / A104C). [Figure 45B] Same as above. [Figure 45C] Same as above. [Figure 46A]A is a graph showing a direct binding ELISA for α-synuclein. Human α-synuclein was coated onto an ELISA plate and treated with multiple dilutions of reagent, and antibody binding at the indicated concentrations was detected with an HRP-conjugated secondary anti-hIgG and measured by absorbance at 450 nm. EC50 values ​​calculated from fitting the data to a sigmoidal curve are shown. hFC5.2 is FC5-H62(T33C / A104C). B is a bar graph of SVARBEC transwell assay results recorded by measuring bottom well antibody concentration by MRM. Data are plotted as the ratio of PAPP of test antibody to anti-HEL mIgG1 control in the well (mean ± standard deviation for n=3 replicates). PAPP for anti-HEL was <1.5×10-5 cm / min in all wells. hFC5.2 is FC5-H62(T33C / A104C). [Figure 46B] Same as above. [Figure 47A] A shows the results of a direct binding ELISA for tau. Human tau was coated onto an ELISA plate, treated with multiple dilutions of reagent, and bound antibody was detected with an HRP-conjugated secondary anti-hIgG and measured by absorbance at 450 nm. EC50 values ​​calculated from fitting the data to a sigmoidal curve are shown. hFC5.2 is FC5-H62(T33C / A104C). B is a bar graph of SVARBEC transwell assay results recorded by measuring bottom well antibody concentration by MRM. Data are plotted as the ratio of PAPP of test antibody to anti-HEL mIgG1 control in the well (mean ± standard deviation for n=3 replicates). PAPP for anti-HEL was <1.5×10-5 cm / min in all wells. hFC5.2 is FC5-H62(T33C / A104C). [Figure 47B] Same as above. [Figure 48]Validation of reduced susceptibility of disulfide engineered FC5 antibody fusions to pepsin digestion. Left: SDS-PAGE analysis of FC5-H62(T33C / A104C) fused to Li81 hIgG1 agly, 12F4 hIgG1 and 12F4 hIgG1 agly with and without pepsin digestion for 1 h at 37° C. FC5-H32-Li81 hIgG1 agly shows increased digestion as indicated by lower molecular weight fragments (arrows). Right: SDS-PAGE analysis of pepsin digestion (2.5 h at 37° C.) of FC5-H62(T33C / A104C) or FC5-H32 fused to Li81 hIgG1 agly and 12F6A hIgG1. [Figure 49] 1 is a schematic representation of non-limiting exemplary bivalent FC5 fusion formats. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Delivery of drugs to the central nervous system (CNS) has been a central issue in the treatment of neurological diseases, since the blood-brain barrier (BBB) ​​is the barricade that prevents most therapeutic molecules from entering the brain. Without access to the brain, the therapeutic potential of these molecules may be compromised or lost.

[0080] The present disclosure is based in part on molecules including humanized FC5 and disulfide engineered FC5. These molecules are useful for transporting "cargo" of interest, such as antibodies, antigen-binding fragments, peptides, enzymes, and nucleic acids (e.g., antisense oligonucleotides) to the CNS. In some embodiments, these molecules are transcytosed across the BBB. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express (or have been modified not to express) TMEM30A. In some embodiments, these molecules have reduced instability in human serum compared to wild-type FC5 (SEQ ID NO:1). Each of the FC5 therapeutic (e.g., antibody) formats, including the monovalent FC5 format depicted generally in FIG. 48, is encompassed by the present disclosure.

[0081] Humanized FC5 FC5 is a camelid single domain antibody that can transcytose across the BBB and deliver bioactive molecules to the CNS (see, e.g., Muruganandam et al., FASEB J., 16:240-242 (2002); Abulrob et al., J Neurochem., 95:1201-1214 (2005); Haqqani et al., Mol. Pharmaceutics, 10:1542-1556 (2013); Farrington et al., FASEB J., 28:4764-4778 (2014); U.S. Patent No. 9,676,849). The amino acid sequence of wild-type FC5 is provided in SEQ ID NO:1.

[0082] FC5 is species cross-reactive and can bind to rat, mouse and human brain endothelial cells. The present disclosure features, inter alia, humanized FC5 antibody variable domains. These humanized FC5 antibody variable domains reduce the potential risk of human immunogenicity of FC5 while maintaining solubility and stability. The FC5 sequence antibody variable domains have varying degrees of homology to the human VH3 consensus framework.

[0083] Camelid VHH domain frameworks are highly homologous to the heavy chain variable domain frameworks of the human VH3 family. To construct the human VH3 consensus framework, a multiple sequence alignment (MSA) was obtained by Clustal Omega (www.ebi.ac.uk / Tools / msa / clustalo / ), which summarized all human functional ORF VH3 genes deposited in IMGT as of August 25, 2014. The consensus framework was defined as the sequence of the most frequent amino acids at each position of the MSA, combined with the first allele of the human JH4 gene (IGHJ4*01, IMGT nomenclature) to obtain the framework of the complete VH domain. In framework regions 1, 2 and 3, the consensus framework is identical to that of the fourth allele of the human VH3 gene IGHV3-23*04. FC5 has 15 framework residues that differ from the human VH3 consensus framework. Camelid residues 37, 44, 45 and 47 are on the surface that corresponds to the VH / VL interface in human IgG. These residues can strongly influence the solubility, expression levels or binding affinity of single domain antibodies, although these effects also depend on the CDRs of the particular single domain antibody.

[0084] The complementarity determining regions (CDRs) of wild-type FC5 are located at positions 23-35 (CDR1), 50-66 (CDR2), and 97-111 (CDR3) of SEQ ID NO:1, and are provided below. Wild type FC5 CDR1: AASGFKITHYTMG (SEQ ID NO: 47) Wild type FC5 CDR2: RITWGGDNTFYSNSVKG (SEQ ID NO: 50) Wild type FC5 CDR3: AAGSTSTATPLRVDY (SEQ ID NO:51).

[0085] In some embodiments, the FC5 polypeptides of the disclosure have a CDR1 selected from one of the amino acid sequences listed below. [ka]

[0086] In certain embodiments, the FC5 polypeptide of the disclosure has a CDR3 selected from one of the amino acid sequences listed below. [ka] [ka]

[0087] In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:47 with no more than two (e.g., 2, 1, 0) amino acid substitutions, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50 with no more than two amino acid substitutions, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51 with no more than two amino acid substitutions. In some embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:47 with no more than two (e.g., 2, 1, 0) amino acid substitutions, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:51 with no more than two amino acid substitutions. In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47 with one or zero amino acid substitutions, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 50 with one or zero amino acid substitutions, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51 with one or zero amino acid substitutions. In some embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47 with one or zero amino acid substitutions, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 51 with one or zero amino acid substitutions. The antibody variable domain may be humanized. In some instances, in all of the above embodiments, the amino acid substitution occurs at one or more of positions 33, 34, 102, 103, 104, or 105 (e.g., 1, 2, 3, 4, 5, 6) of SEQ ID NO: 1. In some embodiments, these molecules are transcytosed across the BBB.In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells that express TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0088] In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:57, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:57, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:57, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In other embodiments, an antibody variable domain of the present disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:57, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, these molecules are transcytosed across the BBB. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0089] In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In other embodiments, an antibody variable domain of the present disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:48, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, these molecules are transcytosed across the BBB. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0090] In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In other embodiments, an antibody variable domain of the present disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, these molecules are transcytosed across the BBB. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0091] In certain embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:49, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:52. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:49, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:53. In other embodiments, an antibody variable domain of the disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:49, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:54. In other embodiments, an antibody variable domain of the present disclosure comprises a polypeptide comprising a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:49, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:50, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:55. In some embodiments, these molecules are transcytosed across the BBB. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express (or have been modified not to express) TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0092] In some instances, the humanized FC5 polypeptide of the present disclosure comprises an amino acid sequence that is at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%) identical to the sequence set forth in SEQ ID NO:1. In one instance, the humanized FC5 polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:1. These humanized FC5 polypeptides can translocate across the blood-brain barrier and thus transport "cargo" (e.g., biologically active moieties such as antibodies, small molecule drugs, antisense oligonucleotides, enzymes, peptides or antigen-binding fragments thereof) to the CNS. In some embodiments, these molecules can bind directly or indirectly to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells that express TMEM30A than to cells that do not express (or are modified not to express) TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1). In some embodiments, these FC5 polypeptides have amino acid substitutions at 30 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids of SEQ ID NO:1 compared to SEQ ID NO:1. In certain embodiments, these FC5 polypeptides have amino acid substitutions compared to SEQ ID NO:1 at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. The present disclosure encompasses FC5 antibody variable domains with substitutions at all permutations of the above nine positions of SEQ ID NO:1.Non-limiting examples of positions in SEQ ID NO:1 that may be substituted include: 5 and 6; 5, 6, and 14; 5, 6, 14, and 75; 5, 6, 14, 75, and 87; 5, 6, 14, 75, 87, and 88; 5, 6, 14, 75, 87, 88, and 93; 5, 6, 14, 75, 87, 88, 93, and 114; 5, 6, 14, 75, 87, 88, 93, 114, and 117; 5 and 14; 5, 14, and 75; 5, 14, 75, and 87; 5, 14, 75, 87, and 88; 5, 14, 75, 87, 88, and 93; 5, 14, 75, 87, 88, 93, and 114; 5, 14, 75, 87, 88, 93, and 114; 5, 14, 75, 87, 88, 93, 114, and 117. ;5 and 75;5, 75, and 87;5, 75, 87, and 88;5, 75, 87, 88, and 93;5, 75, 87, 88, 93, and 114;5, 75, 87, 88, 93, 114, and 117;5 and 87;5, 87, and 88;5, 87, 88, and 93;5, 87, 88, 93, and 114;5, 87, 88, 93, 114, and 117;5 and 88;5, 88, and 93;5, 88, 93, and 114;5, 88, 93, 114, and 117;5 and 93;5, 93, and 114;5, 93, 114, and 117;5 and 114;5, 114, and 117; and 5 and 117. In some embodiments, the antibody variable domains further comprise a CDR1 comprising the sequence set forth in SEQ ID NO:47, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51. In other embodiments, the antibody variable domains further comprise a CDR1 comprising the sequence set forth in SEQ ID NO:57, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51.In some embodiments, the antibody variable domain comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) of a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; or a leucine at a position corresponding to position 117 of SEQ ID NO:1. The antibody variable domain may comprise additional amino acid substitutions, such as those described below.

[0093] In certain embodiments, the antibody variable domain has amino acid substitutions, relative to SEQ ID NO:1, at one or more (e.g., 1, 2, 3, 4, 5, or 6) of the positions corresponding to positions 1, 37, 44, 45, 47, and 79 of SEQ ID NO:1. The present disclosure covers antibody variable domains with substitutions at all permutations of the above six positions of SEQ ID NO:1.

[0094] In some instances, the antibody variable domain further comprises an amino acid substitution at one or more of positions corresponding to positions 44 and 45 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises a glycine at a position corresponding to position 44 of SEQ ID NO:1 and / or a leucine at a position corresponding to position 45 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0095] In certain instances, the antibody variable domains of the present disclosure further comprise amino acid substitutions at one or more of positions corresponding to positions 1, 44, and 45 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, or 3) of a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; or a leucine at a position corresponding to position 45 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0096] In other instances, the antibody variable domains of the present disclosure further comprise amino acid substitutions at one or more of positions corresponding to positions 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, or 3) of a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; or a tryptophan at a position corresponding to position 47 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0097] In some instances, the antibody variable domain of the present disclosure additionally comprises an amino acid substitution at one or more of positions corresponding to positions 1, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, 3, or 4) of glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; leucine at a position corresponding to position 45 of SEQ ID NO:1; or tryptophan at a position corresponding to position 47 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0098] In certain instances, the antibody variable domains of the present disclosure further comprise amino acid substitutions at one or more of positions corresponding to positions 37, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, 3, or 4) of a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; or a tryptophan at a position corresponding to position 47 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0099] In some instances, the antibody variable domain of the present disclosure additionally comprises an amino acid substitution at one or more of positions corresponding to positions 1, 37, 44, 45, and 47 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, 3, 4, or 5) of glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; leucine at a position corresponding to position 45 of SEQ ID NO:1; or tryptophan at a position corresponding to position 47 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum compared to wild-type FC5 (SEQ ID NO:1).

[0100] In other instances, the antibody variable domain of the present disclosure further comprises an amino acid substitution at one or more of positions corresponding to positions 37, 44, 45, 47, and 79 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, 3, 4, or 5) of a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; a tryptophan at a position corresponding to position 47 of SEQ ID NO:1; or a leucine at a position corresponding to position 79 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum compared to wild-type FC5 (SEQ ID NO:1).

[0101] In some instances, the antibody variable domains described above further comprise an amino acid substitution at one or more of positions corresponding to positions 1, 37, 44, 45, 47, or 79 of SEQ ID NO:1, as compared to SEQ ID NO:1. In some cases, the amino acid sequence comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) of glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; leucine at a position corresponding to position 45 of SEQ ID NO:1; tryptophan at a position corresponding to position 47 of SEQ ID NO:1; or leucine at a position corresponding to position 79 of SEQ ID NO:1. These antibody variable domains can translocate across the blood-brain barrier. In some embodiments, these molecules can directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum compared to wild-type FC5 (SEQ ID NO:1).

[0102] Illustrative, non-limiting examples of humanized FC5 molecules are described in Example 4 of the present disclosure. In certain instances, the humanized FC5 polypeptide comprises an amino acid sequence that is at least 80%, (85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 10-17, and the humanized FC5 molecule is capable of transcytosing across the BBB. In one embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10. In another embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In another embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12. In yet another embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In one embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In a different embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14. In another embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 15. In yet another embodiment, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the humanized FC5 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. The biological activity of the humanized FC5 variants can be assessed using, for example, an in vitro BBB transwell cell culture test using SV-ARBEC cells as described in Example 2 of the present disclosure.

[0103] Stabilizing FC5 As shown in Example 3 of the present disclosure, FC5 may be unstable in serum. When exposed to serum, proteolysis may occur at one or more sites in FC5. Stabilized FC5 may reduce the susceptibility to proteolytic degradation, so that FC5 can be developed for clinical use. In one embodiment, the FC5 molecule of the present disclosure is stabilized by the introduction of a second disulfide bond in the FC5 molecule.

[0104] FC5, isolated from a naive llama single domain antibody library, contains a single disulfide bond. Approximately half of the VHH germlines of all camelid species contain a third cysteine, a fourth cysteine ​​is acquired during somatic hypermutation, and many camelid VHHs have two disulfides.

[0105] In addition to the cysteines formed by Cys22 and Cys96 common to all immunoglobulin variable domains, several strategies were pursued to add a second disulfide bond to FC5. In the first strategy, FC5 was engineered to contain a second disulfide formed by cysteines at positions 34 and 79 (position numbering based on FIG. 1), connecting β-strands C and E in framework regions 2 and 3, respectively. C34 is the end of CDR-H1 and the beginning of β-strand C. This disulfide is located in the interior of the protein globule and may improve the thermal melting temperature (Tm) between 4 and 18° C. The second strategy involved combining spatially close internal positions that can accommodate a disulfide-forming cysteine. This strategy relies on positions 49 and 70 (position numbering based on FIG. 1), again connecting strands C' and E in framework regions 2 and 3. For this strategy, the Tm can be improved between 6 and 19° C. The third disulfide stabilization strategy is to connect the externally facing position 33 in CDR-H1 with various positions in CDR-H3 (positions 102, 103, 104, and 105), which in the VHH are folded onto a side that often corresponds to the VH / VL interface in conventional antibodies. Thus, this interloop disulfide bond is partially solvent exposed.

[0106] The present disclosure features antibody variable domains that translocate across the blood-brain barrier and are at least 80% identical (e.g., 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 95%, at least 97%, or 100% identical) to SEQ ID NO:1. In some instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:47, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51, wherein the amino acid sequence comprises cysteines at positions corresponding to positions 49 and 70 of SEQ ID NO:1. In other instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:48, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51, wherein the amino acid sequence comprises a cysteine ​​at a position corresponding to position 79 of SEQ ID NO:1. In certain instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 49, a CDR2 comprising the sequence set forth in SEQ ID NO: 50, and a CDR3 comprising the sequence set forth in SEQ ID NO: 52. In some instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 58, a CDR2 comprising the sequence set forth in SEQ ID NO: 50, and a CDR3 comprising the sequence set forth in SEQ ID NO: 52. In certain instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 49, a CDR2 comprising the sequence set forth in SEQ ID NO: 50, and a CDR3 comprising the sequence set forth in SEQ ID NO: 53. In certain instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 58, a CDR2 comprising the sequence set forth in SEQ ID NO: 50, and a CDR3 comprising the sequence set forth in SEQ ID NO: 53. In some instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:49, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:54.In certain instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:58, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:54. In certain instances, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:49, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:55. In yet another instance, the antibody variable domain comprises a CDR1 comprising the sequence set forth in SEQ ID NO:58, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:55. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells expressing TMEM30A than to cells that do not express or have been modified to not express TMEM30A. In some embodiments, these molecules have reduced instability in human serum compared to wild-type FC5 (SEQ ID NO:1).

[0107] The antibody variable domain may include 30 or less (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:1. In certain cases, the amino acid substitutions are conservative amino acid substitutions. In some instances, the antibody variable domain includes a substitution at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) of positions corresponding to positions 1, 5, 6, 14, 37, 44, 45, 47, 75, 87, 88, 93, 114, or 117 of SEQ ID NO:1 compared to SEQ ID NO:1. In certain instances, the amino acid sequence includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) of a glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 5 of SEQ ID NO:1; a glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; a proline at a position corresponding to position 14 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; a glycine at a position corresponding to position 44 of SEQ ID NO:1; a leucine at a position corresponding to position 45 of SEQ ID NO:1; a tryptophan at a position corresponding to position 47 of SEQ ID NO:1; a serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; a glutamine at a position corresponding to position 114 of SEQ ID NO:1; or a leucine at a position corresponding to position 117 of SEQ ID NO:1. In some embodiments, these molecules may directly or indirectly bind to human TMEM30A (UniProtKB-Q9NV96). In certain embodiments, these molecules bind better to cells that express TMEM30A than to cells that do not express or have been modified not to express TMEM30A. In some embodiments, these molecules have reduced instability in human serum than wild-type FC5 (SEQ ID NO:1).

