β-glucocerebrosidase enzymes, fusion proteins and complexes comprising the same, and methods of use thereof

Abstract

The present disclosure is generally directed GCase polypeptides and fragments thereof as well as fusion proteins and complexes comprising a GCase polypeptide or fragment thereof and an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc). Such fusion proteins and complexes can transport the GCase polypeptide or fragment thereof across the blood-brain barrier (BBB), e.g., for treatment of GCase deficiencies in the central nervous system.

Description

p-GLUCOCEREBROSIDASE ENZYMES, FUSION PROTEINS AND COMPLEXES COMPRISING THE SAME, AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application Nos. 63 / 627,563, filed January 31, 2024; 63 / 660,910, filed June 17, 2024; and 63 / 743,841, filed January 10, 2025, each of which is herein incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing (Name: 4503_029PC03_Sequencelisting_ST26.xml; Size: 411,353 bytes; and Date of Creation: January 27, 2025) is herein incorporated by reference in its entirety.FIELD OF THE PRESENT DISCLOSURE

[0003] The present disclosure relates to P-Glucocerebrosidase (GCase) polypeptides and fusion proteins and complexes for transporting GCase polypeptides across the blood-brain barrier (BBB) as well as uses (e.g., therapeutic uses) of such GCase polypeptides, fusion proteins, or complexes.BACKGROUND

[0004] The P-glucocerebrosidase (GCase) enzyme is an essential lysosomal enzyme that is required for the production of lipid components of the cell membrane and for cellular energy. The GCase enzyme catalyzes hydrolysis of glucosylceramide (GlcCer) and glucosyl sphingosine (GlcSph) into their lipid and glucose components. Mutations in the gene encoding the GCase enzyme, called GBA L can cause reductions of GCase enzymatic activity and / or protein levels, and they are associated with diseases and disorders.

[0005] For example, mutations in GBA1 are associated with a higher risk of Parkinson’s Disease (PD) and faster progression of the disease. PD patients with mutations in GBA1 have higher GCase substrate levels than idiopathic PD patients. In vitro data shows that accumulation of the substrate GlcSph is linked with higher alpha-synuclein aggregation.

[0006] In addition, compound GBA1 mutations can cause a lysosomal storage disorder called Gaucher Disease. Therefore, a need exists for improved therapies to restore normal GCase activity and / or protein levels.SUMMARY OF THE PRESENT DISCLOSURE

[0007] Provided herein are complexes comprising (i) an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) a GCase polypeptide of fragment thereof.

[0008] In some aspects, the complex comprises a first polypeptide comprising the GCase polypeptide or fragment there and a first Fc domain; a second polypeptide comprising a VH of the antigen-binding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigen-binding domain.

[0009] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the first Fc domain.

[0010] In some aspects, the first polypeptide further comprises an antibody hinge between the GCase polypeptide or fragment thereof and the first Fc domain.

[0011] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the second Fc domain.

[0012] In some aspects, the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation.

[0013] In some aspects, the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

[0014] In some aspects, the second polypeptide is an antibody heavy chain.

[0015] In some aspects, the third polypeptide further comprises a CL domain. In some aspects, the third polypeptide is an antibody light chain.

[0016] In some aspects, the first Fc domain comprises a CH2 and a CH3. In some aspects, the second Fc domain comprises a CH2 and a CH3.

[0017] In some aspects, the complex comprises a first polypeptide comprising an antibody heavy chain and a GCase polypeptide or fragment thereof and a second polypeptide comprising an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise the antigen-binding domain.

[0018] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the antibody heavy chain.

[0019] In some aspects, the complex further comprises a third polypeptide comprising a Fc domain.

[0020] In some aspects, the antibody heavy chain comprises a knob mutation and the Fc domain comprises a hole mutation.

[0021] In some aspects, the antibody heavy chain comprises a hole mutation and the Fc domain comprises a knob mutation.

[0022] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0023] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are contained in a single polypeptide.

[0024] In some aspects, the GCase polypeptide or fragment thereof and the antigen-binding domain are directly connected via a peptide bond.

[0025] In some aspects, the GCase polypeptide or fragment thereof and the antigen-binding domain are connected via a linker.

[0026] In some aspects, the linker is a peptide linker.

[0027] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the antigen-binding domain.

[0028] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the antigen-binding domain.

[0029] In some aspects, the complex further comprises an Fc domain.

[0030] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0031] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are both N-terminal to the Fc domain.

[0032] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are both C-terminal to the Fc domain.

[0033] In some aspects, the antigen-binding domain is N-terminal to the Fc domain and the GCase polypeptide or fragment thereof is C-terminal to the Fc domain or fragment thereof.

[0034] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the Fc domain or fragment thereof and the antigen-binding domain is C-terminal to the Fc domain or fragment thereof.

[0035] In some aspects, the Gcase polypeptide or fragment thereof comprises one or more mutations.

[0036] In some aspects, the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of: L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N,Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, H184F, H184L, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, H262N, H262Y, D263N, E272L, E272Q, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, H313N, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, W351C, F355A, L356F, K360A, K360N, M361E, S364A, T369E, N370D, L372N, H374W, V376T, A380C, A380T, N386D, R395K, H404K, 1406 A, I406T, I407C, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, D482C, I483V, D484C, H490K, S494C, H495R, S503C, R534C, R534H, R534N, and combinations thereof as compared to SEQ ID NO:241.

[0037] In some aspects, the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of V78I, N102E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E; H374W, and combinations thereof as compared to SEQ ID NO:241.

[0038] In some aspects, the GCase polypeptide or fragment thereof does not comprise any of the following mutations L34P, K224G, K224N, T369E and N370D relative to SEQ ID NO: 241.

[0039] In some aspects, the GCase polypeptide or fragment thereof comprises the mutations W351C and A380C relative to SEQ ID NO: 241.

[0040] In some aspects, the GCase polypeptide or fragment thereof comprises the sequence of SEQ ID NO:265.

[0041] In some aspects, the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:241 when expressed under the same conditions.

[0042] In some aspects, the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:243 when expressed under the same conditions.

[0043] In some aspects, the GCase polypeptide or fragment thereof has increased protein expression in CHO cells as compared to wildtype human GCase.

[0044] In some aspects, provided herein is a a human P-Glucocerebrosidase (GCase) polypeptide or fragment thereof, comprising one of more of the following mutations relative to SEQ ID NO:241 : V78I, N102E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E; and H374W. In some aspects, provided herein is a complex comprising a human P- Glucocerebrosidase (GCase) polypeptide or fragment thereof, comprising one of more of the following mutations relative to SEQ ID NO:241 : V78I, N102E, I130T, A168S, V191M, R21 IN, Q226T, L241I, S242P, V343T, M361E; and H374W. In some aspects, the human GCasepolypeptide or fragment thereof does not comprise any of the following mutations relative to SEQ ID NO: 241 : L34P; K224N / G, T369E and N370D.

[0045] In some aspects, the GCase polypeptide or fragment thereof comprises the V78I mutation.

[0046] In some aspects, the GCase polypeptide or fragment thereof comprises the N102E mutation.

[0047] In some aspects, the GCase polypeptide or fragment thereof comprises the I130T mutation.

[0048] In some aspects, the GCase polypeptide or fragment thereof comprises the A168S mutation.

[0049] In some aspects, the GCase polypeptide or fragment thereof comprises the VI 9 IM mutation.

[0050] In some aspects, the GCase polypeptide or fragment thereof comprises the R21 IN mutation.

[0051] In some aspects, the GCase polypeptide or fragment thereof comprises the Q226T mutation.

[0052] In some aspects, the GCase polypeptide or fragment thereof comprises the L241I mutation.

[0053] In some aspects, the GCase polypeptide or fragment thereof comprises the S242P mutation.

[0054] In some aspects, the GCase polypeptide or fragment thereof comprises the V343T mutation.

[0055] In some aspects, the GCase polypeptide or fragment thereof comprises the M361E mutation.

[0056] In some aspects, the GCase polypeptide or fragment thereof comprises the H374W mutation.

[0057] In some aspects, provided herein is a GCase polypeptide or fragment thereof, comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs:242, 245, 247, and 267-279. In some aspects, provided herein is a complex comprising a GCase polypeptide or fragment thereof, comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs:242, 245, 247, and 267-279.

[0058] In some aspects, the GCase polypeptide or fragment thereof comprises the amino acid sequence any one of SEQ ID NOs: 242, 245, 247, and 267-279.

[0059] In some aspects, the GCase polypeptide or fragment thereof does not comprise the amino acid sequence of SEQ ID NO: 246.

[0060] In some aspects, the GCase polypeptide or fragment thereof is capable of hydrolyzing glucosylceramide.

[0061] In some aspects, the GCase polypeptide or fragment thereof has greater than 60, 70, 80, or 90% monomer purity after one step of ProA purification.

[0062] In some aspects, the GCase polypeptide or fragment thereof has greater stability in human serum than wildtype human GCase or Cerezyme.

[0063] In some aspects, the GCase polypeptide or fragment thereof has a melting temp (Tm) greater than 60° C.

[0064] In some aspects, the GCase polypeptide or fragment thereof has an aggregation temperature (Tagg) greater than 65° C.

[0065] In some aspects, the complex comprises any of the GCase polypeptides or fragments thereof disclosed herein.

[0066] In some aspects, the antigen binding domain of the complex is a Fab.

[0067] In some aspects, the antigen-binding domain of the complex is an scFv.

[0068] In some aspects, the antigen-binding domain of the complex is a VHH.

[0069] In some aspects, the anti-TfR antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 8, 11, 24; and 40, 55, and 61; respectively;SEQ ID NOs: 8, 11, 25; and 41, 55, and 61; respectively;SEQ ID NOs: 8, 12, 26; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 12, 27; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 13, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 14, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 15, 25; and 43, 55, and 61; respectively;SEQ ID NOs: 8, 16, 25; and 42, 55, and 61; respectively;SEQ ID NOs: 8, 17, 25; and 44, 55, and 61; respectively;SEQ ID NOs: 9, 18, 28; and 45, 56, and 62; respectively;SEQ ID NOs: 9, 19, 28; and 45, 56, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 9, 20, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 9, 21, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 21, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 46, 57, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 9, 22, 28; and 47, 59, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 30; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 31; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 32; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 33; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 34; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 35; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 36; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 37; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 38; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 39; and 46, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 49, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 50, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 51, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 52, 58, and 62; respectively;SEQ ID NOs: 10, 22, 28; and 53, 58, and 62; respectively; or SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively.

[0070] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:SEQ ID NOs: 64 and 129, respectively;SEQ ID NOs: 65 and 130, respectively;SEQ ID NOs: 66 and 131, respectively;SEQ ID NOs: 67 and 130, respectively;SEQ ID NOs: 68 and 131, respectively;SEQ ID NOs: 69 and 130, respectively;SEQ ID NOs: 70 and 131, respectively;SEQ ID NOs: 71 and 130, respectively;SEQ ID NOs: 72 and 131, respectively;SEQ ID NOs: 73 and 130, respectively;SEQ ID NOs: 74 and 131, respectively;SEQ ID NOs: 75 and 132, respectively;SEQ ID NOs: 76 and 131, respectively;SEQ ID NOs: 77 and 132, respectively;SEQ ID NOs: 77 and 133, respectively;SEQ ID NOs: 78 and 134, respectively;SEQ ID NOs: 77 and 135, respectively;SEQ ID NOs: 75 and 136, respectively;SEQ ID NOs: 77 and 137, respectively;SEQ ID NOs: 77 and 138, respectively;SEQ ID NOs: 79 and 131, respectively;SEQ ID NOs: 77 and 139, respectively;SEQ ID NOs: 77 and 131, respectively;SEQ ID NOs: 80 and 140, respectively;SEQ ID NOs: 81 and 141, respectively;SEQ ID NOs: 82 and 131, respectively;SEQ ID NOs: 83 and 142, respectively;SEQ ID NOs: 77 and 143, respectively;SEQ ID NOs: 75 and 131, respectively;SEQ ID NOs: 75 and 144, respectively;SEQ ID NOs: 77 and 145, respectively;SEQ ID NOs: 84 and 131, respectively;SEQ ID NOs: 75 and 146, respectively;SEQ ID NOs: 85 and 131, respectively;SEQ ID NOs: 86 and 138, respectively;SEQ ID NOs: 79 and 139, respectively;SEQ ID NOs: 77 and 147, respectively;SEQ ID NOs: 75 and 148, respectively;SEQ ID NOs: 87 and 131, respectively;SEQ ID NOs: 88 and 131, respectively;SEQ ID NOs: 75 and 149, respectively;SEQ ID NOs: 89 and 150, respectively;SEQ ID NOs: 90 and 151, respectively;SEQ ID NOs: 77 and 152, respectively;SEQ ID NOs: 79 and 153, respectively;SEQ ID NOs: 77 and 139, respectively;SEQ ID NOs: 91 and 131, respectively;SEQ ID NOs: 92 and 131, respectively;SEQ ID NOs: 79 and 154, respectively;SEQ ID NOs: 93 and 155, respectively;SEQ ID NOs: 80 and 131, respectively;SEQ ID NOs: 94 and 131, respectively;SEQ ID NOs: 95 and 131, respectively;SEQ ID NOs: 66 and 156, respectively;SEQ ID NOs: 97 and 138, respectively;SEQ ID NOs: 95 and 156, respectively;SEQ ID NOs: 98 and 157, respectively;SEQ ID NOs: 99 and 157, respectively;SEQ ID NOs: 100 and 157, respectively;SEQ ID NOs: 101 and 157, respectively;SEQ ID NOs: 102 and 158, respectively;SEQ ID NOs: 103 and 157, respectively;SEQ ID NOs: 104 and 159, respectively;SEQ ID NOs: 105 and 160, respectively;SEQ ID NOs: 106 and 161, respectively;SEQ ID NOs: 107 and 162, respectively;SEQ ID NOs: 106 and 163, respectively; SEQ ID NOs: 108 and 164, respectively; SEQ ID NOs. 106 and 165, respectively; SEQ ID NOs: 108 and 166, respectively; SEQ ID NOs: 109 and 165, respectively; SEQ ID NOs: 110 and 167, respectively; SEQ ID NOs: 111 and 168, respectively; SEQ ID NOs: 112 and 160, respectively; SEQ ID NOs: 113 and 169, respectively; SEQ ID NOs: 113 and 170, respectively; SEQ ID NOs: 113 and 171, respectively; SEQ ID NOs: 114 and 169, respectively; SEQ ID NOs: 114 and 171, respectively; SEQ ID NOs: 115 and 169, respectively; SEQ ID NOs: 115 and 170, respectively; SEQ ID NOs: 115 and 171, respectively; SEQ ID NOs: 116 and 169, respectively; SEQ ID NOs: 116 and 170, respectively; SEQ ID NOs: 116 and 171, respectively; SEQ ID NOs: 117 and 170, respectively; SEQ ID NOs: 118 and 170, respectively; SEQ ID NOs: 119 and 170, respectively; SEQ ID NOs: 120 and 170, respectively; SEQ ID NOs: 121 and 170, respectively; SEQ ID NOs: 122 and 170, respectively; SEQ ID NOs: 123 and 170, respectively; SEQ ID NOs: 124 and 170, respectively; SEQ ID NOs: 125 and 170, respectively; SEQ ID NOs: 126 and 170, respectively; SEQ ID NOs: 127 and 170, respectively; SEQ ID NOs: 117 and 173, respectively; SEQ ID NOs: 117 and 174, respectively;SEQ ID NOs: 117 and 175, respectively;SEQ ID NOs: 117 and 176, respectively;SEQ ID NOs: 117 and 177, respectively; orSEQ ID NOs: 117 and 178, respectively.

[0071] In some aspects, the anti-CD98hc antigen-binding domain comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:SEQ ID NOs: 181, 185, 191, 194, 198, and 201; respectively;SEQ ID NOs: 182, 186, 191, 195, 199, and 202; respectively;SEQ ID NOs: 183, 187, 192, 196, 200, and 203; respectively;SEQ ID NOs: 9, 188, 192, 196, 200, and 203; respectively;SEQ ID NOs: 184, 189, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 190, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 232, 193, 197, 198, and 204; respectively;SEQ ID NOs: 184, 233, 193, 197, 198, and 204; respectively; orSEQ ID NOs: 184, 234, 193, 197, 198, and 204; respectively.