[0108] Disulfide stabilization strategies can be used with wild-type FC5 or humanized FC5 molecules. Example 5 of the present disclosure provides non-limiting examples of disulfide stabilized antibody variable domains. The present disclosure encompasses antibody variable domains comprising an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOs:20-28, wherein the antibody variable domain is capable of transcytosing through the BBB. In one embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:20. In another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:21. In another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:22. In yet another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:23. In another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:24. In some embodiments, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:25. In another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:26. In another embodiment, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:27. In certain embodiments, the disulfide stabilized antibody variable domain has the amino acid sequence set forth in SEQ ID NO:28.

[0109] Chimeric molecules The present disclosure also features chimeric molecules comprising any of the antibody variable domains disclosed herein (e.g., humanized antibody variable domains, disulfide stabilized antibody variable domains, or humanized disulfide stabilized antibody variable domains). In certain embodiments, the chimeric molecules comprise a dimeric portion (e.g., Fc) or other fusion protein (e.g., human serum albumin (HSA), it is noted that albumin is not dimeric but may be fused at its N-terminus and / or C-terminus). In some instances, the C-terminus of the antibody variable domain disclosed herein is linked directly or indirectly to the N-terminus of each member of the dimeric portion. In certain cases, the chimeric molecules comprise an antibody, an antigen-binding fragment of an antibody, a single chain antibody, a peptide, an enzyme, a nucleic acid (e.g., an antisense oligonucleotide), or a small molecule drug, or a liposome or lipid nanoparticle encapsulating a bioactive moiety (such as a nucleic acid, a small molecule drug, or a peptide). Non-limiting examples of chimeric fusions of the present disclosure are provided in diagrammatic form in FIG. 48.

[0110] Fc area In some instances, the chimeric molecule comprises an antibody Fc region. As used herein, the term "Fc region" is defined as the portion of an immunoglobulin formed by the dimeric association of the Fc domains (or Fc portions) of each of the two heavy chains. A native Fc region comprises two polypeptide chains in homodimerization. The Fc region of the present disclosure can be a homodimer (identical Fc domains) or a heterodimer (non-identical Fc domains). As used herein, the term "Fc domain" or "Fc portion" refers to a single polypeptide chain of an immunoglobulin, including a CH2 domain, a CH3 domain, or a variant thereof. In some embodiments, the Fc region is from human IgG1, human IgG2, human IgG3, or human IgG4. In certain embodiments, the Fc region is not glycosylated (e.g., N297Q or T299A). In certain embodiments, the Fc region is a human IgG1 agly Fc region. In certain embodiments, the Fc region contains a human IgG4P agly CH2 domain and a human IgG1 CH3 domain. In certain instances, the Fc region or Fc domain has reduced effector function compared to its wild-type counterpart. IgG antibodies exist in various allotypes and isoallotypes, each of which is encompassed by the present disclosure. In certain embodiments, the Fc region of the present disclosure comprises an IgG1 heavy chain Fc having an allotype of G1m1; nG1m2; G1m3; G1m17,1; G1m17,1,2; G1m3,1; or G1m17. These allotypes or isoallotypes are characterized by the following amino acid residues at the indicated positions (EU numbering) in the IgG1 heavy chain constant region (Fc): G1m1: D356, L358; nG1m1: E356, M358; G1m3: R214, E356, M358, A431; G1m17,1: K214, D356, L358, A431; G1m17,1,2: K214, D356, L358, G431; G1m3,1: R214, D356, L358, A431; and G1m17: K214, E356, M358, A431. Exemplary Fc domain sequences are provided below. IgG1 Fc (IgG1m3): APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 61) Fc of IgG1 agly (it will be understood that other agly versions such as T299A may be used): APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62) IgG2 Fc: APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63) IgG2 SAA Fc: APPAAAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64) It is noted that the "SAA" version also contains a C222S mutation in the hinge. ERKSCVECPPCP (SEQ ID NO: 95) IgG3 Fc: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 65) IgG4 Fc: APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG (SEQ ID NO: 66) IgG4 agly / G1 Fc: APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 67)

[0111] In certain instances, the Fc domain may comprise one or more mutations compared to the wild-type Fc domain. For example, one or more mutations may be made to reduce effector function, increase stability, improve pharmacokinetics, and / or increase half-life. In certain cases, the most C-terminal residue of each IgG1 Fc domain is deleted (i.e., the last lysine is removed), because it may be this C-terminal lysine that is subject to splicing and / or enzymatic cleavage that may lead to product heterogeneity. In certain instances, the most C-terminal residue of the IgG1 Fc domain is a lysine residue. In certain instances, the most C-terminal residue of the IgG1 Fc domain is changed from a lysine residue to a cysteine ​​residue. This allows for C-terminal site conjugation. In some instances, the Fc domain comprises an S442C mutation (numbering according to the EU index). In some cases, a mutation(s) can be introduced to enhance heterodimerization (including, for example, knob-into-hole mutations, electrosteering mutations, DuoBody mutations, etc.). Such mutations are well known in the art. See, for example, Ridgway et al., Protein Engineering 9(7):617-21(1996); US5,807,706; US9,862,778; WO2017 / 106462; Labrijn et al, PNAS, 110(13):5145-5150(2013); Gramer et al. mAbs, 5(6):962-973(2013); Labrijn et al. Nature Protocols, 9(10):2450-63(2014). Fc domain containing knob-into-hole mutations: [ka] Fc domain containing electrosteering mutations: [ka] DuoBody mutations involving the Fc domain: Pair 1: Kuhlman Demarest 20.8.34: "a" side [ka] Kuhlman Demarest 20.8.34: "b" side [ka] Pair 2: Kuhlman Demarest 7.8.60: "a" side [ka] Kuhlman Demarest 7.8.60: "b" side [ka] Pair 3: MP43a:S364K / K409L [ka] MP43b:K370S / F405K [ka] Pair 4: ZW1a: T350V / L351Y / F405A / Y407V [ka] ZW1b: T350V / T366L / K392L / T394W [ka]

[0112] In certain instances, the Fc region has an Fc domain that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any one of SEQ ID NOs: 61-101. In certain embodiments, the Fc region has reduced effector function relative to the corresponding wild-type Fc region.

[0113] Hinge Area In certain instances, the chimeric molecule may include an antibody hinge region. In some instances, the chimeric molecule includes an antibody hinge region and an antibody Fc region. The hinge region may be fused to the Fc moiety directly or via a linker. For example, the hinge connects the C-terminus of the antibody variable domain described above and the N-terminus of the CH2 domain of the Fc domain. The hinge region may be any flexible peptide sequence 1-30 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) amino acids in length. In some instances, the hinge region is 1-20 amino acids in length. In other instances, the hinge region is 1-18 amino acids in length. In some cases, the hinge region is a hinge from human IgG1, human IgG2, human IgG3, or human IgG4. In certain cases, these hinge regions can be engineered to include one or more amino acid substitutions (e.g., S228P in IgG4). In certain instances, the hinge region comprises the amino acid sequence AEPKSCD (SEQ ID NO:56). In other instances, the hinge region comprises the amino acid sequence AEPKSSD (SEQ ID NO:59). In still other instances, the hinge region comprises the amino acid sequence KTHTCPPCP (SEQ ID NO:19). In certain instances, the hinge region comprises the amino acid sequence AEPKSCDKTHTCPPCP (SEQ ID NO:74). In other instances, the hinge region comprises the amino acid sequence AEPKSSDKTHTCPPCP (SEQ ID NO:75). In other instances, the hinge region comprises the amino acid sequence GGGGSDKTHTCPPCP (SEQ ID NO:76). In some instances, the hinge region comprises the amino acid sequence VERKCCVECPPCP (SEQ ID NO: 102). In other instances, the hinge region comprises the amino acid sequence VESKYGPPCPSCP (SEQ ID NO: 103). In other instances, the hinge region comprises the amino acid sequence ELKTPLGDTTHTCPRCP (SEQ ID NO: 104). In yet other instances, the hinge region comprises the amino acid sequence EPKSCDTPPPCPRCP (SEQ ID NO: 105).

[0114] In certain embodiments, the chimeric molecule homodimerizes or heterodimerizes with another chimeric molecule. For example, if the Fc domains of the chimeric molecules are identical, they homodimerize to form a homodimeric Fc region. However, if the Fc domains of the chimeric molecules are not identical (e.g., they contain knob-into-hole, electrosteering, or DuoBody mutations), they heterodimerize to form a heterodimeric Fc region. The C-terminus of the antibody variable domain of the present disclosure can be linked, directly or via an intervening amino acid sequence, to the N-terminus of the hinge region, which in turn can be linked via its C-terminus to the N-terminus of the CH2 domain of the Fc domain.

[0115] Linker In some examples, the chimeric molecule comprises one or more linkers. There is no particular limitation on the linker that links different regions of the chimeric molecule (e.g., linking an antibody variable domain to the N-terminus of the VH domain or VL domain of a therapeutic antibody; linking an antibody variable domain to a therapeutic moiety; linking the C-terminus of the Fc domain of an Fc region to the N-terminus of a therapeutic antibody or fragment thereof; linking the C-terminus of the Fc domain of an Fc region to a therapeutic moiety; linking an antibody variable domain to a hinge region). In some embodiments, the linker is a peptide linker. Any arbitrary single-chain peptide containing about 1 to 30 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues) can be used as a linker. Examples of such peptide linkers include Gly; Ser; Gly Ser; Gly Gly Ser; Ser Gly Gly; Gly Gly Gly Ser (SEQ ID NO: 77); Ser Gly Gly Gly (SEQ ID NO: 78); Gly Gly Gly Gly Ser (SEQ ID NO: 5); Ser Gly Gly Gly Gly (SEQ ID NO: 79); Gly Gly Gly Gly Gly Ser (SEQ ID NO: 80); Ser Gly Gly Gly Gly Gly (SEQ ID NO: 81); Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 82); Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 83); (Gly Gly Gly Gly Ser) n (SEQ ID NO:5) n (wherein n is an integer of 1 or more); and (Ser Gly Gly Gly Gly) n (SEQ ID NO:79) n (wherein n is an integer of 1 or more).

[0116] In other embodiments, the linker peptide is modified so that the amino acid sequence GSG, which occurs at the junction of a conventional Gly / Ser linker peptide repeat, is absent. For example, the peptide linker is (GGGXX) n GGGGS (SEQ ID NO: 84) and GGGGS (XGGGS) n (SEQ ID NO:85), where X is any amino acid that cannot be inserted into the sequence to result in a polypeptide containing the sequence GSG, and n is 0 to 4. In one embodiment, the linker peptide has the sequence (GGGX1X2) n GGGGS, X1 is P, X2 is S, and n is 0 to 4 (SEQ ID NO: 86). In another embodiment, the sequence of the linker peptide is (GGGX1X2) n GGGGS, X1 is G, X2 is Q, and n is 0 to 4 (SEQ ID NO: 87). In another embodiment, the sequence of the linker peptide is (GGGX1X2) n In yet another embodiment, the sequence of the linker peptide is GGGGS (XGGGS), X1 is G, X2 is A, and n is 0 to 4 (SEQ ID NO: 88). nwherein X is P and n is 0 to 4 (SEQ ID NO: 89). In one embodiment, the linker peptide of the invention comprises or consists of the amino acid sequence (GGGGA)2GGGGS (SEQ ID NO: 90). In another embodiment, the linker peptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS (SEQ ID NO: 91). In yet another embodiment, the linker peptide comprises or consists of the amino acid sequence (GGGPS)2GGGGS (SEQ ID NO: 92). In a further embodiment, the linker peptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2 (SEQ ID NO: 93).

[0117] In certain embodiments, the linker is a synthetic compound linker (chemical cross-linker). Examples of crosslinkers include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES), sulfo-SMCC series and bismaleimidohexane (BMH) series crosslinkers.

[0118] XTEN In some instances, the chimeric molecule comprises an XTEN sequence. "XTEN sequence" refers to an extended length polypeptide of a non-naturally occurring substantially non-repetitive sequence composed primarily of small hydrophilic amino acids, which sequence has a low degree of structure or no secondary or tertiary structure under physiological conditions. As a partner of the chimeric molecule, XTEN acts as a carrier and confers certain desired pharmacokinetic, physicochemical, and pharmaceutical properties when linked to a clotting factor, a clotting factor heavy chain, a clotting factor light chain, a targeting moiety, or other sequences or molecules on the chimeric molecule. Such desired properties include, but are not limited to, enhanced pharmacokinetic parameters and solubility characteristics.

[0119] In some embodiments, the XTEN sequences of the invention are peptides or polypeptides having more than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, or 2000 amino acid residues. In certain embodiments, the XTEN is a peptide or polypeptide having from about 20 to more than about 3000 amino acid residues, from 30 to more than about 2500 residues, from 40 to more than about 2000 residues, from 50 to more than about 1500 residues, from 60 to more than about 1000 residues, from 70 to more than about 900 residues, from 80 to more than about 800 residues, from 90 to more than about 700 residues, from 100 to more than about 600 residues, from 110 to more than about 500 residues, or from 120 to more than about 400 residues.

[0120] The table below provides non-limiting amino acid sequences of XTEN. Exemplary XTEN Sequences [Table 14-1] [Table 14-2] [Table 14-3]

[0121] In some instances, XTEN is selected from the group consisting of AE42, AG42, AE42_2, AE42_3, AE48, AM48, AE72, AE72_2, AE72_3, AG72, AE108, AG108, AE144, AF144, AE144_2, AE144_3, AG144, AE180, AG180, AE216, AG216, AE252, AG252 , AE288, AG288, AE295, AE324, AG324, AE360, AG360, AE396, AG396, AE432, AG432, AE468, AE504 , AG504, AF504, AE540, AG540, AF540, AD576, AE576, AF576, AG576, AE612, AG612, AE624, AE648, AG648, AG684, AE720, AG720, AE756, AG756, AE792, AG792, AE828, AG828, AD836, AE864, AF864, AG864, AE872, AE884, AM875, AE912, AM923, AM1318, BC864, BD864, AE948, AE1044, AE1140, AE12 36, AE1332, AE1428, AE1524, AE1620, AE1716, AE1812, AE1908, AE2004A, AG948, AG1044, AG1140, AG1236, AG1332, AG1428, AG1524, AG1620, AG1716, AG1812, AG1908, and AG2004. In some embodiments, the XTEN is selected from the group consisting of AE42, AE864, AE576, AE288, AE144, AE288, AG864, AG576, AG288, and AG144. In some embodiments, the XTEN is selected AE144. In some embodiments, the XTEN is AE288.

[0122] Additional examples of XTEN polypeptides that may be used in accordance with the present disclosure are described in U.S. Pat. Nos. 7,855,279 and 7,846,445, U.S. Patent Publication Nos. 2009 / 0092582, 2010 / 0239554, 2010 / 0323956, 2011 / 0046060, 2011 / 0046061, 2011 / 0077199A1, 2011 / 0172146 No. 2012 / 0178691; No. 2012 / 0263701; or No. 2013 / 0017997, International Patent Publication Nos. WO2010091122, WO2010144502, WO2010144508, WO2011028228, WO2011028229, WO2011028344; and WO2013130683.

[0123] Human serum albumin In some instances, the chimeric molecule comprises human serum albumin (HSA) as a fusion moiety in place of the Fc region. It is noted that HSA is not a dimerization moiety, although N- and / or C-terminal fusions are possible. In some embodiments, the C-terminus of an antibody variable domain of the present disclosure is linked directly or indirectly (e.g., via a linker or a therapeutic agent (e.g., an antibody, antibody fragment, peptide, enzyme, antisense oligonucleotide)) to the N-terminus of an HSA polypeptide.

[0124] HSA has many desirable pharmaceutical properties. These include a serum half-life of 19-20 days; a solubility of about 300 mg / mL; good stability; ease of expression; no effector functions; low immunogenicity; and a circulating serum level of about 45 mg / mL. Crystal structures of HSA without and with ligands (including biologically important molecules such as fatty acids and drugs) or complexed with other proteins are well known in the art. See, for example, Universal Protein Resource Knowledgebase P02768; He et al., Nature, 358:209-215 (1992); Sugio et al., Protein Eng., 12:439-446 (1999). X-ray crystallographic studies of HSA have shown that the polypeptide forms a heart-shaped protein with dimensions of approximately 80×80×80 Å and a thickness of 30 Å. It has about 67% α-helices but no β-sheets and can be divided into three homologous domains (I-III). Each of these three domains is composed of two subdomains (A and B). The A and B subdomains have 6 and 4 α-helices, respectively, connected by flexible loops. The major regions of ligand binding to human serum albumin are located in the cavities of subdomains IIA and IIIA, which are formed mainly by hydrophobic and positively charged residues and show similar chemical properties. All but one of the 35 cysteine ​​residues in the molecule participates in the formation of 17 stabilizing disulfide bonds. The structures and amino acid sequences of bovine, equine, rabbit, and hares albumins are also known. See, e.g., Majorek et al., Mol. Immunol., 52:174-182 (2012); Bujacz, Acta Crystallogr. D Biol. Crystallogr., 68:1278-1289 (2012). Numerous genetic variants of human serum albumin are known in the art.See, for example, the albumin websites maintained by the University of Aarhus, Denmark and the University of Pavia, Italy at albumin.org / genetic-variants-of-human-serum-albumin and albumin.org / genetic-variants-of-human-serum-albumin-reference-list.

[0125] In one embodiment, the human serum albumin used in the chimeric molecule comprises or consists of the amino acid sequence set forth below. (SEQ ID NO:94)

[0126] In another embodiment, the human serum albumin used in the chimeric molecule is an HSA variant and has an amino acid sequence that is at least 85%, 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 to the amino acid sequence set forth in SEQ ID NO:94. The percent identity between amino acid sequences can be determined using the BLAST 2.0 program. Sequence comparison can be performed using ungapped alignments and using the default parameters (Blossom 62 matrix, gap existence cost of 11, gap cost per residue of 1, and lambda ratio of 0.85). The mathematical algorithm used in the BLAST program is described in Altschul et al., 1997, Nucleic Acids: Research 25:3389-3402.