[0072] In some aspects, the antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of:SEQ ID NOs: 205 and 211, respectively;SEQ ID NOs: 206 and 212, respectively;SEQ ID NOs: 207 and 213, respectively;SEQ ID NOs: 208 and 214, respectively;SEQ ID NOs: 209 and 215, respectively;SEQ ID NOs: 210 and 216, respectively;SEQ ID NOs: 235 and 216, respectively;SEQ ID NOs: 236 and 216, respectively; orSEQ ID NOs: 237 and 216, respectively.

[0073] In some aspects, the antigen-binding domain specifically binds to human TfR with an affinity of about 751 nM to about 10,000 nM. In some aspects, the antigen-binding domain specifically binds to human TfR with an affinity of about 51 nM to about 750 nM. In someaspects, the antigen-binding domain specifically binds to human TfR with an affinity of about 0.01 nM to about 50 nM.

[0074] In some aspects, the antigen-binding domain specifically binds to human CD98hc with an affinity of about 500 nM to about 10,000 nM. In some aspects, the antigen-binding domain specifically binds to human CD98hc with an affinity of about 100 nM to about 499 nM. In some aspects, the antigen-binding domain specifically binds to human CD98hc with an affinity of less than about 100 nM.

[0075] In some aspects, the affinity is measured using surface plasmon resonance.

[0076] In some aspects, provided herein is a host cell comprising any of the GCase polypeptides or fragments thereof disclosed herein, or any of the complexes disclosed herein.

[0077] In some aspects, provided herein is a composition comprising any of the GCase polypeptides or fragments thereof disclosed herein, or any of the complexes disclosed herein.

[0078] In some aspects, provided herein is a pharmaceutical composition comprising (a) any of the GCase polypeptides or fragments thereof disclosed herein, or any of the complexes disclosed herein and (b) a pharmaceutically acceptable carrier, excipient or stabilizer.

[0079] In some aspects, provided herein is a polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding any of the GCase polypeptides or fragments thereof disclosed herein, or any of the complexes disclosed herein.

[0080] In some aspects, provided herein is a method of transporting a GCase polypeptide or fragment thereof across the blood-brain barrier of a subject comprising administering to the subject any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotide or combination of polynucleotides disclosed herein.

[0081] In some aspects, provided herein is a method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject any of the GCase polypeptides or fragments thereof disclosed herein, any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein.

[0082] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease.

[0083] In some aspects, provided herein is a method of treating a CNS disease or disorder in a subject comprising administering to the subject any of the GCase polypeptides or fragments thereof disclosed herein, any of the complexes disclosed herein, any of the compositionsdisclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein.

[0084] In some aspects, the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia.

[0085] In some aspects, provided herein are any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein for use in any of the methods disclosed herein.

[0086] In some aspects, provided herein is the use of any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein in any of the methods disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1 shows exemplary formats for exemplary fusion proteins and complexes disclosed herein. FIG. 1 includes exemplary 1+1 formats ((i)-(iv)), 2+1 formats ((v)-(vi)), and 2+2 formats ((vii)-(viii)). The 1+1 format in FIG. 1 (i) shows a GCase - Fc domain - anti-BBB scFv fusion protein. The 1+1 format in FIG. 1 (ii) shows an anti-BBB antigen-binding domain (Fv) - Fc domain - GCase complex. The 1+1 format in FIG. 1 (iii) shows an anti-BBB antigenbinding domain (Fv) - Fc region (i.e. a Fc domain in a hole format and a second Fc domain in a knob format) - GCase complex. The 1+1 format in FIG. 1 (iv) shows a complex comprising GCase linked to the N-terminal of a Fc domain in a knob format, and an anti-BBB antigenbinding domain (Fv) linked to the N-terminal of a Fc domain in a hole format. The 2+1 format in FIG. 1 (v) shows a complex comprising an anti-BBB antigen-binding domain (Fv) linked to the N-terminal of a Fc domain in a knob format, a GCase linked to the C-terminal of the Fc domain in the knob format, and a GCase linked to the C-terminal of a Fc domain in a hole format. The 2+1 format in FIG. 1 (vi) shows a complex comprising a GCase linked to the N-terminal of a Fc domain in a knob format, a GCase linked to the N-terminal of a Fc domain in a hole format, and an anti-BBB scFv linked to the C-terminal of the Fc domain in the knob format. The 2+2 format in FIG. 1 (vii) shows a complex comprising an anti-BBB antibody and a GCase linked to the C- terminal of both Fc domains of the anti-BBB antibody. The 2+2 format in FIG. 1 (viii) shows a complex comprising a GCase linked to the N-terminal of a Fc domain in a knob format, an anti- BBB scFv linked to the C-terminal of the Fc domain in the knob format, a GCase linked to the N-terminal of a Fc domain in a hole format, and an anti-BBB scFv linked to the C-terminal of the Fc domain in the hole format.

[0088] FIGs. 2A-2D show formats of GCase-BBB complexes described herein. FIG. 2A shows a complex in a N-terminal monozyme format (or “mono”), where the complex comprises a first polypeptide comprising a GCase linked to the N-terminal of a Fc domain in a knob format; a second polypeptide comprising a VH and CHI of an anti-BBB antigen-binding domain (Fab) linked to the N-terminal end of a Fc domain in a hole format; and a third polypeptide comprising a VL and CL of the anti-BBB antigen-binding domain (Fab). FIG. 2B shows a complex in a C- terminal monozyme (monoval ent-Fc or “mv-Fc”) format, wherein the complex comprises a first polypeptide comprising a GCase linked to the C-terminal of a Fc domain and a VH and CHI of an anti-BBB antigen-binding domain (Fab) linked to the N-terminal of the Fc domain; and a second polypeptide comprising a VL and CL of the anti-BBB antigen-binding domain (Fab). FIG. 2C shows a complex in a C terminal monozyme (Fab knob) format, where the complex comprises a first polypeptide comprising a GCase linked to the C-terminal of a Fc domain in a knob format; a second polypeptide comprising a VH and CHI of an anti-BBB antigen-binding domain (Fab) linked to the N-terminal end of the Fc domain in the knob format; and a third polypeptide comprising a VL and CL of the anti-BBB antigen-binding domain (Fab). FIG. 2D shows a complex in a C terminal monozyme (Fab hole) format, where the complex comprises a first polypeptide comprising a GCase linked to the C-terminal of a Fc domain in a knob format; a second polypeptide comprising a VH and CHI of an anti-BBB antigen-binding domain (Fab) linked to the N-terminal end of a Fc domain in a hole format; and a third polypeptide comprising a VL and CL of the anti-BBB antigen-binding domain (Fab).

[0089] FIGs. 3A-3B show nonspecific binding to baculovirus particles (BVP) of anti-TfR antibodies (H6-4, L7-2, L10-1, L10-8, H3-7, LIO-16, L-35, 42Q, 24A, and 24A_42Q), Isotype control, negative control antibody, and positive control antibody at different concentrations of antibody (FIG. 3 A) and nonspecific binding (BVP) of anti-TfR antibodies (42Q, 24A, L-21, L-6, 39.38, and L-19), negative control antibody, and positive control antibody at different concentrations of antibody (FIG. 3B).

[0090] FIGs. 4A-4B show nonspecific binding to double stranded DNA (dsDNA) of anti-TfR antibodies (H6-4, L7-2, L10-1, L10-8, H3-7, LIO-16, L-35, 42Q, 24A, and 24A_42Q), Isotype control, negative control antibody, and positive control antibody at at 10 pg / mL (FIG. 4 A) andnonspecific binding (dsDNA) of anti-TfR antibodies (42Q, 24A, L-21, L-6, 39.38, and L-19), negative control antibody, and positive control antibody at 10 pg / mL (FIG. 4B).

[0091] FIG. 5 shows cell microscopy of anti-TfR antibodies (H6-4, H3-7, 24A, L7-2, LIO-16, 24A.42Q, L10-1, L-35, L10-8, and 42Q) and isotype control assessing cell uptake in hCMEC / D3 cells.

[0092] FIGs. 6A-6B show brain uptake in vessel depleted brains for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, and 24A_42Q) and isotype control) after 24 hours of 5 mg / kg of antibody is administered in mice with data shown as ng / mg tissue (FIG.6A) and fold-change over isotype control (FIG. 6B).

[0093] FIG. 7 shows the ratio of antibody concentration in vessel depleted brain to whole brain for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, 24A_42Q) and isotype control after 24 hours of 5 mg / kg of antibody is administered in mice.

[0094] FIG. 8 shows absolute reticulocyte (K / pL) and % of reticulocytes in whole blood for anti-TfR antibodies (42Q, H6-4, L7-2, L10-1, L10-8, L-35, 24A, LIO-16, H3-7, 24A 42Q) and isotype control after 24 hours of 5 mg / kg of antibody is administered in mice.

[0095] FIG. 9 shows TfR levels normalized to GAPDH in mice treated with 5 mg / kg of anti- TfR antibodies (42Q, 24A, L-21, L-6, L-35, and 39.38) and isotype control).

[0096] FIGs. 10A-10B shows purity profiles (FIG. 10A) of GCase proteins and activity levels (FIG. 10B) of GCase protein preparations with more and less monomer. GC2 contains the sequence of “dGCl” in Pokorna et al., Design of a stable human acid-P-glucosidase: towards improved Gaucher disease therapy and mutation classification. FEBS J. 2023 Jul; 290(13):3383- 3399 (SEQ ID NO:242). GC3 contains the sequence of “dGC3” in Pokorna et al. (SEQ ID NO:243).

[0097] FIG. 11 shows fluorescent activity of modified GCase proteins as measured by Abeam GC Activity Assay Kit.

[0098] FIG. 12 shows fluorescent activity of modified GCase proteins (constructs GC3 and GC4) after incubation in Human Serum for 1 hour. Cerezyme was used as a control.

[0099] FIGs. 13A-13B show fluorescent activity of modified GCase proteins (constructs GC3 and GC4) after incubation in Human Serum for 10 days. Cerezyme was used as a control. FIG. 13 A shows Cerezyme Activity (Left), GC3 Activity (Center), and GC4 Activity (Right). FIG. 13B shows all three modified GCase protein activities in one graph.

[0100] FIG. 14 shows fluorescent activity of complexes comprising GCase proteins (constructs mTfRl_3GS-wtGC; mTfRl_3GS-GC3; mvFc_PD-GC15v2a_V343T; mvFc PD- GC15v2a_R21 IN; mvFc_PD-GC15v2a_V191M; N-term monozyme wtGC; N-term monozyme GC3; and N-term monozyme WCAC) before and after 3 weeks of storage at -80°C. wtGC = wild-type GCase.

[0101] FIGs. 15A-15B show melting temperatures of GCase proteins as measured by Differential scanning fluorimetry (DSF).

[0102] FIGs. 16A-16B show recovery levels of various GCase-BBB constructs from a small- scale (20 mL) purification study.

[0103] FIG. 17 shows median fluorescence intensity (MFI) values as measured by FACS for cells treated with various GCase complexes (N-Term Mono WT GCase, N-Term Mono GC3, and N-Term Mono WCAC) or GCase proteins (GC3-C4 and WCAC-C4).

[0104] FIG 18 shows GCase variants that were screened in a FACS enzymatic assay in GBA- / - HEK293T cells.

[0105] FIG. 19A shows GCase activity in a rescue assay in Neuro-2A GBA knockout cells with various GCase-BBB-binding complexes (N-term monozyme WCAC mTfRl, N-term monozyme WCAC mTfRl_21, N-term monozyme WCAC mTfRl_33, N-term monozyme WCAC mTfRl_12, and N-term monozyme WCAC mTfRl_44).

[0106] FIG. 19B shows a relationship between anti-TfR dissociation constant as measured by SPR (x-axis) against the concentration required for full GCase activity rescue (y-axis).

[0107] FIG. 20 shows the concentration (nM) of GCase-TfR-binding complexes in serum over time after administering 2.5 mg / kg of N-term monozyme wtGC mTfRl, N-term monozyme GC3 mTfRl, and N-term monozyme WCAC mTfRl.

[0108] FIG. 21 shows GCase activity (AU / minute) over time after administering 2.5 mg / kg of N-term monozyme wtGC mTfRl, N-term monozyme GC3 mTfRl, and N-term monozyme WCAC mTfRl.

[0109] FIG. 22 shows GCase activity in a rescue assay in SH-SY5Y GBA knockout cells with various GCase-TfR-binding complexes (N-term monozyme WCAC hTfR15.WH8.1.42Q, N-term monozyme WCAC hTfR9.1B.39.27.L35, and N-term monozyme WCAC hTfR15.WH8.1.24A) and mFc_PD-WCAC.

[0110] FIG. 23 shows confocal microscopy images of SH-SY-5Y GBA knockout cells showing the GCase-TfR-binding Complex (upper right), early endosomal marker (EEA-1;bottom left), late endosomal / lysosomal marker (LAMP-1; bottom right), and overlay of all markers (upper left).

[0111] FIG. 24 shows the concentration of GCase-TfR-binding complexes in serum over time after administering 10 mg / kg of N-term monozyme WCAC mTfRl, N-term monozyme WCAC mTfR.1 21, and N-term monozyme WCAC mTfRl_33.

[0112] FIG. 25 shows GCase activity for N-term monozyme WCAC mTfRl, N-term monozyme WCAC mTfRl_21, and N-term monozyme WCAC mTfRl_33 normalized to mTfRl-mvFc.

[0113] FIGs. 26A and 26B shows increased brain uptake of anti-CD98hc.04.048.WHl in mice (FIG. 26A) and fold-change compared to isotype control antibody (FIG. 26B).

[0114] FIG. 27 shows immunohistochemistry analysis of brain tissues of mice administered anti-CD98hc.04.048.WHl compared to isotype control antibody.

[0115] FIG. 28 shows no significant changes in red blood cell count in mice following administration of anti-CD98hc.04.048.WHl compared to isotype control antibody.

[0116] FIG. 29A shows increased brain uptake in mice of anti-CD98hc.04.048.WHl antibody variants of the present disclosure.

[0117] FIG. 29B shows increased brain uptake in mice of anti-CD98hc.04.048.WHl antibody variants of the present disclosure, shown as fold-change compared to isotype control antibody.

[0118] FIG. 30 shows no decrease in amino acid uptake in cells treated with anti- CD98hc.04.048.WHl antibody variants of the present disclosure.

[0119] FIG. 31 shows activity of GCase constructs in parenchyma-enriched brain fractions of GB A D409V / D409V homozygous mice. Wild-type mice and GBA D409V / D409V homozygous mice were treated with PBS as controls.

[0120] FIG. 32A and FIG. 32B show GlcSph concentrations in brain (FIG. 32A) and liver (FIG. 32B) after the indicated treatments in GBA D409V / D409V mice. Wild-type mice and GBA D409V / D409V homozygous mice were treated with PBS as controls.

[0121] FIG. 33 shows brain GlcSph levels after treatment with GCase constructs in brain fractions of GBA D409V / D409V homozygous mice at indicated time points after treatment.

[0122] FIGs. 34A and 34B show levels of brain uptake of various anti-CD98hc.04.048.WHl monovalent Fab variants of the present disclosure presented as ng Ab / mg total protein and foldchange over control, respectively.

[0123] FIG. 35A shows serum clearance of antibodies containing an anti- CD98hc.04.048.WHl binding domain following 3 weekly doses in mice.

[0124] FIG. 35B shows increased levels of antibodies containing an anti-CD98hc.04.048.WHl binding domain in vessel-depleted mouse brain fractions compared to that observed with an isotype control antibody.

[0125] FIGs. 36A and 36B show serum pharmacokinetic (PK) measurements of various anti- CD98hc.04.048.WHl antibody variants in mice dosed with 20 mg / kg and 3 mg / kg, respectively.