[0127] In certain embodiments, the human serum albumin used in the chimeric molecule is an HSA variant that may have an N-terminal and / or C-terminal deletion in the sequence of SEQ ID NO:94 (e.g., 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 consecutive amino acids may be deleted at the N-terminus and / or C-terminus). In some instances, the HSA variant has the same or substantially the same desirable pharmaceutical properties of HSA having the amino acid sequence of SEQ ID NO:94 (e.g., a serum half-life of 19-20 days; solubility of about 300 mg / mL; good stability; ease of expression; no effector functions; low immunogenicity; and / or circulating serum levels of about 45 mg / mL). In some instances, the HSA used in the chimeric molecule is a genetic variant of HSA. In some instances, the HSA variant is any one of the 77 variants disclosed in Otagiri et al., 2009, Biol. Pharm. Bull. 32(4), 527-534 (2009). In certain embodiments, the HSA used in the chimeric molecule is a mutated version of HSA that has improved affinity for the neonatal Fc receptor (FcRn) compared to the HSA of SEQ ID NO:94 (e.g., US9,120,875; US9,045,564; US8,822,417; US8,748,380; Sand et al., Front. Immunol., 5:682 (2014); Andersen et al., J. Biol. Chem., 289(19):13492-502 (2014); Oganesyan et al., J. Biol. Chem., 289(11):7812-24 (2014); Schmidt et al., J. Biol. Chem., 289(11):7812-24 (2014); Schmidt et al., J. Immunol ... al., Structure, 21(11):1966-78 (2013); WO2014 / 125082A1; WO2011 / 051489, WO2011 / 124718, WO2012 / 059486, WO2012 / 150319; WO2011 / 103076; and WO2012 / 112188, all of which are incorporated by reference herein.) In certain instances, the HSA mutation is an E505G / V547A mutation.In certain instances, the HSA mutation is a K573P mutation, which is an HSA with improved affinity for FcRn that can be used to increase the half-life of the chimeric molecule.

[0128] Therapeutic Agents The chimeric molecule may include a therapeutic agent(s). Examples of therapeutic agents include antibodies, antigen-binding fragments of antibodies, single chain antibodies (e.g., scFv, sc(Fv)2), diabodies, nanobodies, antibody fragments, nucleic acids (e.g., antisense oligonucleotides), peptides, or enzymes, used alone or in combination therapy according to standard of care treatment. The chimeric molecules of the present disclosure can transport therapeutic agents across the BBB to the CNS. Thus, the chimeric molecules can be used to treat and / or prevent neurological disorders. Exemplary neurological disorders include Alzheimer's disease, Parkinson's disease, frontotemporal dementia, ALS, Huntington's chorea, multiple sclerosis, spinal muscular atrophy, muscular dystrophies, spinal cord injuries, stroke, ophthalmologic conditions, acute or chronic optic neuritis, psychiatric disorders, Tourette's disease, brain trauma, brain tumors, and epilepsy.

[0129] Exemplary therapeutic agents for the treatment of Alzheimer's disease include caprylic triglyceride, anti-tau antibodies, anti-β amyloid antibodies, anti-DKK1 antibodies, APOE antagonist antibodies, donepezil, quinidine, serotonin 6 receptor antagonists, β secretase inhibitors, RAGE antagonists, BACE inhibitors, amyloid beta protein inhibitors, phosphodiesterase 9A inhibitors, bisnorcymeserine, bryostatin 1, α-7 potentiators, purinergic receptor P2Y6 agonists, tau protein aggregation / TDP-43 aggregation inhibitors, N3pG-Aβ These include mAbs, mGlu2 agonists, quinazolinones, mitochondrial protein stimulators, amyloid precursor protein secretase inhibitors, 5HT6 antagonists, R-phenserine, amyloid beta / tau protein inhibitors, MAO-B inhibitors, Lp-PLA2 inhibitors, 5-HT6 receptor antagonists, BET protein inhibitors, antigen fibril AB mAbs, nomethiazole, histamine H3 receptor antagonists, PPAR delta / gamma agonists, abeotaxane, and p38 mitogen-activated protein kinase inhibitors.

[0130] Exemplary therapeutic agents for the treatment of ALS include anti-SOD1 antibodies, anti-DR6 antibodies, anti-DPR antibodies, dexpramipexole, arimoclomal, GM6, ibudilast, macrophage modulators, NOGO-A inhibitors, and troponin complex stimulators.

[0131] Exemplary therapeutic agents for the treatment of brain trauma include apomorphine, cytokine inhibitors / neuropeptide receptor modulators, and progesterone receptor agonists.

[0132] Exemplary therapeutic agents for the treatment of brain tumors include IDH1 inhibitors, doxorubicin, paclitaxel, anti-EGFRvIII antibody drug conjugates, bevacizumab, FGF-R kinase inhibitors, PI3K inhibitors, cabozantinib, iodine I131 derlotuximab biotin, PDGFR inhibitors, orotate carboxyamidotriazole, non-neurotoxic derivatives of penclomidine, golvatinib, dexanabinol, TGF-β1 kinase inhibitors, afatinib, IDO inhibitors, cabazitaxel, Srckinase inhibitors, and the like. These include: endothelin A / B receptor antagonists, proteasome inhibitors, T-type calcium channel antagonists, thapsigargin analogs, irinotecan, nivolumab, CSF-1R inhibitors, pelareolep, EGFR antagonists, exportin-1 protein inhibitors / nucleoprotein inhibitors, BIRC5 protein inhibitors, evofosfamide, abeotaxane, ENG protein inhibitors, trans-sodium crocetinate, N7-alkylating agents, targeted antiangiogenic agents, and veliparib.

[0133] Exemplary therapeutic agents for the treatment of epilepsy include everolimus, eslicarbazepine acetate, alprazolam, brivaracetam, carbamazepine, cannabidiol, 4-aminobutyric acid transaminase inhibitors, perampanel, GABA-A receptor agonists, synthetic huperzine, pregabalin, clobazam, diazepam, GABA A These include modulators of synaptic and extrasynaptic receptors, topiramate IV, lacosamide, and serotonin receptor agonists.

[0134] Exemplary therapeutic agents for the treatment of genetic disorders (e.g., Friedrich's ataxia, late-onset childhood neuronal ceroid, spinal-bulbar muscular atrophy, ataxia-telangiectasia, pantothenate kinase-associated neurodegeneration, spinal muscular atrophy, familial amyloid polyneuropathy, Rett's syndrome, Leigh's syndrome, Wilson's disease) include NF / E2-related factor 2 stimulators, interferon gamma-1b, rhTPP1 enzyme replacement therapy, vatiquinone, deferiprone, Spinraza®, ISIS-TTRRX, serotonin 1A receptor agonists, cytokine inhibitors / neuropeptide receptor modulators, siRNA inhibitors targeting TTR, phosphopantothenate supplementation, DcpS inhibitors, cysteamine bitartrate, indolepropionic acid, transthyretin dissociation inhibitors, and bis-choline tetrathiomolybdate.

[0135] Exemplary therapeutic agents for the treatment of headaches include anti-CGRP mAbs, CGRP receptor antagonist mAbs, sumatriptan, dextromethorphan / quinidine, onabotulinumtoxinA, serotonin-1F receptor agonists, nNOS inhibitors / 5HT, dihydroergotamine, cyclobenzaprine, and aspirin / sumatriptan combinations.

[0136] Exemplary therapeutic agents for the treatment of Huntington's disease include laquinimod, a PDE10 inhibitor, pridopidine, cysteamine bitartrate, and aVMAT2 inhibitors.

[0137] Exemplary therapeutic agents for the treatment of multiple sclerosis include natalizumab, monomethyl fumarate prodrugs, anti-LINGO-1 antibodies, Nck protein modulators, S1PR-1 / 5 receptor agonists, fingolimod, anti-CD52 mAbs, idebenone, PPARγ agonists / modulators, laquinimod, tyrosine kinase inhibitors, anti-CD19 mAbs, ibudilast, guanabenz, anti-CD20 mAbs, interferon beta-1b, IL-7 receptor inhibitors, S1P1 receptor agonists, myelin protein stimulators, estriol succinate, imilecleucel-T, anti-VLA2 mAbs, BAFF-R modulators, CD100 antigen inhibitors, anti-DR6 antibodies, and NF-κB inhibitors.

[0138] Exemplary therapeutic agents for the treatment of muscular dystrophies include myostatin inhibitors, drisapersen, eteplirsen, halofuginone, idebenone, ISIS-DMPKR X , steroid receptor agonists, GAPDH inhibitors, transcription inhibitors, tadalafil, ataluren, and glucocorticoid receptor agonists.

[0139] Exemplary therapeutic agents for the treatment of pain include neublastin, P2X3 purinergic receptor antagonists, SNARE protein antagonists, oxycodone-naltrexone core (abuse-resistant), amitriptyline / ketamine, lintatrimod, cannabinoid receptor CB2 agonists, non-erythropoietic peptides, PPARγ agonists, glycogen phosphorylase inhibitors, NMDA receptor antagonists, zoledronic acid, early growth response protein 1 inhibitors, histamine-3 receptor antagonists, and the like. receptor antagonists, buprenorphine, cytokine inhibitors, cebranopadol, celecoxib, arachidonic acid analogs, synthetic capsaicin, Nav1.7 sodium channel inhibitors, opioid kappa receptor agonists, duloxetine, nerve growth factor stimulators, dexmedetomidine, voltage-gated sodium channel inhibitors, bupicaine, angiotensin 2 receptor antagonists, nerve growth factor inhibitors, p38 inhibitors, rapastinel, levorphanol, CGRP These include mAbs, pregabalin, mGlu2 / 3 receptor agonists, CACNA2D1 protein modulators, bone resorption inhibitors, neublastin, μ-opioid analgesics, nNOS inhibitors, O-desmethyltramadol, palmitoylethanolamide, GABA A agonists, TRPV-1 receptor agonists, nabiximols, cyclooxygenase 2 inhibitors, nerve growth factor modulators, cyclobenzaprine, flurbiprofen, fatty acid amide hydrolase inhibitors, and ibuprofen / phosphatidylcholine.

[0140] Exemplary therapeutic agents for the treatment of Parkinson's disease include amantadine, apomorphine, alpha7 nicotinic acetylcholine receptor partial agonists, anti-alpha-synuclein antibodies, alpha-synuclein inhibitors, levodopa, D1 potentiators, zipraglant, serotonin 1A / 1B partial agonists, fipamezole, GM6, retinoid X receptor agonists, istradefylline, rotigotine, pramipexole / rasagiline, R-phenserine, serotonin 2A / 6 receptor antagonists, adenosine A2A receptor antagonists, safinamide, and dopamine receptor agonists.

[0141] Exemplary therapeutic agents for the treatment of spasticity include baclofen, onabotulinumtoxinA, abobotulinumtoxinA, arbaclofen, nabiximols, and incobotulinumtoxinA.

[0142] Exemplary therapeutic agents for the treatment of spinal cord injury include anti-Lingo-1 antibodies, anti-NgR1 antibodies, neublastin, nervous system modulators, Rho GTP-binding protein inhibitors, and fibroblast growth factor receptors.

[0143] Exemplary therapeutic agents for the treatment of stroke include natalizumab, a recombinant mutant form of human wild-type activated protein C, ticagrelor, dalfampridine, aspirin, nimodipine microparticles, GM6, PARP inhibitors, PDZ domain inhibitors, beta amyloid inhibitors, dabigatran, and sodium nitrite.

[0144] Exemplary therapeutic agents for the treatment of Tourette's syndrome include histamine-3 receptor antagonists, 4-aminobutyric acid transaminase inhibitors, abobotulinumtoxin A, ecopipam, VMAT2 inhibitors, acamprosate, and vigabatrin.

[0145] Other exemplary therapeutic agents for the treatment of other neurological disorders include myostatin inhibitors, NF / E2-related factor 2 stimulators, anti-tau antibodies, myeloperoxidase inhibitors, mitochondrial permeability transition pore inhibitors, belimumab, type II-B activin receptor modulator mAb, C1 esterase inhibitor, ferric carboxymaltose, amifampridine, fingolimod, monoamine oxidase B inhibitors, neurotransmitter modulators, dopamine receptor agonists, anti-CD19 mAb, VMAT2 inhibitors, CD20 mAb, thymosin beta-4, anti-IL-6 receptor mAb, eculizumab, AMPA receptor modulators, steroid hydroxylase inhibitors, pyridoxal phosphate, abeotaxane, aceneuraminic acid, and sodium oxybate.

[0146] In one example, the therapeutic agent is an antibody. Non-limiting examples of CDRs of exemplary therapeutic antibodies are provided below. [Table 15] [Table 16-1] [Table 16-2]

[0147] Non-limiting examples of VH and VL of exemplary therapeutic antibodies are provided below.

[0148] Anti-LINGO-1: VH: EVQLLESGGG LVQPGGSLRL SCAASGFTFS AYEMKWVRQA PGKGLEWVSV IGPSGGFTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCATEG DNDAFDIWGQ GTTVTVSS (SEQ ID NO: 183) VL: DIQMTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ RSNWPMYTFG QGTKLEIK (SEQ ID NO: 184)

[0149] Anti-TWEAK: VH: EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYAMSWVRQA PGKGLEWVAE ISSGGSYPYY PDTVTGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARVL YYDYDGDRIE VMDYWGQGTL VTVSS (SEQ ID NO: 185) VL: DVVMTQSPLS LPVTPGEPAS ISCRSSQSLV SSKGNTYLHW YLQKPGQSPQ LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDVGV YYCSQSTHFP RTFGGGTKVE IK (sequence number: 186)

[0150] Anti-β-amyloid: VH: QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWFDGTKKYYTDSVKGRFTISRDNSKNTLYLQMNTLRAEDTAVYYCARDRGIGARRGPYYMDVWGKGTTVTVSS (SEQ ID NO: 187) VL: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIKR (SEQ ID NO: 188)

[0151] Anti-tau: VH: [ka] VL: SYELTQPPSVSVSPGQTARITCSGDALPKRYVYWYQQKSGQAPVLVIYEDSKRPSGIPETFSGSSSGTMATLTISGAQVEDEADYYCYSTDSNGHHWVFGGGTKLTVL (SEQ ID NO: 190)

[0152] Anti-tau: VH: EVQLVESGGGLVQPGGSLRLSCAASGFTFPNYVMTWVRQAPGKGLEWVSGISGSGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRVEDTALYYCAKGSGGIRGQGTMVTVSS (SEQ ID NO: 191) VL: QSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWHQQHPGKAPKLLIYDVTKRPSGVPDRFSGSKSGNTAALTISGLQAEDEADYYCHSYVGSYTLVFGGGTKLTVL (SEQ ID NO: 192)

[0153] Anti-tau: VH: EVHLVESGGA LVKPGGSLRL SCAASGFSFS KYGMSWVRQA PGKGLEWVAT ISSSGSRTYY PDSVKGRFTI SRDNAKNTLY LQMNSLRAED TAMYYCSISW DGAMDYWGQG TTVTVSS (SEQ ID NO: 193) VL: DVVMTQSPLS LPVTLGQPAS ISCKSSQSIV HSNGNTYLEW YLQKPGQSPQ LLVYKVSNRFSGVPDRFSGS GSGTDFTLKI SRVEAEDVGT YYCFQGSLVP WAFGGGTKVE IK (SEQ ID NO: 194)

[0154] Anti-α-synuclein: VH: EVQLVESGGG LVEPGGSLRL SCAVSGFDFE KAWMSWVRQA PGQGLQWVAR IKSTADGGTT SYAAPVEGRF IISRDDSRNM LYLQMNSLKT EDTAVYYCTS AHWGQGTLVT VSS (SEQ ID NO: 195) VL: SYELTQPPSV SVSPGQTARI TCSGEALPMQ FAHWYQQRPG KAPVIVVYKD SERPSGVPER FSGSSSGTTA TLTITGVQAE DEADYYCQSP DSTNTYEVFG GGTKLTVL (SEQ ID NO: 196)

[0155] Anti-C9orf72 DPR: VH1: QVQLVESGGGVVQPGRSLRLSCAASGFTFSNHAMHWVRQAPGKGLEWVAVISYDGENTYYADSIEGRFTISRDNFKNTLFLQMYSLTADDTAMYFCARGGRRGHFTSYYLDYWGQGTLVTVSS (SEQ ID NO: 197) VL1: DIQMTQSPSSLSASVGDRVTITCRASQNIDKYLNWYQQIPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFSLTISSLQPEDFAIYYCQQSYSSFRTFGQGTKLEIK (SEQ ID NO: 198) VH2: QVQLQESGPGLVKPSETLSLTYTVLGGSVSDYYWSCIRQPAGKGLEWIGRTYTNGKTTYTYNPSLESRLSLSIDTSMNQFSLKLTSVTAADTAVYYCARWGAVTGDYYYGMDVWGPGTLVTVSS (SEQ ID NO: 199) VL2: EIVLTQSPLSLSVTPGEPASISCRSPRSLLHTNGYTYLDWYLQRPGQSPQLLIFLASNRASGVPDRFSGSGSGTNFTLRISGVEADDVGVYYCMQGLQPSWTFGQGTKVEIK (SEQ ID NO: 200)

[0156] In some embodiments, the chimeric molecule is a fusion polypeptide comprising an antibody variable domain as described above and a whole antibody (therapeutic agent) (see Figure 48). The C-terminus of an antibody variable domain described herein is linked, either directly or via a linker (e.g., G, GG, G4S (SEQ ID NO:5), 3xG4S (SEQ ID NO:60)), to the N-terminus of both VH domains or both VL domains of the antibody. In certain embodiments, the whole antibody is an anti-beta amyloid antibody, an anti-tau antibody, an anti-alpha synuclein antibody, an anti-TDP-43 antibody, an anti-LINGO-1 antibody, an anti-LINGO-2 antibody, an anti-LINGO-3 antibody, an anti-LINGO-4 antibody, an anti-TREM2 antibody, an anti-C9orf72 dipeptide repeat poly-GA antibody (i.e., an antibody capable of binding a dipeptide repeat (DPR) of poly-glycine-alanine (GA) having at least six repeats (GA)6 translated from the chromosome 9 open reading frame 72 (C9orf72) gene), an anti-TWEAK antibody, or an anti-TWEAK-R antibody.

[0157] In some embodiments, the chimeric molecule comprises an antibody variable domain, a hinge region, an Fc region, and a Fab (therapeutic agent) as described above (see FIG. 48). The C-terminus of the antibody variable domain is linked directly or indirectly to the N-terminus of each hinge region, which in turn is linked directly or indirectly to an Fc region. The C-terminus of each Fc domain of the Fc region is linked via a linker (e.g., G, GG, G4S (SEQ ID NO:5), 3xG4S (SEQ ID NO:60)) to the N-terminus of the VH or VL domain of the Fab (therapeutic agent). In certain embodiments, the Fab is an anti-beta amyloid Fab, an anti-tau Fab, an anti-alpha synuclein Fab, an anti-TDP-43 Fab, an anti-LINGO-1 Fab, an anti-LINGO-2 Fab, an anti-LINGO-3 Fab, an anti-LINGO-4 Fab, an anti-TREM2 Fab, an anti-C9orf72 dipeptide repeat poly-GA Fab, an anti-TWEAK Fab, or an anti-TWEAK-R Fab.