[0126] FIGs. 36C and 36D show antibody concentrations in vessel-depleted brain samples from mice following administration of various anti-CD98hc.04.048.WHl antibody variants at 20 mg / kg and 3 mg / kg, respectively.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0127] Recombinant GCase polypeptides should theoretically be useful in treating conditions associated with deficiencies in GCase activity. The present disclosure relates to the identification of GCase polypeptides with improved functional properties, such as increased monomer purity. GCase enzymes function in the central nervous system (CNS), and passive transfer of substances from blood to brain is restricted by the blood-brain barrier (BBB). The BBB provides precise control of CNS homeostasis allowing for proper neuronal function and also protecting neural tissue from toxins and pathogens. However, the BBB poses a problem with regard to delivering therapeutics to the CNS. While recombinant proteins and antibody therapeutics have shown much success outside the CNS, such biologies do not cross the BBB efficiently. As a result, delivery of certain therapeutics to the CNS has relied on injection of the therapeutic directly into the CNS; an invasive procedure with limited efficacy due to rapid export of cerebral spinal fluid (CSF) containing the therapeutic from the brain to the blood. Alternatively, a therapeutic intended for the CNS may be administered systemically at doses high enough to allow for sufficient penetration of the BBB by the therapeutic. However, this approach may result in unintended effects due to the high dose in the periphery or increased manufacturing and formulation burdens to achieve the high dose. Accordingly, and in view of the need for improved methods of delivering GCase polypeptides across the BBB where they can function in the CNS, the present disclosure also relates to fusion proteins and complexes comprising GCase polypeptides that are capable of crossing the BBB as a result of binding to transferrin receptor (TfR) or CD98 heavy chain (CD98hc).

[0128] In some aspects, a “1+1 format” comprising a TfR-binding domain refers to a format comprising (i) one antigen-binding domain that binds to TfR (e.g., human) and (ii) a GCase polypeptide or fragment thereof. Such a “1+1” format can also comprise an Fc domain. In some aspects, a “1+1 format” comprising a CD98hc antibody refers to a format comprising (i) an antigen-binding domain that binds to CD98hc (e.g., human) and (ii) a GCase polypeptide or fragment thereof. Such a “1+1” format can also comprise an Fc domain. Exemplary 1+1 formats are shown in parts (i) to (iv) of Fig. 1.

[0129] In some aspects, a “2+1 format” comprising a TfR-binding domain refers to a trivalent format comprising (i) a single antigen-binding domain that binds to TfR (e.g., human) and (ii) two copies of a GCase polypeptide or fragment thereof. Such a “2+1” format can also comprise an Fc domain. In some aspects, a “2+1 format” comprising a CD98hc-binding domain refers to a trivalent format comprising (i) a single antigen-binding domain that binds to CD98hc (e.g., human) and (ii) two copies of a GCase polypeptide or fragment thereof. Such a “2+1” format can also comprise an Fc domain. Exemplary 2+1 formats are shown in parts (v) and (vi) of Fig. 1. In some aspects, a “2+1 format” comprising a TfR-binding domain refers to a trivalent format comprising (i) two antigen-binding domains that bind to TfR (e.g., human) and (ii) a single copy of a GCase polypeptide or fragment thereof. Such a “2+1” format can also comprise an Fc domain. In some aspects, a “2+1 format” comprising a CD98hc-binding domain refers to a trivalent format comprising (i) two antigen-binding domain that bind to CD98hc (e.g., human) and (ii) a single copy of a GCase polypeptide or fragment thereof. Such a “2+1” format can also comprise an Fc domain.

[0130] In some aspects, a “2+2 format” comprising a TfR-binding domain refers to a tetravalent format comprising (i) two antigen-binding domains that bind to TfR (e.g., human) and (ii) two copies of two copies of a GCase polypeptide or fragment thereof. Such a “2+2” format can also comprise an Fc domain. In some aspects, a “2+2 format” comprising a CD98hc-binding domain refers to a tetravalent format comprising (i) two antigen-binding domains that bind to Cd98hc (e.g., human) and (ii) two copies of two copies of a GCase polypeptide or fragment thereof. Such a “2+2” format can also comprise an Fc domain. Exemplary 1+1 formats are shown in parts (vii) and (viii) of Fig. 1.

[0131] The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies such as those described in Sambrook etal. Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000).Definitions

[0132] The terms “central nervous system” or “CNS” refer to the complex of nerve tissues that control bodily function and includes the brain and spinal cord.

[0133] The terms “blood-brain barrier” or “BBB” refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions.

[0134] A “central nervous system antigen” or “CNS antigen” is an antigen expressed in the CNS, including the brain.

[0135] A “brain antigen” is a CNS antigen expressed in the brain.

[0136] The terms “BBB target”, “BBB protein”, “BBB receptor”, and “BBB antigen” refer to a target / protein / receptor / antigen expressed on blood-brain barrier cells (e.g., TfR or CD98hc). In some aspects, an antigen-binding domain (e.g., in an antibody, scFv, or Fab) that binds to the BBB target / protein / receptor / antigen allows transportation of a molecule or compound associated with said antigen-binding domain across the BBB.

[0137] A “neurological disorder” as used herein refers to a disease or disorder which affects the CNS and / or which has an etiology in the CNS. Exemplary CNS diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, an ocular disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, seizure, behavioral disorders, and a lysosomal storage disease.

[0138] A “lysosomal storage disorder” or “LSD” as used herein refers to an inherited metabolic disease characterized by the accumulation of substrates, such as undigested or partially digested macromolecules, in excess in various cells of organs, which ultimately results in cellular dysfunction and clinical abnormalities. LSDs have been defined as deficiencies in lysosomal function generally classified by the accumulated substrate and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, neuronal ceroid lipofuscinoses, and others. LSDs may also include other deficiencies or defects in proteins that result in accumulation of macromolecules, such as proteins necessary for normal post-translational modification of lysosomal enzymes, or proteins important for proper lysosomaltrafficking. LSDs are diseases caused by defects in single genes. Enzyme defects cause nearly seventy percent of the LSDs, and the rest are defects in enzyme activator or associated proteins.

[0139] “Protein replacement therapy” or “PRT” refers to a medical treatment or therapy that supplements or replaces a protein in an individual in whom that particular protein is deficient, non-functional, or absent.

[0140] An “enzyme replacement therapy enzyme” or “ERT enzyme” refers to an enzyme that is deficient in a lysosomal storage disorder or other disorder associated with enzyme deficiency or malfunction. An “ERT enzyme variant” refers to a functional variant, including allelic and splice variants, of a wild-type ERT enzyme or a fragment thereof, where the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or fragment thereof, e.g., when assayed under identical conditions. A “catalytically active fragment” of an ERT enzyme refers to a portion of a full-length ERT enzyme or a variant thereof, where the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or variant thereof, e.g., when assayed under identical conditions.

[0141] The terms “P-Glucocerebrosidase”, “GCase”, “Lysosomal acid glucosylceramidase”, “Acid beta-glucosidase”, or “Beta-glucosylceramidase 1” are used interchangeably herein to refer to any GCase from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full- length,” unprocessed GCase, as well as any form of GCase that results from processing in the cell.

[0142] The terms “Transferrin receptor,” “TfR,” “TfR polypeptide,” and “TfR protein” are used interchangeably herein to refer to any native TfR from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. TfR is also referred to as transferrin receptor protein 1, TR, tfRl, Trfr, T9, and p90. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed TfR, as well as any form of TfR thatresults from processing in the cell. Full-length transferrin receptor protein includes a short N- terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. In some aspects, the TfR is human TfR. As used herein, the term “human TfR” refers to a polypeptide with the following amino acid sequence:MMDQARS AF SNLFGGEPLS YTRF SLARQ VDGDNSHVEMKL AVDEEENADNNTKANVT KPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPA ARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFK LSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHA NFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELS FFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSD WKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPG AAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSL HLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVE KLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEV AGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFR ATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLP ALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 1).

[0143] As used herein, the terms “CD98hc,” “CD98hc polypeptide,” and “CD98hc protein” are used interchangeably herein to refer to any native CD98hc from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys (cynos)) and rodents (e.g., mice and rats), unless otherwise indicated. CD98hc is also referred to as 4F2 cell-surface antigen heavy chain, 4F2hc, 4F2 heavy chain antigen, lymphocyte activation antigen 4F2 large subunit, solute carrier family 3 member 2, and CD98. CD98hc protein is encoded by the SLC3A2 gene and is part of the large amino acid transporter (LAT) complex. In some aspects, the term encompasses both wild-type sequences and naturally occurring variant sequences, e.g., splice variants or allelic variants. In some aspects, the term encompasses “full-length,” unprocessed CD98hc, as well as any form of CD98hc that results from processing in the cell. In some aspects, the CD98hc is human CD98hc. As used herein, the term “human CI)98hc" refers to a polypeptide with the following amino acid sequence:MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPSASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNEL EPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSP GWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAF QGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFD SLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDI ENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGST GEHTKSL VTQ YLNATGNRWC SWSLSQARLLTSFLP AQLLRL YQLMLFTLPGTPVF S YGD EIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQ RSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASAS LPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (SEQ ID NO: 2).

[0144] As used herein, the terms “antibody” and “immunoglobulin” are used interchangeably and refer to an antibody molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing (e.g., a glycoprotein), through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The term “antibody” encompasses monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multi-specific (e.g., bispecific) antibodies, and any other immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy-chain constant regions referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of antibodies have different and well-known subunit structures and three-dimensional configurations. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0145] The terms “anti-TfR antibody,” “antibody that binds to TfR,” and “antibody that specifically binds TfR” refer to an antibody that is capable of binding TfR with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting TfR. In some aspects the anti-TfR antibody is capable of transporting another diagnostic and / or therapeutic agent into the brain. In certain aspects, an anti-TfR antibody binds to an epitope of TfR that is conserved among TfR from different species.

[0146] The terms “anti-TfR antigen-binding domain,” “antigen-binding domain that binds to TfR,” “anti-TfR antigen-binding region,” “antigen-binding region that binds to TfR,” and “TfR binding domain” refer to an antigen-binding domain that binds to TfR with sufficient affinity such that the antigen-binding domain is useful as a diagnostic and / or therapeutic agent in targeting TfR. In one aspect, the extent of binding of an anti-TfR antigen-binding domain to an unrelated, non-TfR polypeptide is less than about 10% of the binding of the antigen-binding domain to TfR as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to TfR has a dissociation constant (KD) of < 20 pM, <15 pM, <12 pM, <10 pM, < 7.5 pM, < 5 pM, <2.5 pM, < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or< 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). In certain embodiments, an anti-TfR antigen-binding domain binds to an epitope of TfR that is conserved among TfR from different species.

[0147] The terms “anti-CD98hc antibody,” “antibody that binds to CD98hc,” and “antibody that specifically binds CD98hc” refer to an antibody that is capable of binding CD98hc with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD98hc. In some aspects, the anti-CD98hc antibody is capable of transporting another diagnostic and / or therapeutic agent into the brain. In certain aspects, an anti-CD98hc antibody binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0148] The terms “anti-CD98hc antigen-binding domain,” “antigen-binding domain that binds to CD98hc,” “anti-CD98hc antigen-binding region,” “antigen-binding region that binds to CD98hc,” and “CD98hc binding domain” refer to an antigen-binding domain that binds to CD98hc with sufficient affinity such that the antigen-binding domain is useful for targeting CD98hc and / or useful as a diagnostic agent, a therapeutic agent, or for transporting a molecule or compound across the BBB. In one aspect, the extent of binding of an anti-CD98hc antigenbinding domain to an unrelated, non-CD98hc polypeptide is less than about 10% of the binding of the antigen-binding domain to CD98hc as measured, e.g., by a radioimmunoassay (RIA). In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of < 0.1 pM,< 1 pM, <10 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., IO’8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M). In certain aspects, an anti- CD98hc antigen-binding domain binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0149] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant regions can be native sequence constant regions (e.g., human native sequence constant regions) or amino acid sequence variants thereof. In some cases, the intact antibody can have one or more effector functions. The C-terminal lysine (residue 447 according to the EU numbering system) of an intact antibody can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of “full-length antibodies,” “intact antibodies,” or “whole antibodies” can comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0150] The term “native IgG antibodies” refers to heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (“L”) chains and two identical heavy (“H”) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[0151] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0152] As used herein, the term “heavy chain” when used in reference to an antibody can refer to any distinct type, e.g., alpha (a), delta (5), epsilon (a), gamma (y), and mu (p), based on the amino acid sequence of the constant region, which give rise to IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgGi, IgG2, IgGs, and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0153] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0154] As used herein, the term “light chain” when used in reference to an antibody can refer to any distinct type, e.g., kappa (K) or lambda (X) based on the amino acid sequence of the constant regions. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0155] The terms “variable region” or “variable domain” refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. Generally, the variable region or variable domain is typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain.

[0156] The term “Fv” or “variable fragment” refers to the minimum antibody fragment which comprises a complete antigen-binding site and consists of a dimer of one heavy-chain variable region (VH) and one light-chain variable region (VL). From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody.

[0157] As used herein, the term “constant region” is a region of an antibody that is not the variable region of the antibody, e.g., a carboxyl terminal portion of a light and / or heavy chain which is not directly involved in binding of an antibody to antigen, but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. In certain aspects, an antibody or antigen-binding fragment comprises a constant region or portion thereof that is sufficient for antibody-dependent cell- mediated cytotoxicity (ADCC).

[0158] A “constant domain” means a domain within a constant region that is capable of forming an immunoglobulin fold. Constant domains include the CHI, CH2, CH3, and CL domains.

[0159] The term “antibody fragment” refers to a portion of an antibody. An “antigen-binding fragment” of an antibody refers to a portion of an antibody that binds to an antigen. An antigenbinding fragment of an antibody can comprise the antigenic determining regions of an antibody(e.g., the complementarity determining regions (CDRs)). Examples of antigen-binding fragments of antibodies include, but are not limited to Fab, Fab’, F(ab’)2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody can be monovalent or multi-valent (e.g., bi-valent). An antigen-binding fragment of an antibody can be monospecific or multi-specific (e.g., bi-specific.) An antigen-binding fragment of an antibody can be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0160] The term “Fab” or “fragment antigen-binding region” refers to a region on an antibody that binds to antigens. It is composed of one constant domain, one variable domain of the heavy chain and one variable domain of the light chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site.

[0161] The term “F(ab’)2 fragment” refers to antibody fragments that are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab’)2 fragments have two antigen-binding F(ab) portions linked together by disulfide bonds, and therefore are divalent with a molecular weight of about 110 kDa. Fab’ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group.

[0162] As used herein, a “Fc fragment,” “fragment crystallizable region,” or “Fc region” is composed of two or more polypeptides, each being an antibody heavy chain fragment and each containing at least one (e.g., two or three) heavy chain constant domains. In some aspects, an Fc region is composed of two heavy chain fragments, each containing a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region can be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, an Fc region may not contain any K447 residues, may contain at least one polypeptide containing a K447 residue and at least one polypeptide that does not contain a K447 residue, or may only contain polypeptides that include a K447 residue. Suitable native-sequence Fc regions for use in the present disclosureinclude human IgGl, IgG2, IgG3 and IgG4. In a native antibody, an Fc region refers to the region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. However, as used herein, an Fc region can be modified to increase, decrease, or eliminate interaction with Fc receptors and / or proteins of the compliment system. In native IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, derived from the second and third constant domains of the antibody’s two heavy chains. However, as used herein, the two or more polypeptides in an “Fc region” do not need to have identical sequences. In some aspects, an “Fc region” comprises a first polypeptide comprising an Fc domain (e.g., IgGl Fc domain) with a knob mutation and a second polypeptide comprising an Fc domain (e.g., IgGl Fc domain) with a hole mutation. In native IgM and IgE antibody isotypes, the Fc region contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain.

[0163] The term “Fc domain” refers to one or more constant region domains within an Fc region, such as a CH2 or CH3 domain, in a single polypeptide. In some aspects, the Fc domain includes at least one amino acid deletion, addition, or substitution as compared to the amino acid sequence of a native Fc domain, such as by including a set of “knob-into-hole” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc domain to favor attractive interactions among different polypeptide chains. In some aspects, the Fc domain is in a “knob” format. In some aspects, the Fc domain is in a “hole” format.

[0164] The term “single-chain Fv”, also abbreviated as “sFv” or “scFv”, refers to antibody fragments that comprise the VH and VL antibody domains that form a single polypeptide chain. In some aspects, the scFv polypeptide comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0165] The term “diabodies” refers to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between the VH and VL domains, such that inter-chain but not intra-chain pairing of the variable domains is achieved, thereby resulting in a bivalent fragment (i.e., a fragment having two antigen-binding sites). Bispecific diabodies are heterodimers of two “crossover” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.

[0166] The term “CDR” or “complementarity determining region” refers to hypervariable regions in the variable region of an immunoglobulin that determine antibody diversity and antigen specificity.

[0167] The term “Kabat numbering” and like terms are recognized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In certain aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two additional amino acids, following 35 (referred to in the Kabat numbering scheme as 35 A and 35B) (CDRH1), amino acid positions 50 to 65 (CDRH2), and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDRL1), amino acid positions 50 to 56 (CDRL2), and amino acid positions 89 to 97 (CDRL3).