[0158] In certain embodiments, the chimeric molecule comprises an antibody variable domain, a therapeutic agent, a hinge region, and an Fc region as described herein. The C-terminus of the antibody variable domain is linked, either directly or via a linker (e.g., G, GG, G4S (SEQ ID NO:5), 3xG4S (SEQ ID NO:60)), to the N-terminus of a therapeutic agent (e.g., an antibody fragment (e.g., Fab), an enzyme, a peptide). The C-terminus of the therapeutic agent is linked, either directly or via a linker, to the N-terminus of the hinge region. The C-terminus of the hinge region is linked to the N-terminus of each of the Fc domains of the Fc region. In some cases, the

[0159] In some embodiments, the chimeric molecule comprises an antibody variable domain, a therapeutic agent, a hinge region, and an Fc region as described herein. The C-terminus of the antibody variable domain is linked, either directly or via a linker, to the N-terminus of the hinge region. The C-terminus of the hinge region is linked to the N-terminus of each Fc domain of the Fc region. The N-terminus of a therapeutic agent (e.g., an antibody fragment (e.g., Fab), an enzyme, a peptide) is linked, either directly or via a linker (e.g., G, GG, G4S (SEQ ID NO:5), 3xG4S (SEQ ID NO:60)) to the C-terminus of one or both Fc domains of the Fc region.

[0160] In certain embodiments, the chimeric molecule comprises an antibody variable domain, an ASO (therapeutic agent), a hinge region, and an Fc region as described herein. The C-terminus of the antibody variable domain is linked to the N-terminus of the hinge region, either directly or via a linker. The C-terminus of the hinge region is linked to the N-terminus of each Fc domain of the Fc region. The ASO is linked to the hinge region. In certain instances, the ASO is a splice-switching oligonucleotide. In other instances, the ASO is a gapmer.

[0161] In some cases, the chimeric molecule comprises an antibody variable domain described herein, a hinge region, an Fc region, and a lipid nanoparticle, liposome, or polymeric nanocarrier encapsulating a therapeutic agent (e.g., an ASO (e.g., a splice-switching oligonucleotide, or a gapmer), a small molecule drug, a nucleic acid, a peptide). The C-terminus of the antibody variable domain is linked, either directly or via a linker, to the N-terminus of the hinge region. The C-terminus of the hinge region is linked to the N-terminus of each Fc domain of the Fc region. The liposome may be linked to the C-terminus of one or both Fc domains of the Fc region. Methods of nanoscale delivery are well known in the art. See, e.g., Juliano et al., Nucl. Acids Res., 44(14) (2016) and references cited therein, all of which are incorporated herein by reference in their entireties.

[0162] It will be understood that the chimeric molecules described above can contain an HSA portion in place of the Fc region.

[0163] In addition, the present disclosure covers monovalent, bispecific, and tetravalent designs, as shown in FIG.

[0164] Nucleic acids, vectors, host cells, and methods for producing polypeptides The present disclosure also covers the nucleic acid encoding the antibody variable domain disclosed above.In addition, the nucleic acid encoding the fusion polypeptide described herein or the nucleic acid is covered.The nucleic acid(s) can be inserted into a vector(s) (e.g., an expression vector).

[0165] Nucleic acids encoding antibody variable domains and fusion polypeptides comprising the same as described above can be expressed in any desired host cell (e.g., bacterial cells, yeast cells, mammalian cells). In certain embodiments, the polypeptide is secreted from the host cell. In one embodiment, the host cell is a mammalian cell. In certain embodiments, the host cell is a CHO cell or cell line. In some instances, the antibody variable domain polypeptide coding sequence (e.g., humanized FC5, or disulfide-stabilized humanized FC5) is fused to an Fc (e.g., IgG1, agly IgG1, IgG2, IgG3, IgG4) coding sequence via an intervening amino acid sequence. In some instances, the intervening amino acid sequence comprises a hinge sequence (e.g., a hinge from IgG1, IgG4, etc.). In some instances, the intervening amino acid sequence comprises a Fab and a hinge sequence.

[0166] If the polypeptide is to be expressed in a bacterial cell (e.g., E. coli), the expression vector should have characteristics that allow the vector to be amplified in the bacterial cell. Additionally, when E. coli (such as JM109, DH5α or HB101, or XL1-Blue) is used as the host, the vector must have a promoter that can allow efficient expression in E. coli, such as the lacZ promoter (Ward et al., Nature, 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. Examples of such vectors include, for example, M13-series vectors, pUC-series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21, which expresses T7 RNA polymerase). The expression vector may contain a signal sequence for secretion. For production into the periplasm of E. coli, the pelB signal sequence (Lei et al., J. Bacteriol., 169:4379 (1987) can be used as a signal sequence for secretion. For bacterial expression, the calcium chloride method or electroporation method can be used to introduce the expression vector into bacterial cells.

[0167] The polypeptide is present in yeast cells (e.g. Saccharomyces cerevisiae, Saccharomyces italicus, Saccharomyces rouxii, Pichia pastoris, Pichia angusta, Pichia anomala, Pichia capsulate, Kluyveromyces lactis, or yeasts of the genera Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschunikowia, Rhodosporidium, Leucosporidium, Borryoascus, Sporidiobolus, or Endomycopsis, the expression vector will include a promoter to drive expression of the polypeptide in a yeast cell and / or a signal sequence effective to direct secretion in the yeast. Suitable promoters for the genus Saccharomyces include those associated with the genes PGK1, GAL1 or GAL10, CYC1, PHOS, TRP1, ADH1, ADH2, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase, α-mating factor pheromone, PRB1 promoter, GUT2 promoter, GPD1 promoter, and hybrid promoters including hybrids of portions of the 5'regulatory region of other promoters or portions of the 5'regulatory region with upstream activation sites (e.g., promoters described in EP-A-258 067). Suitable promoters for the genus Pichia include AOX1, AOX2, MOX1, and FMD1. In some instances, the signal sequence is a yeast-derived signal sequence (e.g., one that is homologous to the yeast host).

[0168] If the polypeptide is to be expressed in cells (CHO cells, COS cells, and NIH3T3 cells), the expression vector contains a promoter necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter. In addition to the nucleic acid sequence encoding the polypeptide, the recombinant expression vector may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to a drug (such as G418, hygromycin, or methotrexate) to a host cell into which the vector has been introduced. Examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0169] The polypeptides can also be expressed in human cells, such as HEK-293 cells.

[0170] Variant FC5 polypeptides (and fusions thereof) can be constructed using any of several methods known in the art. One such method is site-directed mutagenesis, in which a specific nucleotide (or a small number of specific nucleotides, if desired) is altered to change a single amino acid (or a small number of predetermined amino acid residues, if desired) in the encoded variant FC5 polypeptide. Many site-directed mutagenesis kits are commercially available. One such kit is the "Transformer Site Directed Mutagenesis Kit" sold by Clontech Laboratories (Palo Alto, Calif.). Kit".

[0171] FC5 polypeptides and fusions thereof can be produced and isolated using methods well known in the art. In some embodiments, such polypeptides are produced by recombinant DNA techniques. For example, a nucleic acid molecule(s) encoding an FC5 polypeptide or a fusion thereof can be inserted into a vector(s) (e.g., an expression vector) and the nucleic acid can be introduced into a cell. Suitable cells include, for example, mammalian cells (such as human cells or CHO cells), fungal cells, yeast cells, insect cells and bacterial cells. When expressed in recombinant cells, the cells are preferably cultured under conditions that allow expression of the polypeptide. If desired, the polypeptide can be recovered from the cell suspension. As used herein, "recovered" means that the mutated polypeptide is removed from those components of the cell or the culture medium present prior to the recovery process. The recovery process can include one or more refolding or purification steps. Methods for isolation and purification commonly used for protein purification can be used for the isolation and purification of the polypeptides described herein and are not limited to any particular method. Polypeptides can be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, metal chelate chromatography, reversed phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). Chromatography can be carried out using liquid phase chromatography (such as HPLC and FPLC).Columns used for affinity chromatography include Protein A and Protein G columns, Capture Select HSA, and heparin Sepharose. Examples of columns using Protein A columns include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences). The present disclosure also encompasses highly purified polypeptides using these purification methods.

[0172] Treatment methods The antibody variable domains described herein can be used to transport cargo (e.g., therapeutic agents) useful for treating and / or preventing a disease or disorder. The therapeutic agent can be, for example, an antibody, an antibody fragment (e.g., Fab), a nanobody, a diabody, a single-chain antibody, a small molecule drug, a peptide, an enzyme, or a nucleic acid (e.g., ASO). In certain instances, the therapeutic agent is directly or indirectly linked to the antibody variable domain. In other instances, the therapeutic agent is encapsulated in a liposome, lipid nanoparticle, or polymer nanocarrier, which is then directly or indirectly linked to the antibody variable domain. By linking a therapeutic agent targeted to the central nervous system to the antibody variable domains described herein, such a fusion can transport the therapeutic agent across the BBB.

[0173] In certain embodiments, the disease or disorder is a neurological disorder. In some instances, the disease or disorder is a tauopathy. In some instances, the disease or disorder is a synucleinopathy.

[0174] In some instances, the disease or disorder is Alzheimer's disease, Parkinson's disease, multiple sclerosis, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), spinal cord injury (SCI), spinal muscular atrophy (SMA), neurological cancer, acute or chronic optic neuritis, amyotrophic lateral sclerosis / Parkinsonism dementia complex, argyrophilic grain dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcification, Down's syndrome, syndrome, frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, subacute sclerosing panencephalitis, dementia with neurofibrillary tangles only, or multi-infarct dementia.

[0175] In other instances, the disease or disorder is selected from the group consisting of stroke, chronic traumatic encephalopathy, traumatic brain injury (TBI), concussion, seizures, epilepsy, or acute lead encephalopathy.

[0176] Exemplary therapeutic antibodies that may be used are disclosed in U.S. Patent Nos. 8,906,367, 9,605,059; 9,598,484; 9,587,014; 9,777,058; 9,567,395; 8,128,926; 8,058,406; 8,048,422; and U.S. Application No. 2017 / 0247471-A1, all of which are incorporated by reference in their entirety herein.

[0177] Exemplary therapeutic ASOs that may be used are disclosed in U.S. Pat. Nos. 8,361,977 and 8,980,853, both of which are incorporated by reference in their entireties.

[0178] The therapeutically effective amount of the composition can be administered to a human subject in need thereof in a dosing regimen that can be ascertained by a person skilled in the art. The dosing frequency for the composition is within the skill and clinical judgment of a physician. Typically, the dosing regime is established by clinical trials that can establish optimal dosing parameters. However, the practitioner can vary such dosing regime according to the age, health, weight, sex, and medical condition of the subject. The dosing frequency can also vary between acute and chronic treatments for a disease or disorder. In addition, the dosing frequency can vary depending on whether the treatment is preventive or therapeutic.

[0179] Assay The present disclosure also features an assay for assessing the instability of antibody variable domains.In certain embodiments, the antibody variable domain is a single domain antibody.In one embodiment, the single domain antibody is FC5 or a variant thereof (e.g., humanized, disulfide stabilized, or humanized and disulfide stabilized).

[0180] The method includes providing adding an antibody variable domain to a serum sample to form a mixture; incubating the mixture; purifying the antibody variable domain; and performing peptide mapping. In some instances, the serum sample is rat serum. In other instances, the serum sample is human serum.

[0181] In certain embodiments, the antibody variable domain is in serum at a concentration of 0.01 to 0.5 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of about 0.1 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.05 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.1 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.2 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.3 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.4 mg / mL. In some embodiments, the antibody variable domain is in serum at a concentration of 0.5 mg / mL.

[0182] In certain cases, the mixture is incubated for 10-100 hours at 25-50° C. In certain cases, the mixture is incubated for about 70 hours at about 37° C. In one embodiment, the mixture is incubated at 37° C. for 70 hours.

[0183] Example 3 of the present disclosure provides a non-limiting method for performing this assay.

[0184] The following examples are provided to more fully illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without the exercise of inventive ability and without departing from the scope of the invention. EXAMPLES

[0185] Example 1: Production of FC5 anti-LINGO-1 antibody (Li81) fusion protein To assess whether a FC5 Li81 bispecific antibody containing FC5 transport activity and Li81 function could be generated, nine different versions, listed in Table 1, were designed, expressed, purified, and characterized. A schematic of the design is shown in the column labeled Structure, and the ratio of FC5 Vhh to Li81 valency in each construct is listed in the column labeled FC5:Li81. The constructs contained one, two, or four FC5 Vhh moieties and one or two Li81 Fabs. In the first four constructs listed, FC5 was added to the N-terminus of the heavy chain, light chain, or both of Li81, using one or three G4S (SEQ ID NO:5) linkers to connect the domains as indicated. In the fifth and sixth constructs, FC5 was added directly to the hinge-Fc of Li81 mAb, and Li81 Fab was fused to the C-terminus of the Fc via its heavy chain. Two different versions of the hinge, designated long and short, were used to connect the FC5Vhh and Fc domains. The four other versions were heterodimers, as indicated by each arm carrying a different functional group. Table 1. List of constructs [Table 1]

[0186] The FC5 Li81 construct was expressed in CHO cells. For preparation of conditioned medium, transfected CHO cells were expanded in serum-free medium, supplemented and grown to high density and shifted to reduced temperature. Cultures were maintained at the reduced temperature for 11-14 days and then harvested by centrifugation and clarified by 0.45 micron filtration.

[0187] For purification, the clarified filtered culture medium was loaded onto a Protein A column and washed with 20 mM NaH2PO4 pH 7.4, 100 mM NaCl and then with 25 mM NaH2PO4 pH 5.5, 100 mM NaCl. FC5-Li81 was eluted from the column with 25 mM Na2HPO4 pH 2.8, 100 mM NaCl pH 2.8 and neutralized with 25 mM Na2HPO4 pH 8.6 diluted from a 0.5 M stock solution. The protein content of the eluted samples was estimated from the absorbance at 280 nm using an extinction coefficient of 1.4 for a 1 mg / mL solution. Figure 3 shows SDS-PAGE analysis of the first three constructs in which FC5 was fused to the N-terminus of Li81 mAb by a 3xG4S (SEQ ID NO:6) linker connecting FC5 to the N-terminus of the HC (ID#3014), LC (ID#3015), or both (ID#3016). This analysis confirms the expression of HC and LC and their assembly into the characteristic HC2LC2 tetrameric complex under non-reducing conditions. The slightly larger size of the protein in lane 3 is consistent with the presence of four copies of FC5 versus two copies in the other samples. Figure 2 shows the amino acid sequences of the heavy and light chains used in the three constructs. Intact mass analysis of reduced heavy chain 3014 (predicted mass 62784.5 Da, observed mass 62783 Da), reduced light chain 3014 (predicted mass 23628.4 Da, observed mass 23628 Da), reduced heavy chain 3015 (predicted mass 48608.9 Da, observed mass 48608 Da), reduced light chain 3015 (predicted mass 37804.0 Da, observed mass 37804 Da), reduced heavy chain 3016 (predicted mass 62784.5 Da, observed mass 62782 Da), and reduced light chain 3016 (predicted mass 37804.0 Da, observed mass 37804 Da) showed that the major components in all samples were the predicted light and heavy chains. The corresponding deconvoluted spectra are shown in Figures 4, 5 and 6. No unexpected modifications of the major components were observed.

[0188] FIG. 7 (left panel) shows SDS-PAGE analysis under non-reducing conditions of six other products described in Table 1. Versions 3438, 3440, 3441 contained the characteristic HC2LC2 dimer structure characteristic of the antibody and were >95% pure after Protein A purification. In contrast, versions 3442, 3443 and 3444 after Protein A purification were highly heterogeneous. They contained the expected mixture of components and would be expected for heteromeric designs reflecting the three different possibilities of generating the desired heteromers and homomers from either arm. Size exclusion chromatography (SEC) was used to enrich the heteromers from the mixture. For SEC fractionation, the Protein A eluate was concentrated and then SEC was performed on a 24 mL Superdex 200 column at a flow rate of 0.5 mL / min in 20 mM Na2HPO4, 150 mM NaCl pH 7.4. Column fractions were collected and analyzed by SDS-PAGE, and those containing the heteromer were pooled, filtered, aliquoted, and stored at -70°C. Figure 7 (right panel) shows the SEC fractionation of sample 3443. The fractions shown in lanes 7 and 8 (indicated by arrows) were pooled to produce the sample shown in lane 7 of the left panel.

[0189] Figure 8 shows the amino acid sequences of the heavy and light chains for the various FC5-(G4S)1-Li81 constructs. Intact mass analysis of reduced heavy chain 3438 (predicted mass 62,153.9 Da, observed mass 62155 Da), reduced heavy chain 3440 (predicted mass 62,897.8 Da, observed mass 62899 Da), reduced heavy chain 3441 (predicted mass 62,167.0 Da, observed mass 62168 Da), reduced heavy chain 3442 (predicted mass 62,897.8 Da, observed mass 62899 Da), reduced heavy chain 3443 (predicted mass 62,167.0 Da, observed mass 62169 Da), reduced heavy chain 3444 (predicted mass 62,167.0 Da, observed mass 62169 Da). All of the constructs contained the Li81 light chain (reduced light chain predicted mass 23,628.4, observed mass 23629 Da). These analyses showed that the major components in all samples contained the predicted light and heavy chains. The corresponding deconvoluted spectrum for the heavy chain is shown in Figure 9. No unexpected modifications of the major components were observed.

[0190] To test whether the FC5 fusions affected Li81 function, samples were analyzed for binding to LINGO-1 in an ELISA format, where 96-well plates were coated with LINGO-1 and then treated with serial dilutions of the FC5-Li81 bispecific. Figure 10 shows the ELISA data for the six FC5-(G4S)1-Li81 constructs. All three bivalent constructs (3438, 3440, 3441) were equipotent with Li81 without FC5 addition, indicating that the addition of FC5 did not affect the binding of the constructs to LINGO-1. In contrast, the three heteromeric versions (3442, 3443, and 3444) containing a single copy of the Li81 Fab showed reduced signal, consistent with the presence of one binding site vs. two binding sites present in the bivalent constructs. The three FC5-(G4S)3-Li81 constructs were also tested in ELISA and were equipotent to Li81 without FC5 addition. Nine samples were also tested for function in the oligodendrocyte differentiation assay and all were active. The EC50s for enhancing differentiation in the potency assay were all approximately 3-fold lower than Li81 without FC5 addition. Samples were characterized by ELISA for activity and in differentiation assays using methods previously described for characterization of the Li81 antibody (Pepinsky et al., Journal of Pharmacology and Experimental Therapeutics, 339:519-529 (2011)).