[0168] The term “Chothia” refers to the location of the structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol 273: 927- 948; Chothia C et al., (1992) J Mol Biol 227: 799-817; Tramontane A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). In some aspects, the Chothia residues are numbered as shown in the table below.

[0169] The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops and are used by Oxford Molecular’s AbM antibody modeling software.

[0170] In some aspects, the CDRs can be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures.

[0171] The residues from each of these CDRs are noted below.Loop Kabat AbM Chothia ContactLI L24-L34 L24-L34 L26-L32 L30-L36L2 L50-L56 L50-L56 L50-L52 L46-L55L3 L89-L97 L89-L97 L91-L96 L89-L96Hl H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering)H2 H50-H65 H50-H58 H52-H56 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

[0172] CDRs can comprise “extended CDRs” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (Hl), 50-65 or 49-65 (H2), and 93-102, 94- 102, or 95-102 (H3) in the VH. The variable-domain residues are numbered according to Kabat et al., supra, for each of these extended-CDR definitions.

[0173] CDRs can also be identified according to the IMGT numbering system as described in Lefranc M-P, (1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0174] The term “monoclonal” when referring to an antibody or antigen-binding fragment thereof refers to a homogeneous antibody or antigen-binding fragment population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal” antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) mutants, fusion proteins or complexes comprising an antibody or antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, a “monoclonal” antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0175] The term “chimeric” antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof wherein the amino acid sequence is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability, while the constant regions are homologous to the sequences in antibodies or antigen-binding fragments thereof derived from another (usually human) to avoid eliciting an immune response in that species.

[0176] The term “humanized” antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues from the complementarity determining regions (CDRs) are replaced by residues from the CDRs of a molecule originating from a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (“CDR grafted”) (Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)). The humanized antibody or antigen-binding fragment thereof can be further modified by the substitution of additional residues either in the Fv framework region and / or within the replaced non-human residues to refine and optimize the specificity, affinity, and / or capability of the antibody or antigen-binding fragment thereof. In general, the humanized antibody or antigenbinding fragment thereof will comprise VH and VL that comprise substantially all of at least one, and typically two or three, of the CDR regions that correspond to the non-human immunoglobulin, whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody or antigen-binding fragment thereof can also comprise at least a portion of an immunoglobulin constant region or Fc region, typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539; Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996). In some aspects, a “humanized antibody” is a resurfaced antibody.

[0177] The term “human” antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin gene locus, where such antibody or antigen-binding fragment is made using any technique known in the art. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0178] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

[0179] An “acceptor human framework” as used herein is a framework comprising the amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework can comprise the same amino acid sequence thereof, or it can comprise pre-existing amino acid sequence changes. In some aspects, the number of pre-existing amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Where pre-existing amino acid changes are present in a VH, in some aspects those changes occur at only three, two, or one of positions 71H, 73H and 78H; for instance, the amino acid residues at those positions can by 71 A, 73T and / or 78A. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0180] A “human consensus framework” is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for the VL, the subgroup can be subgroup kappa I, kappa II, kappa III or kappa IV as in Kabat et al., supra. Additionally, for the VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0181] An “amino-acid modification” at a specified position, e.g., of an antibody of the present disclosure, refers to the substitution or deletion of the specified residue, or the insertion of at least one amino acid residue adjacent the specified residue. Insertion “adjacent” to a specified residue means insertion within one to two residues thereof. The insertion can be N-terminal or C- terminal to the specified residue. In some aspects, an amino acid modification is a substitution.

[0182] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody and vary with the antibody isotype.

[0183] A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human IgGl Fc region (non- A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region.

[0184] A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, in some aspects two or more amino acid substitutions. In some aspects, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parentpolypeptide, e.g., from about one to about ten amino acid substitutions, and in some aspects from about one to about five amino acid substitutions compared to a native sequence Fc region or in the Fc region of the parent polypeptide. In some aspects, the variant Fc region possesses at least 80% homology with a native sequence Fc region and / or with an Fc region of a parent polypeptide, at least 90% homology therewith, or at least 95% homology therewith.

[0185] “Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. In some aspects, an FcR is a native sequence human FcR. In some aspects, a FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors, FcyRII receptors include FcyRIIA (an “activating receptor”) and FcyRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (“IT AM”) in its cytoplasmic domain. Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (“ITIM”) in its cytoplasmic domain. Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. FcRs can also increase the serum half-life of antibodies.

[0186] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured and / or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of k0ff / k0n, whereas KA is calculated from the quotient of k0n / k0ff. Konrefers to the association rate constant of, e.g., an antibody or antigen-binding fragment thereof to an antigen, and koir refers to the dissociation rate constant of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The konand koir can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA. Dissociation constants may also be determined through any analytical technique, including any biochemical or biophysical technique such as ELISA, surface plasmon resonance (SPR), bio-layer interferometry see, e.g., Octet System by ForteBio),isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), circular dichroism (CD), stopped-flow analysis, and colorimetric or fluorescent protein melting analyses. (See, e.g., Estep et al, (2013) Mabs 5(2):270-8.)

[0187] With regard to the binding of an antibody to a target molecule, the term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of about any of 10'4M or lower, 10'5M or lower, 10'6M or lower, 10'7M or lower, 10'8M or lower, 10'9M or lower, IO'10M or lower, 10'11M or lower, 10'12M or lower or a KD in the range of 10'4M to 10'6M or 10'6M to IO'10M or 10'7M to 10'9M. As will be appreciated by the skilled artisan, affinity and KD values are inversely related. A high affinity for an antigen is measured by a low KD value. In some aspects, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.

[0188] The term “linker” or “linked” refers to the covalent linkage between two polypeptides or two heterologous molecules. In some aspects, a linker is a chemical linker. In some aspects, the linker comprises a peptide bond, and the two polypeptides or two heterologous molecules are linked to each other either directly to or via one or more additional amino acids. A glycine linker is one that comprises one or more glycines, but no other amino acids, e.g., GGGG (SEQ ID NO: 3). A glycine-rich linker is one that comprises one or more glycines and can contain other amino acids as long as glycine is the predominant species in the linker e.g., GGGNGG (SEQ ID NO: 4), wherein N is any amino acid. A glycine-serine linker is one which contains both glycine and serine in any proportion, e.g., GGGS (SEQ ID NO: 5), (G4S)x3 (SEQ ID NO: 179), GGSGG (no repeats) (SEQ ID NO: 180), or (GGSGG)x3 (SEQ ID NO: 217). Similarly, a proline linker is one that comprises one or more prolines but no other amino acids. A proline-rich linker is one thatcomprises one or more prolines and can contain other amino acids so long as proline is the predominant species in the linker.

[0189] As used herein, “percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as identical matches. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full-length of the sequences being compared.

[0190] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0191] As used herein, “substantially pure” refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0192] The term “expression system” refers to one or more nucleic acid molecules comprising coding sequence and control sequence(s) in operable linkage, along with a host cell and / or other in vitro transcription and translation machinery, such that one or more proteins encoded by the nucleic acid molecule(s) are capable of being produced.

[0193] The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterialvectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or simply, “expression vectors.” In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.

[0194] “Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction.

[0195] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention. In some aspects, the host cell is an isolated host cell.

[0196] “ Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed.

[0197] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated”, for example, if one or more symptoms associated with a particular disease, disorder, or condition are mitigated or eliminated.

[0198] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that can be used to deliver a drug, e.g., an anti -human antibody or antigenbinding fragment thereof, to the desired site of biological action.

[0199] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. An effective amount can be provided in one or more administrations. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be or is achieved.

[0200] As used herein, the terms “subject” and “patient” are used interchangeably. The subject can be a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a cynomolgus monkey. In some aspects, the subject is a human.

[0201] As used herein, administration “in conjunction” or “in combination” with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a coformulation or administration as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration. In some aspects, administration in conjunction is administration as a part of the same treatment regimen.