[0191] The biological activity of the FC5 moiety of the FC5-Li81 sample was assessed against Simian Virus 40-immortalized adult rat brain endothelial cells (SV-ARBEC) in an in vitro BBB transwell cell culture assay. The molecules were co-applied in paired combinations (control and test molecules) to the upper chamber of an in vitro BBB model and quantified by mass spectrometry quantification (MRM) in the bottom compartment, and the P for each was APPValues ​​were calculated over a 90 min period. The (upper chamber) input concentrations of FC5-Li81 ranged from 1.5 to 3 μM (linear phase) and were co-administered with equimolar inputs of various control antibodies. Results from this analysis are shown in FIG. 11. All of the samples showed an increase in FC5-mediated transport compared to the control, with P APP The improvement in P values ​​ranged from 2- to 15-fold. All three bivalent FC5-(G4S)3-Li81 constructs (3014, 3015, 3016) were equipotent, indicating that the addition of FC5, whether on the HC, LC, or both, did not affect the activity of the constructs. All were P APP The most significant was the fold improvement observed with FC5-(G4S)1-Li81(3438), which showed an approximately three-fold improvement in P APP The FC5-(G4S)3-Li81(3015) and FC5-(G4S)1-Li81(3438) constructs differ only in the length of the G4S sequence (SEQ ID NO:5) connecting FC5 with Li81, and both contain FC5 fused to Li81 HC. The FC5-(G4S)1-Li81(3440) construct, in which FC5 is fused to Fc and Li81 Fab is added to the C-terminus of Fc via a (G4S)1 linker, also showed an approximately 15-fold increase in P value, exceeding approximately 5-fold over the corresponding FC5-(G4S)3-Li81(3015). APP This was a large improvement of approximately 12-fold in P APP The 3440 and 3441 heteromeric versions showed only about a six-fold improvement in transport activity compared to the control. Thus, similar to what was seen with the linker length connecting constructs 3015 and 3438, the hinge design in constructs 3440 and 3441 affected the in vitro transport activity of the constructs. Three heteromeric versions (3442, 3443, and 3444) showed improvements ranging from 2- to 5-fold compared to the control. Although the increases in transport were significant, these constructs were not tested in in vivo studies.

[0192] In vitro transwell assays were performed as follows: SV-ARBECs were plated onto rat tail collagen-coated 0.83 cm 2 Falcon cell inserts (1 μm pore size in 1 mL of SV-ARBEC feeding medium without phenol red) were seeded at 80,000 cells / membrane. The wells (i.e., bottom chambers for transport) of 12-well tissue culture plates contained 2 mL of a 50:50 (v / v) mixture of SV-ARBEC medium without phenol red and rat astrocyte-conditioned medium. Characterization of the model is described in detail in Garberg et al., Toxicol In Vitro, 19(3):299-334 (2005). Cells were cultured at 0.4–0.6 [× 10 -3 The model was used when the kinetic energy of the antibody was between 0.01 and 0.1 cm / min. Transport experiments were performed as described in Haqqani et al., Mol. Pharm., 10:1542-1556 (2012) by adding equimolar mixtures of test samples and controls to the top chamber and collecting 100 μl aliquots (subsequently replenished with 100 μl transport buffer) from the bottom chamber at 15, 30, 60 and 90 min using a multiplex SRM-ILIS method for simultaneous quantification of all antibodies. The apparent permeability coefficient Papp was calculated as previously described (Haqqani et al., 2012 (op. cit.)).

[0193] To test whether the FC5-Li81 constructs exhibited BBB transport activity in vivo, constructs 3015, 3438, and 3440 were tested in the Hargreaves model of inflammatory hyperalgesia. In this model, the constructs were chemically conjugated to dalargin, administered by intravenous (IV) injection, and pain responses were measured over time. The methodology for running the model was as previously described (Farrington et al, A novel platform for engineering blood-brain barrier-crossing bispecific biologics, FASEB J., 28(11):4764-78(2014)). The opioid peptide dalargin does not cross the BBB and is not analgesic after systemic administration. As shown in Figure 12, systemic administration of the FC5-Li81 dalargin conjugate induced a significant analgesic response. The data show that dalargin chemically linked to FC5-Li81 can engage central opioid receptors after systemic administration. The AUC values ​​for the three treatments were similar, ranging from 25-35% of the maximum possible effect (MPE). Figure 13A shows the dose-dependence of the response of the three constructs and Li81 as a control in the model. Figure 13B is a plot of constructs 3438 and Li81. Treatment with FC5-Li81 resulted in a 20% MPE as a % of the area under the curve (AUC) at a dose 10-fold lower than for Li81 alone, thus indicating that the addition of FC5 to Li81 improved its ability to cross the blood-brain barrier and engage opioid receptors.

[0194] The FC5-Li81 fusion and Li81 alone were conjugated to the dalargin analog Tyr-dAla-Gly-Phe-Leu-Arg-cysteamide (Dal-Cys) using the general method as described by Mattson et al., Mol. Biol. Rep., 17:167-183 (1993). Briefly, sulfo-succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC; Pierce Rockland, IL, USA) at 10 mg / mL in N,N-dimethylformamide was added in 7.5-fold molar excess to test antibody at 1-2 mg / mL in PBS. The reaction was incubated at room temperature for 2.5 hours, and Dal-Cys in 50 mM MES (pH 6.0) was added in 4-fold molar excess to SMCC. The Dal-labeled protein was concentrated in a centrifuge and desalted to remove unconjugated Dal-Cys and SMCC. The stoichiometry of dalargin / molecule was determined from intact mass spectrometry analysis of the labeled protein. Figure 11 shows the transport activity of dalargin conjugates 3015 and 3016. Under these conditions, conjugation was associated with a significantly increased P compared to the corresponding protein without added dalargin. APP This resulted in a 20-40% decrease in values.

[0195] Assessment of the dalargin conjugates of Li81 and the FC5-Li81 construct provided clear in vivo evidence that fusions of antibodies to FC5 can improve delivery into the central nervous system (CNS), although the duration of the opioid readout lasted only 4 hours. To obtain a more quantitative readout of the transporter and explore the duration of effect, a pharmacokinetic analysis was performed assessing serum and CNS levels by mass spectrometry at 24, 48, and 72 hours after intravenous administration. Figure 14 shows the results from the analysis of Li81 and three FC5-Li81 constructs (3015, 3438, and 3441) at doses of 20, 65, and 200 nmol / kg. All FC5 fusions at all concentrations led to increased CSF levels at 24 hours compared to those observed with Li81 mAb, with a clear dependency of CSF levels on the administered dose. In contrast, at 72 hours, the difference from the control was reduced. Consistent with this, the CSF / serum ratio for the FC5 fusion decreased over time, whereas the CSF / serum ratio for Li81 remained relatively constant at all time points and all doses. These data confirm that FC5 improves delivery of Li81 to the CNS, but with reduced efficacy over time.

[0196] Example 2: Production of FC5-(G4S)1-Li81 FC5-(G4S)1-Li81 Light chain GC058 [ka] Heavy chain pYL945 [ka]

[0197] FC5-(G4S)1-Li81 was expressed in CHO cells. For preparation of conditioned medium, transfected CHO cells from the unsorted pool were expanded in serum-free medium, supplemented and grown to high density and shifted to reduced temperature. Cultures were kept at this reduced temperature for 14 days, then harvested by centrifugation and clarified by 0.45 micron filtration. 1.8 L of the clarified filtered culture medium (estimated titer of FC5-Li81 of 150 mg / L) was loaded onto a 20 mL Protein A column. The column was washed with 10 mM Na2HPO4 pH 7.0, 150 mM NaCl and FC5-(G4S)1-Li81 was eluted from the column with 25 mM Na2HPO4 pH 2.8, 100 mM NaCl pH 2.8 and neutralized with 25 mM NaH2PO4 pH 8.6 diluted from a 0.5 M stock solution. The protein content of the eluted sample was estimated from the absorbance at 280 nm using an extinction coefficient of 1.4 for a 1 mg / mL solution. 290 mg of purified FC5-(G4S)1-Li81 was recovered. The preparation was filtered, aliquoted and stored at -70°C.

[0198] Example 3: Assessment of the stability of FC5(G4S)1-Li81 in rat serum Pharmacokinetic analysis of FC5-(G4S)1-Li81 in rats measuring serum and CSF levels at 24, 48 and 72 hours after intravenous administration at 30 mg / kg revealed that the transporter activity of FC5 decreased over time, being highest in the CSF at the earliest time point tested, 24 hours, with a CSF / serum ratio of 0.45%, decreasing to a CSF / serum ratio of 0.28% at 48 hours, and further decreasing to a CSF / serum ratio of 0.13% after 72 hours. Results from this analysis are presented in Figure 15. Serum levels of FC5-(G4S)1-Li81 at the 72 hour time point were approximately 150 μg / mL, and therefore did not account for the reduction in protein detected in the CSF.

[0199] To determine whether the loss of transport activity resulted from changes in the biochemical or functional properties of FC5-(G4S)1-Li81, three rats were dosed intravenously with 30 mg / kg FC5-(G4S)1-Li81, and the protein was purified and characterized from serum after 72 hours as follows. After 72 hours, the dosed animals were exsanguinated and serum was prepared. Serum was filtered through a 0.45μ syringe driven filter unit. 12 mL of serum pooled from three rats was batch loaded onto 350 μL Ig-Select (GE Healthcare) for 1.5 hours. The resin was collected in a column and washed with 12 column volumes of 20 mM Na2HPO4 pH 7.0, 150 mM NaCl (PBS), 12 column volumes of 25 mM NaH2PO4 pH 5.5, 100 mM NaCl, then eluted with 25 mM NaH2PO4 pH 2.8, 100 mM NaCl pH 2.8, collecting 200 μL fractions. The protein content of the eluted samples was estimated from the absorbance at 280 nm using an extinction coefficient of 1.4 for a 1 mg / mL solution (fractions 4-7 were pooled and neutralized with 25 mM NaH2PO4 pH 8.6 diluted from a 0.5 M stock solution). The 800 μL elution pool had a concentration of 1.9 mg / mL. Recovery was >90% based on the measured concentration of FC5-(G4S)1-Li81 in serum as determined by MRM. Samples were aliquoted, flash frozen on dry ice, and stored at -70°C. As a control for sample processing, 1.2 mg of FC5-(G4S)1-Li81 was spiked into 10 mL of Wistar rat serum (Innovative Research, Inc.) and the sample purification process was carried out as for samples collected from the in vivo study. Figure 16 shows the analysis of the purified samples by SDS-PAGE under reducing and non-reducing conditions. FC5-Li81 heavy chain (HC) 60 kDa and light chain (LC) 25 kDa bands were observed under reducing conditions, and the characteristic 175 kDa tetrameric 2FC5Li81HC-2LC complex was observed under non-reducing conditions.The Li81 sample eluted as a single prominent peak with a molecular weight of 165 kDa and displayed the expected size exclusion chromatography (SEC) elution profile (Figure 17).

[0200] The biological activity of FC5-(G4S)1-Li81 samples was assessed in an in vitro BBB transwell cell culture test with SV-ARBEC cells. The molecules were co-applied in various pairwise combinations (control and test molecules) to the top chamber of an in vitro BBB model, quantified by MRM in the bottom compartment, and Papp values ​​for each were calculated over a 90-minute period. The (top chamber) input concentrations of FC5-Li81 samples were 1.5-3 μM (linear phase) and were co-administered with equimolar inputs of various control antibodies. The results from this analysis are shown in FIG. 18. The transport activity of FC5-(G4S)1-Li81 was not significantly altered after 72 hours in rats.

[0201] The FC5-(G4S)1-Li81 sample was subjected to extensive characterization by mass spectrometry. First, intact mass analysis under reducing and non-reducing conditions showed that the major component in all samples was the predicted protein (FC5-(G4S)1-Li81) (non-reduced predicted mass 171,528.3 Da; actual mass of dosing solution 171530 Da, spiked sample 171530 Da, 72 hour serum sample 171534 Da: predicted mass of reduced FC5Li81 HC 62,153.9 Da; actual mass of dosing solution 62155 Da, spiked sample 62154 Da, 72 hour serum sample 62154 Da: predicted mass of reduced LC 23,628.4 Da; actual mass of dosing solution 23629 Da, spiked sample 23629 Da, 72 hour serum sample 23629 Da). Results from these analyses are shown in Figures 19, 20 and 21. No unexpected modifications of the major components were observed. For these analyses, approximately 20 pmol of each sample was analyzed under non-reducing and reducing (40 mM DTT in 4 M urea and 10 mM EDTA, 37° C. for 1 hour) conditions on a Xevo G2 mass spectrometer using a MassPREP Micro Desalting 20 μm column (2.1 id×5 mm phenyl, Waters) for separation. Molecular weights were generated by deconvolution using the MaxEnt 1 program. Secondly, disulfide structures for all cysteines were determined by peptide mapping with LC / MS detection. Figure 22 shows the reduced and non-reduced peptide maps for the three test samples. Analysis of disulfide-containing peptides showed that all major disulfide bonds were as expected for each sample (Table 2). Table 2. List of major disulfide-bonded peptides in FC5(G4S)1Li81 [Table 2]

[0202] Notably, the presence of the heavy chain C1-C2 disulfide indicates that the FC5 domain is properly folded in the fusion protein. Based on the peak areas from the extracted ion chromatograms (EIC), the analysis showed that the trisulfide level was about 4.5% at the bond between the heavy chain (Cys348) and the light chain (Cys215) and about 1% in the hinge of the heavy chain in the control and spiked samples. No trisulfides were observed in the 72-hour sample, consistent with previously published trisulfide data for Li81 (mAb1-B in Gu et al. 2010. Anal Biochem. 400(1):89-98). The method used for disulfide peptide mapping of the FC5-(G4S)1-Li81 sample is as follows. Approximately 1.4 μL of a 1:10 dilution of 4-vinylpyridine [in 8M guanidine hydrochloride] was added to a solution containing approximately 24 μg (240 pmol, non-reduced) protein, immediately followed by 60 μL of 8M guanidine HCl (final volume was approximately 70 μL). The solution was kept in the dark at room temperature for 45 minutes. The protein in each vial was recovered by precipitation with 40 volumes of cold ethanol. The mixture was stored at −20° C. for 1 hour and then centrifuged at 14000 g for 12 minutes at 4° C. The supernatant was discarded and the precipitate was washed once with cold ethanol. The protein pellet was redissolved with 100 μL of 0.1% trifluoroacetic acid in 50% acetonitrile and dried in a Speed-Vac for 1.5 hours. The samples were analyzed using two different peptide mapping methods, one utilizing endoproteinase Lys-C and trypsin, and the other endoproteinase AspN. For LysC / trypsin maps, approximately 8 μg of 4-vinylpyridine-treated protein in each sample was digested with 5% (w / w) endoproteinase Lys-C (Wako) in 2 M urea, 0.15 M Tris-HCl, pH 6.7, 2 mM CaCl2, 5 mM methylamine for 5 h at room temperature, then 5% (w / w) trypsin (Promega) was added. The digest was kept at room temperature for an additional 4 h, followed by the addition of 5% (w / w) trypsin, then the digest was kept at room temperature overnight.The total volume was 50 μL. For AspN digestion, approximately 4 μg of 4-vinylpyridine-treated protein in each sample was digested with 5% (w / w) Asp-N (Roche) in 2 M urea, 0.15 M Tris-HCl, pH 6.7, 5 mM MgCl2, 5 mM methylamine for 8 h at room temperature, then 5% (w / w) Asp-N was added and the digests were kept overnight. The total volume was 50 μL. Prior to analysis of the digests by LC-MS, each digest was quenched by the addition of 1 μL of 25% trifluoroacetic acid and 4 M urea. Half of the Lys-C / tryptic digest and all of the Asp-N digest were reduced with 100 mM TCEP in 1 mM EDTA for 1 h at room temperature. Aliquots (approximately 4 μg) of the non-reduced and reduced Lys-C / tryptic digests and the reduced Asp-N digests were analyzed on an LC-MS system consisting of a UPLC-Xevo G2-S system. The columns and separation gradients were the same as those described in the peptide mapping section. Disulfide mapping data were processed using BioPharmaLynx 1.3.3.

[0203] The reduced Lys-C / tryptic and Asp-N maps of the 4-vinylpyridine-treated non-reduced samples were examined to assess free thiol levels and to assess whether there were significant differences in the levels of free thiol in the control, spiked, and 72-hour samples. The amount of free thiol was estimated from extracted ion chromatograms (EICs). The analysis revealed that there was a small amount of free thiol in all samples, but none in the FC5 region. Free thiol levels were 2-3 times higher in the 72-hour samples than in the control and spiked samples.

[0204] A systematic comparison of peptide recovery in peptide maps of the 72 h and spiked samples with those of control samples without exposure to rat serum indicated that N-terminal clipping of the FC5-Li81 heavy chain occurs in serum, since peptide recovery gradually decreased starting from the end of the HC variable region (peptide HC 215-249) to the N-terminus (peptide HC 1-19) (Figure 22). Recovery of the N-terminal peptide (HC residues 1-19) was 60%, indicating that 40% of the protein lacked this peptide. Recovery of the next peptide (HC 29-38) was 70%, and that of peptide 51-65 was 80%. In contrast, recovery of peptides from the interior of the Li81 HC was approximately 100%, indicating that clipping was largely restricted within the FC5 portion of the fusion protein. N-terminal clipping was detected in serum spiked samples, but the extent of clipping was much more extensive in the 72 hour samples than in the spiked samples (Figure 23). In contrast, recovery of the light chain peptide was nearly 100% across the sequence for all samples (Figure 24). These findings indicate that the FC5 portion of the fusion protein is unstable in serum and that proteolysis at multiple sites within the FC5 domain leads to low recovery of the N-terminal peptide.

[0205] Example 4: Humanization of FC5 To maintain solubility and stability but reduce the risk of immunogenicity of FC5 in humans, human V HA series of FC5 sequence variants with various degrees of homology to the 3 consensus framework were designed. For all variants, the following nine humanization mutations were included: Q5V, A6E, A14P, A75S, K87R, P88A, D93V, K114Q, and Q117L. Mutations at the remaining six framework positions that distinguish the different humanization designs are listed in Table 3. These include mutations at the corresponding human IgG VH / VL interface (amino acid positions 37, 44, 45, and 47) and at amino acid positions 1 and 79. Partial humanization of the corresponding interface positions is examined in mutations H31 and H32. Finally, the camelid FC5 wild type and human VH / VL interfaces were compared to determine the human VH / VL mutations at positions 37, 44, 45, and 47, respectively. H Both of the three consensus frameworks contain a methionine (M34) in CDR-H1 that may be partially solvent exposed based on computational modeling of FC5 (see below). The exposed methionine may be a liability for developability due to its potential to be modified by oxidation. Therefore, each humanized design was generated with and without the M34T substitution, as shown in Table 3. In odd-numbered constructs, the methionine at position 34 is retained, and in even-numbered constructs, the methionine is replaced by threonine. Table 3: List of amino acid substitutions that distinguish the FC5 humanized designs [Table 3]

[0206] Table 4 shows the complete VFV from the eight humanized FC5 variant designs listed in Table 3. H The H sequence (residue substitutions in bold) and sequences for the short hinge and long hinge hIgG1-Fc agly designs are provided. Table 4: Humanized FC5 variants [Table 4-1] [Table 4-2]

[0207] A computer model of FC5 was generated by combining the assignment of backbone atoms from the NMR study of FC5 (which revealed the structural features of the FC5 framework and CDR-H3) with the analysis of likely conformations for CDRs H1 and H2 using the PyIgClassify web server (http: / / dunbrack2.fccc.edu / PyIgClassify / ). This information was used to guide the selection of X-ray crystal structures as templates from the Protein Data Bank (PDB). The model combined the coordinates for the framework and CDRs H1 and H2 from the crystal structure with the coordinates for CDR-H3 from the NMR study. Models of humanized variant designs H62 and H12 are shown in Figure 25. The corresponding side chain residues on the surface of the human VH / VL interface are indicated.

[0208] FC5 humanized variants were expressed in DG44 CHO cells as hIgG1 Fc fusions (non-glycosylated) with a short hinge linker (KTHTCPPCP (SEQ ID NO:19)) and purified by Protein A Sepharose affinity chromatography. For preparation of conditioned medium, transfected CHO cells from unsorted pools were expanded in serum-free medium, grown to high density with supplements, and shifted to reduced temperature. Cultures were kept at this reduced temperature for 14 days, then harvested by centrifugation and clarified by 0.45 micron filtration. 1.8 L of clarified filtered culture medium was loaded onto a 25 mL Protein A column (MabSelect SuRe; GE). The column was washed with 20 mM Na2HPO4 pH 7.4, 150 mM NaCl and the FC5-hFc molecules were eluted with 25 mM NaH2PO4 pH 2.8, 100 mM NaCl and neutralized with 12.5 mM NaH2PO4 pH 8.6 diluted from a 0.5 M stock solution. The purified proteins were analyzed for size and homogeneity by denaturing microfluidic capillary electrophoresis (LabChip GXII Protein Express Assay; Perkin Elmer). All molecules showed the expected size of the VHH-hFc dimer under non-reducing conditions and the expected size of the corresponding monomer under reducing conditions (Figure 26) without obvious aggregates or proteolytic products. Analysis of the intact mass spectrometry of the reduced samples confirmed the correspondence between the predicted and measured masses for all FC5-hFc humanized variants (within 5 Da; Figure 27).

[0209] Size-exclusion chromatography analysis was performed on a Biosep s3000 7.8×300 mm silica column (Phenomenex) at 0.6 mL / min in 100 mM Na2HPO4, 200 mM NaCl, pH 6.8 for all FC5-hFc variants. All humanized variants eluted as a single peak (FIG. 28), but H11, H12, H31, H32, and H72 had significantly delayed elution times, indicative of interactions with the silica resin. Notably, H31 and H32 showed extensive peak broadening with shallow upward and downward shoulders.

[0210] Additional testing of molecular stability was performed on the FC5-hFc humanized variants. Thermal stability profiles generated by differential scanning calorimetry (VP-DSC, MicroCal) for all variants were similar, with V H The melting temperatures for the H and CH2 domains ranged from 55-62°C and the CH3 domain melting temperature was >90°C (Table 5). Two major melting transitions were observed by DSC at temperatures Tm1 (characterizing the unfolding of the hFc CH2 domain and the FC5 VHH) and Tm2 (characterizing the unfolding of the hFc CH3 domain). Table 5: Thermal stability of FC5-hFc humanized variants [Table 5]

[0211] After 4 months of storage at 4°C, small amounts of fine precipitates were visible in samples of the H11, H12, and H32 variants.

[0212] The biological activity of FC5-hFc humanized variants was assessed in an in vitro BBB transwell cell culture assay using SV-ARBEC cells as described in Example 2. Molecules were co-applied in paired combinations (A20.1 control antibody and test molecule) at 1.25 μM to the top chamber of an in vitro BBB model and quantified by MRM in the bottom compartment. The apparent permeability (P APP ) values ​​were calculated over 60 min. Each test molecule was tested in triplicate transwell cultures. Results from this analysis for FC5-hFc and the humanized variants are shown in Figure 29. All test samples had a concentration of 130-220 x 10 except for the H72 variant. -6 Average P cm / min APP The results showed that FC5-mediated transport of 100–2000 ng / mL FC5-hFc was significantly higher than that of wild-type FC5-hFc (Table 1). -6 H72 showed significantly lower transport activity than the other humanized variants.

[0213] To test whether the FC5-hFc humanized variants exhibit BBB transport activity in vivo, wild-type FC5-hFc and the H32 and H62 variants were tested in the Hargreaves model of inflammatory hyperalgesia. In this model, the constructs were chemically conjugated to dalargin-cysteine, administered by iv injection, and pain responses were measured over time (as described in Example 1). As shown in Figure 30, systemic dosing of the FC5-hFc dalargin conjugate induced a significant analgesic response that did not differ significantly between the wild-type and either the H32 or H62 humanized variants. The integrated analgesic responses (AUC values) for the three molecules were similar, ranging from 20-30% of the maximum possible effect.

[0214] Example 5: Engineering a second disulfide in FC5 to protect against degradation in rat serum A series of nine constructs (Table 6) were designed to stabilize FC5 from degradation by insertion of a second disulfide at the following sites: S49C-I70C, T34C-V79C, and T33C paired with S102C, T103C, A104C, or T105C in FC5 humanized design H62, and S49C-I70C, T34C-V79C, and T33C-T103C in FC5 humanized design H32. H A molecular model of the engineered disulfides in H is shown in Figure 31. In this model, the engineered cysteines are oriented in positions that allow disulfide formation. T34C-V79C creates a disulfide that connects framework region 2 (between CDR1 and CDR2) to framework region 3 (between CDR2 and CDR3), S49C-I70C creates a different disulfide that connects framework regions 2 and 3, and the S33C series creates a disulfide that connects CDRs 1 and 3. Table 6. Exemplary disulfide-stabilized FC5 sequences [Table 6]

[0215] The constructs were expressed in CHO cells and purified by Protein A Sepharose chromatography. Table 7 shows a list of the nine constructs and the purification yields from 250 mL of conditioned medium. Table 7. List of constructs [Table 7]

[0216] Expression levels of the constructs were estimated by Octet at 7-20 mg / L. For purification, the clarified filtered culture medium was loaded onto a 0.5-0.8 mL Protein A column and washed with 5 column volumes of 20 mM Na2HPO4 pH 7.4, 100 mM NaCl and then with 25 mM NaH2PO4 pH 5.5, 100 mM NaCl. FC5-Li81 was eluted from the column with 25 mM NaH2PO4 pH 2.8, 100 mM NaCl pH 2.8 and neutralized with 25 mM Na2HPO4 pH 8.6 diluted from a 0.5 M stock solution. Protein content of the eluted samples was estimated from the absorbance at 280 nm using an extinction coefficient of 1.4 for a 1 mg / mL solution. SDS-PAGE analysis shown in Figure 32 confirmed the expression of HC and LC and their assembly into the characteristic HC2LC2 tetrameric complex under non-reducing conditions. Sample 4669 showed some disulfide disruption reflected by the band heterogeneity (indicated by arrows) seen under non-reducing conditions. All FC5-Li81 samples showed the expected size-exclusion chromatography profile, each eluting as a single prominent peak with a molecular weight of approximately 165 kDa.

[0217] The biological activity of disulfide-stabilized FC5-(G4S)1-Li81 samples was assessed in an in vitro BBB transwell cell culture assay with SV-ARBEC cells as described in Example 2. The molecules were co-applied in paired combinations (anti-HEL control antibody and test molecule) to the upper chamber of the in vitro BBB model and quantified by MRM in the bottom compartment, and the P for each sample was APP Values ​​were calculated over a 90 min period. (Top chamber) Input concentrations were 1.5-3 μM (linear phase) with equimolar input of control antibody co-administered. Results from this analysis for seven of the disulfide-stabilized constructs are shown in Figure 33. All of the test samples had concentrations between 100-130 x 10 -6 Average P cm / min APPThe transport activity of FC5-(G4S)1-Li81 was not significantly altered by the insertion of the second disulfide.

[0218] To assess whether the inserted cysteines / disulfides stabilize the construct against proteolysis, samples were incubated with pepsin at a ratio of 1:30 (pepsin:sample) for 2.5 hours at 37° C. and analyzed by SDS-PAGE. Figures 34 and 35 show the results from this analysis. Pepsin digestion of Li81 (Figure 34 lane 6) led to the formation of the characteristic Fab'2 fragment (100 kDa) and a low molecular weight Fc fragment, which are routinely seen when antibodies are treated with pepsin and are specifically described for the Li81 mAb (Pepinsky et al., J Pharmacol Exp Ther., 339:519-529 (2011)). Pepsin digestion of FC5-H32-G4S-Li81 (Figure 34 lane 8) led to the formation of FC5 V H Pepsin digestion of the FC5-H62-G4S-Li81 construct (Figure 35) also led to a change in the fragmentation pattern of bands of similar molecular weight, as well as to slightly larger fragments consistent with the presence of H. As seen with the Li81 mAb, pepsin treatment of this construct also led to cleavage of the Fc into low molecular weight fragments. In contrast, FC5-H32(T34C / V79C)-G4S-Li81 and FC5-H32(T33C / T103C)-G4S-Li81 showed less cleavage of the band specific for the FC5 domain, indicating a stabilization of FC5. Pepsin digestion of the FC5-H62-G4S-Li81 construct (Figure 35) also led to a change in the fragmentation pattern of bands of similar molecular weight. Notably, FC5-H62(S49C / I70C)-G4S-Li81, FC5-H62(T34C / V79C)-G4S-Li81, FC5-H62(T33C / T103C)-G4S-Li81 and FC5-H62(T33C / A104C)-G4S-Li81 were more stable than FC5-H62(T33C / S102C)-G4S-Li81 and FC5-H62(T33C / T105C)-G4S-Li81. These findings suggest that the addition of a second disulfide prevents the formation of FC5 V from pepsin treatment. HH can be protected, indicating that the location of the added disulfide can affect the degree to which FC5 is stabilized. As an independent measure of stabilization, samples were also analyzed by differential scanning calorimetry for changes in thermal stability. All of the disulfide engineered constructs showed a 12-13° increase in the Tm of the corresponding FC5 transition, ranging from a value of 60 °C for those without stabilization to 72.7-73.2 °C for all of the constructs with engineered disulfides.

[0219] Samples 4662, 4665, 4667, and 4668 were extensively characterized by mass spectrometry and stability assessment in rat serum. First, intact mass analysis of reduced heavy chain 4662 (predicted mass 62,172.0 Da, observed mass 62172 Da), 4665 (predicted mass 62,172.0 Da, observed mass 62175 Da), 4667 (predicted mass 62,170.0 Da, observed mass 62173 Da), 4668 (predicted mass 62,200.0 Da, observed mass 62202 Da) samples, and light chain (predicted mass 23,628.4 Da, observed mass 23630 Da) showed that the major components in all samples were the predicted light and heavy chains. The corresponding deconvoluted spectra are shown in Figures 36 and 37. No unexpected modifications of the major components were observed.

[0220] Serum stability assessment was performed by incubating 4662, 4665, 4667, 4668 samples in rat serum at 37°C for 70 hours, then purifying them on Ig select and performing peptide mapping on them under reducing and non-reducing conditions as described in Example 3. Samples were spiked into Wistar rat serum at 100 μg / mL and purified on Ig-Select at 50 μL resin / mL serum. Each sample was compared to a no serum treatment control. Analysis of the disulfide peptide maps of the control and 70 hour samples of FC5-H62-G4S-Li81 4662, 4665, 4667, and 4668 constructs showed that the major disulfide bonds were all as expected for each construct (Tables 8-11). Table 8. List of major disulfide-bonded peptides in FC5-H62(S49C / I70C)-G4S-Li81(4662) [Table 8] Table 9. List of major disulfide-bonded peptides in FC5-H62(T34C / V79C)-G4S-Li81(4665) [Table 9] Table 10. List of major disulfide-bonded peptides in FC5-H62(T33C / T103C)-G4S-Li81(4667) [Table 10] Table 11. List of major disulfide-bonded peptides in FC5-H62(T33C / A104C)-G4S-Li81(4668) [Table 11]

[0221] In addition, no misbinding was detected for Cys residues in the heavy chain FC5 region and variable region. Analysis showed that constructs 4667 and 4668 contained trisulfide bonds in the FC5 region (i.e., trisulfide bonds in the disulfide-linked peptide cluster containing C1, C2, C3, and C4 [HC20-28 (C1), HC88-108 (C3, C4), HC29-38 (C2)]). The level of trisulfides was about 11% in the 4667 construct and about 6% in the 4668 construct. The trisulfide bond between the heavy chain (Cys348) and the light chain (Cys215) was highest (13-16%) in the 4807 and 4809 constructs (4807=FC5-H32(E1Q)-G4S-Li81; 4809=FC5-H62(D1Q)-G4S-Li81), but was lower (1-4%) in the remaining constructs 4662, 4665, 4667, and 4668. Only a slight decrease in trisulfide levels was observed in samples incubated in Wistar rat serum for 70 hours when compared to the levels in the control. No other Cys modifications were observed at significant levels in any of the samples. The reduced LysC / trypsin maps of the 4-vinylpyridine-treated non-reduced control and the 70 hour 6-pair constructs were also examined to assess whether there were any significant differences in the levels of free thiols in the native samples. Analysis revealed low amounts of free thiols in all samples with no significant differences between the control and 70 hour samples. Trisulfide levels in samples varied due to different culture conditions and therefore reported levels do not reflect product specific attributes.

[0222] Systematic comparison of peptide recovery in reduced peptide maps of 70 hour samples with those in maps of corresponding control samples indicated that addition of a disulfide bridge in the FC5 region of the heavy chain significantly reduced / eliminated the previously observed N-terminal clipping of the heavy chain (see Example 3). Results from these analyses are shown in Figures 38 and 39. The peptide recoveries of the 70-hour samples of FC5-H62 mutant constructs 4667 (T33C=T103C) and 4668 (T33C=A104C), with disulfide bonds between C1 (first Cys) and C3 (third Cys) and C2 (second Cys) and C4 (fourth Cys), were similar to those of the control samples, whereas the peptide recoveries of the 70-hour samples of FC5-H62 mutant constructs 4662 (S49C=I70C) and 4665 (T34C=V79C), with disulfide bonds between C1 and C4 and C2 and C3, were about 85% and 70% of those of the control, respectively. These findings indicate that the addition of the second disulfide in FC5 protects the FC5 portion of the fusion protein from proteolytic degradation in serum, as observed in FIG. 23 for the wild-type FC5 protein.

[0223] The first amino acid in H62 FC5 is an aspartic acid residue. Because antibodies often start with a pyroglutamate and this modification may provide protection from aminopeptidase activity, a construct (4809) was designed that contains the wild-type H62 FC5 sequence but starts with a glutamine (D1Q), which will form an N-terminal pyroglutamate. Interestingly, when this sample was tested for serum stability (Figure 38), it was highly unstable, with the N-terminal peptide showing only 30% recovery relative to the control. The light chain peptide recovery of the 70 hour sample was very similar to that of the corresponding control sample. A plot of the light chain peptide recovery is shown in Figure 39.

[0224] Based on the improved serum stability of H62-T33C / A104C (4D#4668) observed in FIG. 38, a PK analysis in rats was performed measuring serum and CSF levels after iv administration of 10 mg / kg (approximately 65 nmol / kg). Results from this analysis are shown in FIG. 40. Serum and CSF levels for the disulfide stabilized product were higher than the non-stabilized version at all time points. The CSF / serum shown in the bottom panel of FIG. 40 revealed that disulfide engineering prevented the loss in transport activity over time observed with the wild-type construct. Brain levels were also measured at 24 hours by MRM, where the values ​​for FC5, Li81, and hFc reflected the concentration of the signature peptides from each domain (Table 12). Table 12. Analysis of brain levels of FC5-Li81 samples at 24 hours [Table 12]

[0225] Disulfide stabilization led to an approximately 30% increase in brain levels. To test whether other disulfide stabilized variants improve CNS exposure after systemic administration, H62-S49C / I70C (4D#4662), H62-T34C / V79C (4D#4665), H62-T33C / A104C (4D#4668) were tested in parallel assays. Results from this assay are shown in Figures 41 (serum assay) and 42 (CSF and brain tissue assay). All three forms showed higher serum levels than Li81, leading to increased levels in the CNS. These improvements translated into higher CSF and brain levels.

[0226] In analyzing the different framework designs of Example 1, it was observed that both construct 3438 (in which FC5 was added to the N-terminus of Li81) and construct 3440 (in which FC5 was added to the Fc via a long hinge and Li81 Fab was added at the C-terminus of Fc (Table 1, FIG. 11)) showed good transport activity. Following this finding, in addition to the stabilized version of the 3438 construct described above, FC5 was added to the Fc via a long hinge and Li81 Fab was added at the C-terminus of Fc (Table 1, FIG. 11). A disulfide stabilized version of the 3440 construct was engineered containing the H62 T33C / A104C design (Figure 40). PK analysis in rats of both samples shown in Figure 43 revealed that both led to improved CSF levels, with FC5-H62(T33C / A104C)-Fc-G4S-Li81(VH, CH1) (format 3440) showing higher levels over the first 72 hours after IV administration and FC5-H62(T33C / A104C)-G4S-Li81 huIgG1 (format 3438) showing moderate but more sustained CSF levels over the 168 hours after IV administration. Schematics on the left side of the panel show the two engineered designs.

[0227] Further investigation of FC5-H62(T33C / A104C)-G4S-Li81 (in the format of construct 3438) in cynomolgus monkeys demonstrated that the improved CSF exposure of this bispecific antibody compared to monospecific Li81 translates to non-human primates. Monkeys were implanted with lumbar intrathecal catheters for repeated CSF collection, and 12 naive animals per group were administered 65 nmol / kg of antibody test article via a single IV bolus injection. Antibody levels in serum and CSF samples were quantified by nanoLC-MRM after heat denaturation and trypsin digestion, monitoring two peptides for Li81 and three peptides for FC5-H62(T33C / A104C)-G4S-Li81 relative to protein standards in a matching matrix. Results for serum and CSF antibody levels over a 3-week period following IV administration are shown in FIG. 44. Although serum PK was similar for both molecules, a decrease in serum drug levels of FC5-H62(T33C / A104C)-G4S-Li81 compared to Li81 was observed in some animals after day 10 of dosing. This corresponded to the detection of anti-drug antibodies and a reduction in the mean half-life (Table 13). Compared to Li81, CSF levels for FC5-H62(T33C / A104C)-G4S-Li81 were elevated over the first week after IV administration. The CSF / serum ratio was elevated approximately 3-fold for FC5-H62(T33C / A104C)-G4S-Li81 versus Li81 and persisted from day 4 to day 14 after IV administration. Noncompartmental pharmacokinetic analysis was performed using Pheonix 8.1 software and the pharmacokinetic parameters are reported in Table 13. Table 13. PK parameter estimates from non-compartmental analysis of serum and CSF synergy-time profiles of FC5-Li81 and Li81 in cynomolgus monkeys. [Table 13] C max : Peak concentration observed in serum. t 1 / 2 : Serum half-life. AUC 0-τAUC: Area under the serum concentration-time curve from 0 to the last measured concentration. 0-∞ : area under the serum concentration time curve from 0 to infinity. CL: total body clearance. Estimates are reported as mean ± standard deviation.

[0228] To investigate whether a humanized, disulfide-stabilized form of FC5 (FC5-H62(T33C / A104C); also called hFC5.2) could increase brain endothelial cell permeability of other antibodies against brain targets without interfering with their binding to these targets, fusions of hFC5.2 to anti-amyloid-β (12F6A), anti-α-synuclein (12F4), and anti-tau (6C5) antibodies were generated in different formats. Constructs were expressed in CHO and purified by Protein A affinity chromatography as described for the FC5-Li81 fusion. Size exclusion chromatography was used to remove aggregates and free heavy or light chain only contaminants in samples of molecules in which FC5 was fused to the light chain.

[0229] For antibody 12F6A, FC5-H62(T33C / A104C) was fused to the N-terminus of the heavy chain via a G4S linker (SEQ ID NO:5) to form the FC5-H62(T33C / A104C)-12F6A construct. FC5-H62(T33C / A104C)-G4S-12F6A Heavy chain pYL1446 [ka] Light chain pJD010 [ka]

[0230] This fusion showed binding to Aβ very similar to the 12F6A antibody in an ELISA binding assay (Figure 45A). A construct with the 12F6A Fab fused to the C-terminus of hIgG1 Fc showed a significant loss of binding to Aβ, so the FC5-H62(T33C / A104C) fusion was not pursued in this format. FC5-H62(T33C / A104C)-12F6A also showed increased penetration across rat brain endothelial cells in a SVARBEC transwell assay (Figure 45B), with a 4-8 fold higher P than the control antibody (anti-HEL mouse IgG1; Goldbaum et al., J Immunol., 162:6040-6045 (1999); Genbank AF110316 VH and AY277254.1 VL). APP However, 12F6A alone also showed approximately 2-fold higher P values ​​than the control. APP As a result, the P APP Transcytosis in human brain endothelial cells was also observed in a human pluripotent stem cell (hu-iPSC)-derived transwell BBB model (Ribecco-Lutkiewicz et al. al. Sci Rep., 8(1):1873(2018); Fig. 45C), where a significantly larger P APP values ​​were observed (>10-fold versus control antibody; approximately 5-fold versus 12F6A alone).

[0231] For antibody 12F4, the fusion of FC5-H62(T33C / A104C) was made to the N-terminus of the light chain using a single G4S linker (SEQ ID NO:5) to generate FC5-H62(T33C / A104C)-12F4(LC), since it had previously been determined that modification of the N-terminus of the heavy chain slightly reduced binding to α-synuclein. Light chain pYL1593 [ka]

[0232] To investigate whether FC5-based transcytosis is affected by differences in the Fc region, which regulates binding to Fc receptors, FC5-H62(T33C / A104C)-12F4(LC) was co-expressed with the 12F4 heavy chain of wild-type hIgG1 subclass and three scaffolds with reduced effector function: hIgG1 agly(N297Q), hIgG2 SAA (see Vafa et al. Methods. 65(1):114-26 (2014)), and hIgG4P / G1 agly (see US2012 / 0100140A1). 12F4 hIgG1(HC) EVQLVESGGGLVEPGGSLRLSCAVSGFDFEKAWMSWVRQAPGQGLQWVARIKSTADGGTTSYAAPVEGRFIISRDDSRNMLYLQMNSLKTEDTAVYYCTSAHWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 29) 12F4 hIgG1 agly [ka] 12F4 hIgG2 SAA [ka] 12F4 hIgG4P / G1 [ka]

[0233] In addition, a construct with FC5-H62(T33C / A104C) fused to a long hinge hIgG1 Fc and 12F4 Fab via a G4S linkage from the C-terminus of Fc to the 12F4 light chain (FC5-H62(T33C / A104C)-Fc-12F4 Fab(LC) was generated. FC5-H62(T33C / A104C)-Fc-12F4 Fab(LC), pYL1607 [ka] 12F4(VH-CH1)pYL1595 [ka]

[0234] Analysis of these molecules by α-synuclein direct binding ELISA showed that the FC5-H62(T33C / A104C) domain fused directly to the 12F4 light chain did not impair binding to α-synuclein compared to that of 12F4 hIgG1 (Figure 46A). In contrast, the FC5-H62(T33C / A104C)-Fc-12F4 Fab(LC) molecule showed significantly impaired binding to α-synuclein. A series of effector function variants of the FC5-H62(T33C / A104C)-12F4(LC) hIgG molecule were further tested for functional transcytosis in rat brain endothelial cell (SVARBEC) transwell assays (Figure 46B). All molecules showed improved transport regardless of Fc framework, with 4- to 9-fold higher P than the anti-HELmIgG1 control antibody included in each transwell. APP values, whereas 12F4 without added FC5 showed no significant difference from the control in transcellular permeability.

[0235] To identify a bifunctional anti-tau binding antibody with enhanced BBB penetration, FC5-H62(T33C / A104C)-6C5 hIgG1 was generated as both a heavy and a light chain fusion molecule. FC5-H62(T33C / A104C)-G4S-6C5 hIgG1(HC) Heavy chain pYL1611 [ka] Light chain, pEAG2942 [ka] FC5-H62(T33C / A104C)-G4S-6C5(LC) Light chain, pYL1613 [ka] Heavy chain, pEAG2974 [ka]

[0236] Similarly, the alternative format FC5-H62(T33C / A104C)-Fc-6C5 Fab was generated as a heavy and light chain fusion molecule. FC5-H62(T33C / A104C)-Fc-6C5 Fab(HC) Heavy chain, pYL1612 [ka] Light chain, pEAG2942 [ka] FC5-H62(T33C / A104C)-Fc-6C5 Fab(LC) pYL1614 [ka] pYL1596 [ka]

[0237] FC5-H62(T33C / A104C)-6C5 hIgG1 showed similar binding to tau as 6C5 hIgG1 (Figure 47A), whereas the FC5-H62(T33C / A104C)-Fc-6C5 Fab molecule showed significantly reduced tau binding. FC5-H62(T33C / A104C)-6C5(HC)hIgG1 and FC5-H62(T33C / A104C)-6C5(LC)hIgG1 molecules were further tested for transcytosis in a SVARBEC transwell assay (Figure 47B), showing 4-8 fold higher P than the control antibody. APP 6C5 hIgG1 showed a slightly higher P than the control. APP (1.7-fold increase compared to anti-HELm IgG1).

[0238] Pepsin digestion was performed to verify that disulfide engineered FC5 (fused to 12F4 and 12F6A antibodies) has improved molecular stability relative to non-disulfide engineered variants, as shown for disulfide engineered FC5-H62(T33C / A104C)-Li81 (Figure 35). Using FC5-H62(T33C / A104C)-Li81 hIgG1 agly as an example of a molecule with good stability to pepsin digestion and FC5-H32-Li81 hIgG1 agly as an example of a molecule with low stability, these pepsin digests were compared to digests of disulfide engineered FC5-H62(T33C / A104C)-12F4 (on both wild-type and non-glycosylated hIgG1 scaffolds), FC5-H62(T33C / A104C)-12F6A hIgG1, and non-stabilized FC5-H32-12F6A hIgG1 (Figure 48). All protein digestions were performed in 20 mM sodium acetate, pH 3.6, 1 mg / mL and 0.033 mg / mL pepsin, and incubated at 37°C for either 1 or 2.5 hours. All molecules with an additional disulfide (FC5-H62(T33C / A104C) fusions) showed higher molecular weight bands of higher intensity by SDS-PAGE analysis after digestion compared to molecules that were not disulfide engineered (FC5-H32 fusions). A reduction in the lower molecular weight bands (highlighted by arrows in FIG. 48) was observed with the FC5-H32 fusions (a positive control for protease sensitivity without disulfide stabilization).

[0239] Other embodiments Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. In one embodiment, for example, the following items are provided: (Item 1) 1. An antibody variable domain that translocates across the blood-brain barrier, said antibody variable domain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:1, said amino acid sequence comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR2 comprises the sequence set forth in SEQ ID NO:50, and (i) CDR1 comprises the sequence set forth in SEQ ID NO:47 and CDR3 comprises the sequence set forth in SEQ ID NO:51, said amino acid sequence including cysteines at positions corresponding to positions 49 and 70 of SEQ ID NO:1; (ii) CDR1 comprises the sequence set forth in SEQ ID NO:48 and CDR3 comprises the sequence set forth in SEQ ID NO:51, said amino acid sequence comprising a cysteine ​​at a position corresponding to position 79 of SEQ ID NO:1; (iii) CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:52; (iv) CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:52; (v) CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:53; (vi) CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:53; (vii) CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:54; (viii) CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:54; (ix) CDR1 comprises the sequence set forth in SEQ ID NO:49 and CDR3 comprises the sequence set forth in SEQ ID NO:55; or (x) CDR1 comprises the sequence set forth in SEQ ID NO:58 and CDR3 comprises the sequence set forth in SEQ ID NO:55; The antibody variable domain. (Item 2) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises a substitution at one or more of the positions corresponding to positions 1, 5, 6, 14, 37, 44, 45, 47, 75, 87, 88, 93, 114, or 117 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 3) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 5 of SEQ ID NO:1; Glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; A proline at a position corresponding to position 14 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; A tryptophan at a position corresponding to position 47 of SEQ ID NO:1; A serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; A glutamine at a position corresponding to position 114 of SEQ ID NO:1; or A leucine at a position corresponding to position 117 of SEQ ID NO:1, The antibody variable domain according to item 2, comprising one or more of: (Item 4) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises a substitution at one or more of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, or 117 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 5) The amino acid sequence a valine at a position corresponding to position 5 of SEQ ID NO:1; Glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; A proline at a position corresponding to position 14 of SEQ ID NO:1; A serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; A glutamine at a position corresponding to position 114 of SEQ ID NO:1; or A leucine at a position corresponding to position 117 of SEQ ID NO:1, The antibody variable domain according to item 4, comprising one or more of: (Item 6) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 44 and 45 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 7) The amino acid sequence glycine at a position corresponding to position 44 of SEQ ID NO:1; and A leucine at a position corresponding to position 45 of SEQ ID NO:1, 7. The antibody variable domain according to item 6, comprising: (Item 8) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 1, 44, and 45 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 9) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; and A leucine at a position corresponding to position 45 of SEQ ID NO:1, The antibody variable domain according to item 8, comprising: (Item 10) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 44, 45, and 47 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 11) The amino acid sequence glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 11. The antibody variable domain according to item 10, comprising: (Item 12) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 1, 44, 45, and 47 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 13) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 13. The antibody variable domain according to item 12, comprising: (Item 14) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 37, 44, 45, and 47 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 15) The amino acid sequence a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 15. The antibody variable domain according to item 14, comprising: (Item 16) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 1, 37, 44, 45, and 47 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 17) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 17. The antibody variable domain according to item 16, comprising: (Item 18) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 37, 44, 45, 47, and 79 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 19) The amino acid sequence a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; A tryptophan at a position corresponding to position 47 of SEQ ID NO:1; and A leucine at a position corresponding to position 79 of SEQ ID NO:1, 19. The antibody variable domain of item 18, comprising: (Item 20) 2. The antibody variable domain of item 1, wherein the amino acid sequence comprises substitutions at positions corresponding to positions 1, 37, 44, 45, 47, and 79 of SEQ ID NO:1, compared to SEQ ID NO:1. (Item 21) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; A tryptophan at a position corresponding to position 47 of SEQ ID NO:1; and A leucine at a position corresponding to position 79 of SEQ ID NO:1, 21. The antibody variable domain of item 20, comprising: (Item 22) 2. The antibody variable domain according to item 1, wherein the amino acid sequence is any one of SEQ ID NOs: 20 to 28. (Item 23) 1. An antibody variable domain that translocates across the blood-brain barrier, said antibody variable domain comprising an amino acid sequence that is at least 85% identical to the sequence set forth in SEQ ID NO:1, said amino acid sequence comprising: (i) an amino acid substitution at one or more of positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1, as compared to SEQ ID NO:1; (ii) a complementarity determining region (CDR) 1 comprising the sequence set forth in SEQ ID NO:47 or 57, a CDR2 comprising the sequence set forth in SEQ ID NO:50, and a CDR3 comprising the sequence set forth in SEQ ID NO:51; The antibody variable domain comprising: (Item 24) 24. The antibody variable domain of item 23, wherein the amino acid sequence comprises amino acid substitutions, compared to SEQ ID NO:1, at least four of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. (Item 25) 24. The antibody variable domain of item 23, wherein the amino acid sequence comprises amino acid substitutions, compared to SEQ ID NO:1, at least five of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. (Item 26) 24. The antibody variable domain of item 23, wherein the amino acid sequence comprises amino acid substitutions, compared to SEQ ID NO:1, at least six of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. (Item 27) 24. The antibody variable domain of item 23, wherein the amino acid sequence comprises an amino acid substitution, compared to SEQ ID NO:1, at each of the positions corresponding to positions 5, 6, 14, 75, 87, 88, 93, 114, and 117 of SEQ ID NO:1. (Item 28) 24. The antibody variable domain of claim 23, wherein CDR1 comprises the sequence set forth in SEQ ID NO:47. (Item 29) 24. The antibody variable domain of claim 23, wherein CDR1 comprises the sequence set forth in SEQ ID NO:57. (Item 30) The amino acid sequence a valine at a position corresponding to position 5 of SEQ ID NO:1; Glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; A proline at a position corresponding to position 14 of SEQ ID NO:1; A serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; A glutamine at a position corresponding to position 114 of SEQ ID NO:1; or A leucine at a position corresponding to position 117 of SEQ ID NO:1, 30. The antibody variable domain according to any one of items 23 to 29, comprising one or more of: (Item 31) The amino acid sequence a valine at a position corresponding to position 5 of SEQ ID NO:1; Glutamic acid at a position corresponding to position 6 of SEQ ID NO:1; A proline at a position corresponding to position 14 of SEQ ID NO:1; A serine at a position corresponding to position 75 of SEQ ID NO:1; an arginine at a position corresponding to position 87 of SEQ ID NO:1; an alanine at a position corresponding to position 88 of SEQ ID NO:1; a valine at a position corresponding to position 93 of SEQ ID NO:1; A glutamine at a position corresponding to position 114 of SEQ ID NO:1; and A leucine at a position corresponding to position 117 of SEQ ID NO:1, 30. The antibody variable domain according to any one of items 23 to 29, comprising: (Item 32) 32. The antibody variable domain of any one of items 23 to 31, wherein the amino acid sequence comprises a substitution, compared to SEQ ID NO:1, at one or more of the positions corresponding to positions 1, 37, 44, 45, 47, or 79 of SEQ ID NO:1. (Item 33) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; A tryptophan at a position corresponding to position 47 of SEQ ID NO:1; or A leucine at a position corresponding to position 79 of SEQ ID NO:1, 33. The antibody variable domain of claim 32, comprising one or more of: (Item 34) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises, compared to SEQ ID NO:1, substitutions at positions corresponding to positions 44 and 45 of SEQ ID NO:1. (Item 35) The amino acid sequence glycine at a position corresponding to position 44 of SEQ ID NO:1; and A leucine at a position corresponding to position 45 of SEQ ID NO:1, 35. The antibody variable domain of item 34, comprising: (Item 36) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises, compared to SEQ ID NO:1, substitutions at positions corresponding to positions 1, 44 and 45 of SEQ ID NO:1. (Item 37) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; and A leucine at a position corresponding to position 45 of SEQ ID NO:1, 37. The antibody variable domain of item 36, comprising: (Item 38) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises, compared to SEQ ID NO:1, substitutions at positions corresponding to positions 44, 45 and 47 of SEQ ID NO:1. (Item 39) The amino acid sequence glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 39. The antibody variable domain of item 38, comprising: (Item 40) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises, compared to SEQ ID NO:1, substitutions at positions corresponding to positions 1, 44, 45 and 47 of SEQ ID NO:1. (Item 41) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 41. The antibody variable domain according to item 40, comprising: (Item 42) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises substitutions, compared to SEQ ID NO:1, at positions corresponding to positions 37, 44, 45 and 47 of SEQ ID NO:1. (Item 43) The amino acid sequence a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 43. The antibody variable domain of item 42, comprising: (Item 44) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises substitutions, compared to SEQ ID NO:1, at positions corresponding to positions 1, 37, 44, 45 and 47 of SEQ ID NO:1. (Item 45) The amino acid sequence Glutamic acid at a position corresponding to position 1 of SEQ ID NO:1; a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; and A tryptophan at a position corresponding to position 47 of SEQ ID NO:1, 45. The antibody variable domain according to item 44, comprising: (Item 46) 32. The antibody variable domain according to any one of items 23 to 31, wherein the amino acid sequence comprises substitutions, compared to SEQ ID NO:1, at positions corresponding to positions 37, 44, 45, 47 and 79 of SEQ ID NO:1. (Item 47) The amino acid sequence a valine at a position corresponding to position 37 of SEQ ID NO:1; glycine at a position corresponding to position 44 of SEQ ID NO:1; A leucine at a position corresponding to position 45 of SEQ ID NO:1; A tryptophan at a position corresponding to position 47 of SEQ ID NO:1; and A leucine at a position corresponding to position 79 of SEQ ID NO:1, 47. The antibody variable domain according to item 46, comprising: (Item 48) 24. The antibody variable domain according to Item 23, wherein the amino acid sequence is a sequence set forth in any one of SEQ ID NOs: 10 to 17. (Item 49) 41. The antibody variable domain according to claim 40, wherein the amino acid sequence is the sequence set forth in SEQ ID NO:20. (Item 50) 41. The antibody variable domain according to claim 40, wherein the amino acid sequence is the sequence set forth in SEQ ID NO:26. (Item 51) A chimeric molecule comprising the antibody variable domain according to any one of items 1 to 50. (Item 52) 52. The chimeric molecule of item 51, comprising an antibody Fc region. (Item 53) 53. The chimeric molecule according to item 51 or 52, comprising an antibody, an antigen-binding fragment of an antibody, a peptide, an enzyme, an oligonucleotide, a small molecule drug, or a liposome or lipid nanoparticle encapsulating a nucleic acid, a small molecule drug, or a peptide. (Item 54) the antibody variable domain (i) directly, or (ii) via an intervening amino acid sequence, 52. The chimeric molecule of item 51, wherein the chimeric molecule is linked to the N-terminus of the hinge region of the antibody, the hinge region being fused to the Fc portion. (Item 55) 55. The chimeric molecule of item 54, wherein the hinge region comprises the sequence AEPKSCD (SEQ ID NO: 56), AEPKSSD (SEQ ID NO: 59), or KTHTCPPCP (SEQ ID NO: 19). (Item 56) 55. The chimeric molecule of item 54, wherein the intervening amino acid sequence comprises one of: an antibody heavy chain variable region; an antibody light chain variable region; an enzyme; a peptide; or a linker comprising the amino acid sequence 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G. (Item 57) 55. The chimeric molecule according to item 54, wherein the Fc portion is selected from the group consisting of hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P) / G1 agly Fc, hIgG4(S228P)Fc, and hIgG4(S228P)agly Fc. (Item 58) 58. The chimeric molecule according to any one of items 54 to 57, wherein the Fc portion is linked, directly or via a second intervening amino acid sequence, to a polypeptide comprising a second antibody variable domain; a Fab; an scFv; a single domain antibody; a peptide; an enzyme; a nucleic acid; or an oligonucleotide. (Item 59) 55. The chimeric molecule of item 54, wherein the hinge region is linked to a nucleic acid. (Item 60) 60. The chimeric molecule of item 59, wherein the nucleic acid is an antisense oligonucleotide. (Item 61) the antibody variable domain (i) directly, or (ii) via an intervening amino acid sequence, 51. A chimeric molecule comprising the antibody variable domain according to any one of items 1 to 50, linked to a liposome or lipid nanoparticle. (Item 62) 62. The chimeric molecule of item 61, wherein the liposome or lipid nanoparticle encapsulates a small molecule or a nucleic acid. (Item 63) 62. The chimeric molecule of item 61, wherein the liposome or lipid nanoparticle encapsulates a nucleic acid that is an antisense oligonucleotide. (Item 64) 64. The chimeric molecule of item 63, wherein the antisense oligonucleotide is a gapmer or a splice-switching antisense oligonucleotide. (Item 65) the antibody variable domain (i) directly, or (ii) via an intervening amino acid sequence, 51. A chimeric molecule comprising the antibody variable domain of any one of items 1 to 50 linked to a whole antibody. (Item 66) 53. The chimeric molecule of item 51 or 52, further comprising human serum albumin (HSA). (Item 67) (1) A chimeric protein comprising (i) the antibody variable domain according to any one of items 1 to 50 and (ii) a first protein comprising a first hinge region and a first Fc portion of an antibody, wherein the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the first hinge region; (2) a second protein comprising a second hinge region and a second Fc portion of an antibody, the C-terminus of the second hinge region being linked to the N-terminus of the second Fc portion, such that the second protein pairs with the first protein; and (3) therapeutic agents, A composition comprising: (Item 68) (1) A first chimeric protein comprising: (i) a first antibody variable domain according to any one of items 1 to 50; and (ii) a first protein comprising a first hinge region and a first Fc portion of an antibody, wherein the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the first antibody variable domain is fused to the N-terminus of the first hinge region, either directly or via an intervening amino acid sequence; (2) A second chimeric protein comprising: (i) a second antibody variable domain according to any one of items 1 to 50; and (ii) a second protein comprising a second hinge region and a second Fc portion of an antibody, wherein the C-terminus of the second hinge region is linked to the N-terminus of the second Fc portion and the C-terminus of the second antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the second hinge region, and wherein the second protein is paired with the first protein; and (3) therapeutic agents, A composition comprising: (Item 69) (1) A first chimeric protein comprising: (i) a first antibody variable domain according to any one of items 1 to 50; and (ii) a first protein comprising a first hinge region and a first Fc portion of an antibody, wherein the C-terminus of the first hinge region is linked to the N-terminus of the first Fc portion and the C-terminus of the first antibody variable domain is fused to the N-terminus of the first hinge region via a first intervening amino acid sequence; and (2) A second chimeric protein comprising: (i) a second antibody variable domain according to any one of items 1 to 50; and (ii) a second protein comprising a second hinge region and a second Fc portion of an antibody, wherein the C-terminus of the second hinge region is linked to the N-terminus of the second Fc portion and the C-terminus of the second antibody variable domain is fused to the N-terminus of the second hinge region via a second intervening amino acid sequence, and wherein the second protein is paired with the first protein. Includes; The first and second intervening amino acid sequences are Fabs that act as therapeutic agents; composition. (Item 70) 70. The composition according to any one of items 67 to 69, wherein the hinge region comprises the sequence AEPKSCD (SEQ ID NO: 56) or AEPKSSD (SEQ ID NO: 59). (Item 71) 70. The composition according to any one of items 67 to 69, wherein the hinge region comprises the sequence KTHTCPPCP (SEQ ID NO: 19). (Item 72) 70. The composition according to any one of items 67 to 69, wherein the first and the second Fc portions have identical amino acid sequences. (Item 73) 70. The composition according to any one of items 67 to 69, wherein the first and the second Fc portions have different amino acid sequences. (Item 74) 70. The composition according to any one of items 67 to 69, wherein the first and the second Fc moieties are not glycosylated. (Item 75) 70. The composition according to any one of items 67 to 69, wherein the first and the second Fc moieties are selected from the group consisting of hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P)Fc, hIgG4(S228P) / G1 agly Fc, and hIgG4(S228P)agly Fc. (Item 76) 70. The composition according to any one of items 67 to 69, wherein the first and second hinge regions have the same amino acid sequence. (Item 77) 77. The composition according to any one of items 67 to 76, wherein the therapeutic agent is a binding molecule comprising an antibody variable domain. (Item 78) 78. The composition of claim 77, wherein the binding molecule is a Fab and the N-terminus of the VH or VL domain of the Fab is linked to the C-terminus of the first Fc portion. (Item 79) 77. The composition according to any one of items 67 to 76, wherein the therapeutic agent is an antisense oligonucleotide. (Item 80) 80. The composition of claim 79, wherein the antisense oligonucleotide is linked to the first and / or second hinge region. (Item 81) 77. The composition of any one of items 67 to 76, wherein the therapeutic agent is a small molecule drug or a nucleic acid encapsulated in a liposome or lipid nanoparticle. (Item 82) (1) a chimeric protein comprising (i) an antibody variable domain according to any one of items 1 to 50 and (ii) an antibody light chain, wherein the C-terminus of the antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the antibody light chain; and (2) a heavy chain of the antibody that pairs with the light chain of the antibody; A composition comprising: (Item 83) (1) a chimeric protein comprising (i) an antibody variable domain according to any one of items 1 to 50 and (ii) an antibody heavy chain, wherein the C-terminus of the antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the antibody heavy chain; and (2) a light chain of the antibody that pairs with the heavy chain of the antibody; A composition comprising: (Item 84) A composition comprising: (1) a first chimeric protein comprising (i) a first antibody variable domain according to any one of items 1 to 50 and (ii) an antibody light chain, wherein the C-terminus of the first antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody light chain; and (2) a second chimeric protein comprising (i) a second antibody variable domain according to any one of items 1 to 50 and (ii) an antibody heavy chain, wherein the C-terminus of the second antibody variable domain is fused, either directly or via an intervening amino acid sequence, to the N-terminus of the antibody heavy chain. (Item 85) 85. The composition according to any one of items 82 to 84, wherein the intervening amino acid sequence is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G. (Item 86) A composition comprising a chimeric protein comprising: (i) the antibody variable domain according to any one of items 1 to 50; (ii) a protein comprising an antibody hinge region and an Fc portion, wherein the C-terminus of the antibody variable domain is fused directly or via an intervening amino acid sequence to the N-terminus of the hinge region of a protein; and (iii) a therapeutic agent. (Item 87) 87. The composition of claim 86, wherein the composition comprises a dimer of the chimeric protein. (Item 88) 88. The composition of claim 86 or 87, wherein the hinge region comprises the sequence AEPKSCD (SEQ ID NO: 56) or AEPKSSD (SEQ ID NO: 59). (Item 89) 88. The composition of claim 86 or 87, wherein the hinge region comprises the sequence KTHTCPPCP (SEQ ID NO: 19). (Item 90) 90. The composition according to any one of items 86 to 89, wherein the Fc portion is not glycosylated. (Item 91) 90. The composition according to any one of items 86 to 89, wherein the Fc portion is selected from the group consisting of hIgG1 Fc, hIgG2 Fc, hIgG3 Fc, hIgG4 Fc, hIgG1 agly Fc, hIgG2 SAA Fc, hIgG4(S228P) / G1 agly Fc, hIgG4(S228P)Fc, and hIgG4(S228P)agly Fc. (Item 92) 92. The composition of any one of items 86-91, wherein the C-terminus of the antibody variable domain is fused directly to the N-terminus of the hinge region of the protein. (Item 93) 93. The composition according to any one of items 86 to 92, wherein the therapeutic agent is a binding molecule comprising a second antibody variable domain. (Item 94) 88. The composition of claim 86 or 87, wherein the binding molecule is a Fab and the N-terminus of the VH or VL domain of the Fab is linked to the C-terminus of the Fc portion. (Item 95) 93. The composition according to any one of items 86 to 92, wherein the therapeutic agent is an antisense oligonucleotide. (Item 96) 96. The composition of claim 95, wherein the antisense oligonucleotide is a splice-switching oligonucleotide or a gapmer. (Item 97) 97. The composition of claim 95 or 96, wherein the antisense oligonucleotide is linked to the hinge region. (Item 98) 93. The composition of any one of items 86-92, wherein the therapeutic agent is a small molecule or a nucleic acid encapsulated in a liposome or lipid nanoparticle. (Item 99) A pharmaceutical composition comprising the antibody variable domain according to any one of items 1 to 50, the chimeric molecule according to any one of items 51 to 66, or the composition according to any one of items 67 to 98, and a pharma- ceutically acceptable carrier. (Item 100) A nucleic acid encoding the antibody variable domain according to any one of items 1 to 50. (Item 101) A vector comprising the nucleic acid according to item 100. (Item 102) A host cell comprising the vector described in item 101. (Item 103) A method for producing an antibody variable domain, comprising culturing a host cell according to item 102 in a cell culture medium under conditions which result in the expression of said antibody variable domain; and isolating the antibody variable domains from the cell culture; The method comprising: (Item 104) 51. A method of treating Alzheimer's disease in a human subject in need of such treatment, comprising administering to the subject the antibody variable domain of any one of items 1 to 50, which is linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human β-amyloid. (Item 105) 105. The method of claim 104, wherein the intervening amino acid sequence is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G linker. (Item 106) 105. The method of claim 104, wherein the intervening amino acid sequence comprises an antibody hinge region fused to an Fc portion. (Item 107) 106. The method of claim 104 or 105, wherein the antigen-binding fragment is a Fab. (Item 108) 108. The method of any one of items 104 to 107, wherein the antibody or antigen-binding fragment comprises the six CDRs of 12F6A. (Item 109) 108. The method according to any one of items 104 to 107, wherein the antibody or antigen-binding fragment comprises a VH and a VL of 12F6A. (Item 110) 51. A method of treating a synucleinopathy in a human subject in need of such treatment, comprising administering to the subject an antibody variable domain according to any one of items 1 to 50, which is linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human alpha-synuclein. (Item 111) 111. The method of claim 110, wherein the intervening amino acid sequence is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G linker. (Item 112) 111. The method of claim 110, wherein the intervening amino acid sequence comprises an antibody hinge region fused to an Fc portion. (Item 113) 112. The method of claim 110 or 111, wherein the antigen-binding fragment is a Fab. (Item 114) 114. The method of any one of items 110 to 113, wherein the antibody or antigen-binding fragment comprises the six CDRs of 12F4 or 21D11. (Item 115) 114. The method of any one of items 110 to 113, wherein the antibody or antigen-binding fragment comprises a VH and a VL of 12F4 or 21D11. (Item 116) 51. A method of treating a tauopathy in a human subject in need of such treatment, comprising administering to the subject the antibody variable domain of any one of items 1 to 50, which is linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human tau. (Item 117) 117. The method of claim 116, wherein the intervening amino acid sequence is 3x(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G linker. (Item 118) 17. The method of claim 116, wherein the intervening amino acid sequence comprises an antibody hinge region fused to an Fc portion. (Item 119) 118. The method of claim 116 or 117, wherein the antigen-binding fragment is a Fab. (Item 120) 120. The method of any one of items 116-119, wherein the antibody or antigen-binding fragment comprises the six CDRs of 6C5, 40E8, or BMS-986168. (Item 121) 120. The method of any one of items 116 to 119, wherein the antibody or antigen-binding fragment comprises a VH and VL of 6C5, 40E8, or BMS-986168. (Item 122) 51. A method of treating frontotemporal dementia in a human subject in need of such treatment, comprising administering to the subject the antibody variable domain of any one of items 1 to 50, which is linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human TDP-43. (Item 123) 123. The method of claim 122, wherein the intervening amino acid sequence is 3x(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G linker. (Item 124) 123. The method of claim 122, wherein the intervening amino acid sequence comprises an antibody hinge region fused to an Fc portion. (Item 125) 124. The method of claim 122 or 123, wherein the antigen-binding fragment is a Fab. (Item 126) 126. The method of any one of items 122 to 125, wherein the antibody or antigen-binding fragment comprises the six CDRs of 41D1, 14W3, or 51C1. (Item 127) 126. The method of any one of items 122 to 125, wherein the antibody or antigen-binding fragment comprises a VH and VL of 41D1, 14W3, or 51C1. (Item 128) 51. A method of treating multiple sclerosis in a human subject in need of such treatment, comprising administering to the subject an antibody variable domain according to any one of items 1 to 50, which is linked, directly or via an intervening amino acid sequence, to an antibody or antigen-binding fragment thereof that specifically binds human LINGO-1. (Item 129) 129. The method of claim 128, wherein the intervening amino acid sequence is 3×(G4S) (SEQ ID NO: 60), G4S (SEQ ID NO: 5), GG, or G linker. (Item 130) 129. The method of claim 128, wherein the intervening amino acid sequence is a hinge region of an antibody fused to an Fc portion. (Item 131) 130. The method of claim 128 or 129, wherein the antigen-binding fragment is a Fab. (Item 132) 132. The method of any one of items 128 to 131, wherein the antibody or antigen-binding fragment comprises the six CDRs of Li81 or Li113. (Item 133) 132. The method of any one of items 128 to 131, wherein the antibody or antigen-binding fragment comprises a VH and a VL of Li81 or Li113. (Item 134) 51. A method of treating spinal muscular atrophy in a human subject in need of such treatment, comprising administering to the subject the antibody variable domain of any one of items 1 to 50, which is linked to nusinersen, either directly or via an intervening amino acid sequence. (Item 135) Item 135. The method of item 134, wherein the nusinersen is encapsulated in a liposome or lipid nanoparticle. (Item 136) 135. The method of claim 134, wherein the intervening amino acid sequence comprises a hinge region fused to an Fc portion and nusinersen is linked to the hinge region or a non-hinge linkage site. (Item 137) 1. A method for assessing instability of an antibody variable domain, comprising: Providing an antibody variable domain; adding said antibody variable domains to a serum sample to form a mixture; incubating the mixture; purifying the antibody variable domain; and performing peptide mapping; The method comprising: (Item 138) 138. The method of claim 137, wherein the serum sample is rat serum. (Item 139) 139. The method of claim 137 or 138, wherein the antibody variable domain is in serum at a concentration of about 0.1 mg / mL. (Item 140) 139. The method of any one of items 137 to 139, wherein the mixture is incubated at about 37° C. for about 70 hours. (Item 141) 141. The method of claim 140, wherein the antibody variable domain comprises an FC5 variant. (Item 142) 141. The method of claim 140, wherein the antibody variable domain comprises a stabilized form of FC5. (Item 143) 1. A method for screening for a stabilized form of FC5, comprising: providing an antibody variable domain comprising an FC5 variant which differs from SEQ ID NO:1 by one or more amino acids; adding said antibody variable domains to a serum sample to form a mixture; incubating the mixture; purifying the antibody variable domain; performing peptide mapping; and selecting an antibody variable domain that exhibits increased peptide recovery; The method comprising: (Item 144) 144. The method of item 143, wherein the FC5 variant differs from SEQ ID NO: 1 in 1 to 10 amino acids. (Item 145) 145. The method of claim 143 or 144, wherein the serum sample is rat serum. (Item 146) 146. The method of any one of items 143 to 145, wherein the antibody variable domain is in serum at a concentration of about 0.1 mg / mL. (Item 147) 147. The method of any one of items 143 to 146, wherein the mixture is incubated at about 37° C. for about 70 hours. (Item 148) A nucleic acid encoding the chimeric molecule according to any one of items 51 to 66. (Item 149) A vector comprising the nucleic acid according to item 148. (Item 150) A host cell comprising the vector described in item 149. (Item 151) 1. A method for producing a chimeric molecule comprising the steps of: Cultivating the host cell according to item 150 in a cell culture medium under conditions that result in expression of the chimeric molecule; and isolating the chimeric molecule from the cell culture; The method comprising: (Item 152) A nucleic acid encoding the composition according to any one of items 67 to 98. (Item 153) A vector comprising the nucleic acid according to item 152. (Item 154) A host cell comprising the vector according to item 153. (Item 155) 1. A method of producing a composition, comprising: Cultivating the host cell according to item 154 in a cell culture medium under conditions that result in expression of said composition; and isolating said composition from said cell culture; The method comprising: (Item 156) the antibody variable domain (i) directly, or (ii) via an intervening amino acid sequence, 51. A chimeric molecule comprising the antibody variable domain according to any one of items 1 to 50, linked to a viral capsid containing a therapeutic vector. (Item 157) 157. The chimeric molecule of item 156, wherein the viral capsid is AAV.

Claims

[Claim 1] An FC5 single domain antibody that migrates across the blood-brain barrier, wherein the variable domain of the FC5 single domain antibody comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, said amino acid sequence comprising complementarity determining regions CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:49, CDR2 comprises the sequence set forth in SEQ ID NO:50, and CDR3 comprises the sequence set forth in SEQ ID NO:54.

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