[0202] As used herein, a “complex” refers to one or more proteins comprising connected parts. The parts can be connected e.g., via a peptide bond (e.g., in a fusion protein or complex), a linker (e.g., a peptide linker), or via noncovalent protein-protein interactions such as disulfide bonds (e.g., in an antibody). Exemplary parts that can be included in a complex include a GCase polypeptide or fragment thereof, an antigen-binding domain than specifically binds to CD98hc orTfR, an Fc region, and / or an Fc domain. Accordingly, non-limiting examples of a “complex” comprising an antigen-binding domain and a GCase polypeptide or fragment thereof include (a) a fusion protein or complex comprising the antigen-binding domain and the GCase polypeptide in a single polypeptide chain (e.g., as shown in Figure l(i)), and (b) three proteins connected via noncovalent protein-protein interactions, wherein the first protein contains the GCase polypeptide and an Fc domain, the second protein contains a VH of the antigen-binding domain, and the third protein contains a VL of the antigen-binding domain (e.g., as shown in Figure l(iv)). Formats of other exemplary complexes are provided in Figures 1 and 2.

[0203] As used herein, the term “composition” refers to pharmaceutical combination of components. The components can be connected to each other via covalent bonds or non-covalent bonds or can merely be contained in the same mixture or solution. Accordingly, a “composition” comprising a GCase polypeptide or fragment thereof can refer, e.g., to a fusion protein or complex comprising the GCase polypeptide of fragment thereof or can refer to a mixture or solution comprising such a GCase polypeptide or fragment, fusion protein, or complex. In an additional example, a “composition” comprising an antigen-binding domain can refer, e.g., to an antibody, scFv, or Fab comprising the antigen-binding domain, can refer to a fusion protein or complex comprising the antigen-binding domain, or can refer to a mixture of solution comprising such an antigen-binding domain, antibody, scFv, Fab, fusion protein or complex. In some aspects, a composition is a “pharmaceutical composition.”

[0204] As used herein the term “pharmaceutical composition” refers to a combination comprising an active agent (e.g., a GCase polypeptide or fragment thereof, or fusion protein or complex disclosed herein) with at least one inert pharmaceutically acceptable agent (e.g., an excipient or a carrier).

[0205] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range are also provided.

[0206] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise. For example, reference toan “antibody” is a reference to from one to many antibodies, such as molar amounts, and includes equivalents thereof known to those skilled in the art, and so forth.

[0207] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially of’ are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0208] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0209] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.I. P-Glucocerebrosidase (GCase) Polypeptides and Fragments Thereof

[0210] Human P-Glucocerebrosidase (GCase) is a globular protein of 497 amino acids, composed of three domains. Domain I (residues 1-27 and 383-414 of SEQ ID NO:241) is a small three-stranded antiparallel P-sheet; domain II (residues 30-75 and 431-497 of SEQ ID NO:241) is an independent eight-stranded P-barrel; and domain III (residues 76-381 and 416- 430 of SEQ ID NO:241) is an (a / p) and triose-phosphate isomerase (TIM) barrel, containing the active site of GCase. (Romero et al, Proc Natl Acad Sci U S A. 2019 Mar 12; 116(1 l):5086- 5095).

[0211] Enzyme replacement therapy (ERT) for Gaucher disease (causes by biallelic mutations in the GBA1 gene) has been available via periodic intravenous infusions of a recombinantly expressed GCase, of which Cerezyme is the most commonly administered. However, prior ERT for Gaucher disease has not been able to treat Gaucher types 2 and 3, which cause severe neurological disease due to the inability of prior ERT to cross the blood-brain barrier. In addition, Cerezyme has a lower melting temperature compared to wildtype GCase (see Pokorna et al., Design of a stable human acid-P-glucosidase: towards improved Gaucher disease therapy and mutation classification. FEBS J. 2023 Jul; 290(13):3383-3399 at Figure 2D), leading to difficulties in maintaining a stable and active therapeutic product. As such, there is a need forefficacious GCase ERT therapeutics with optimized stability and manufacturability that can cross the blood-brain barrier.

[0212] Certain aspects of the disclosure are related to GCase polypeptides and fragments thereof. Certain GCase polypeptides and fragments thereof are known in the art. For example, exemplary GCase polypeptides and fragments can be found in PCT Publication Nos. WO 2021 / 199039, WO 2022 / 023761, WO 2020 / 161483, and WO 2024 / 163765 as well as in Pokorna S. et al. Design of a stable human acid-P-glucosidase: towards improved Gaucher disease therapy and mutation classification. FEBS J. 2023 Jul; 290(13):3383-3399. Additional GCase polypeptides and fragments thereof, which can have advantageous properties such as superior enzyme activity, half-life, stability, and / or brain pharmacokinetics, are provided herein.

[0213] In some aspects, the GCase polypeptide or fragment thereof comprises the amino acid sequence corresponding to SEQ ID NO: 241. In some aspects, the GCase protein comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 241. In some aspects, the GCase polypeptide or fragment thereof is encoded by a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 241. In some aspects, the GCase polypeptide or fragment thereof is encoded by a nucleic acid sequence encoding an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 241.

[0214] In some aspects, the GCase polypeptide or fragment thereof comprises the amino acid sequence ofNCBI Ref. Seq. NOs: NP_000148.2, NP_001005741.1, NP_001005742.1,NP_001165282.1, or NP_001165283.1. In some aspects, GCase polypeptide or fragment thereof is encoded by transcripts corresponding to the nucleic acid sequence of NCBI Ref. Seq. NOs: NM_000157.4, NM_001005741.3, NM_001005742.3, NM_001171811.2, or NM_001171812.2. In some aspects, the GCase polypeptide or fragment thereof is derived from the glucosylceramidase beta 1 gene (GBA1'). In some aspects, the glucosylceramidase beta 1 gene corresponds to NCBI Gene ID: 2629. In some aspects, the glucosylceramidase beta 1 gene corresponds to NCBI Ref. Seq. No. NG_009783.1.

[0215] All amino acid substitutions of GCase polypeptides or fragments thereof are described with respect to the numbering of SEQ ID NO:241, unless otherwise specified. For example, the GCase substitution W351C refers to the substitution of tryptophan by cysteine at position 351 of SEQ ID NO:241.

[0216] In some aspects, the GCase polypeptide or fragment thereof comprises at least one substitution. In some aspects, the GCase polypeptide or fragment thereof comprises at least one substitution compared to a wild-type GCase polypeptide, such as a wild-type human GCase polypeptide.

[0217] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution selected from the group consisting of L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, H184F, H184L, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, H262N, H262Y, D263N, E272L, E272Q, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, H313N, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, W351C, F355A, L356F, K360A, K360N, M361E, S364A, T369E, N370D, L372N, H374W, V376T, A380C, A380T, N386D, R395K, H404K, 1406 A, I406T, I407C, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, D482C, I483V, D484C, H490K, S494C, H495R, S503C, R534C, R534H, R534N, or any combination thereof.

[0218] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution comprising at least one of the substitutions L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, H184F, H184L, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, H262N, H262Y, D263N, E272L, E272Q, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, H313N, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, W351C, F355A, L356F, K360A, K360N, M361E, S364A, T369E, N370D, L372N, H374W, V376T, A380C, A380T, N386D, R395K, H404K, 1406 A, I406T, I407C, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, D482C, I483V, D484C, H490K, S494C, H495R, S503C, R534C, R534H, or R534N, or any combinations thereof.

[0219] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution selected from the group consisting of L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, L185M, V191M,S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, D263N, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H374W, V376T, N386D, R395K, I406A, I406T, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L4801, 1483 V, H495R, or any combination thereof.

[0220] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution comprising at least one of the substitutions L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, D263N, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, M361E, S364A, T369E, N370D, L372N, H374W, V376T, N386D, R395K, I406A, I406T, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L4801, 1483 V, or H495R, or any combinations thereof.

[0221] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution selected from the group consisting of H184F, H184L, H262N, H262Y, E272L, E272Q, H313N, W351C, F355A, L356F, K360A, K360N, A380C, A380T, H404K, I407C, D482C, D484C, H490K, S494C, S503C, R534C, R534N, R534H, or any combination thereof.

[0222] In some aspects, the GCase polypeptide or fragment thereof comprises a substitution comprising at least one of the substitutions H184F, H184L, H262N, H262Y, E272L, E272Q, H313N, W351C, F355A, L356F, K360A, K360N, A380C, A380T, H404K, I407C, D482C, D484C, H490K, S494C, S503C, R534C, R534N, or R534H, or any combinations thereof.

[0223] In some aspects, the GCase polypeptide or fragment thereof comprises a combination of substitutions as disclosed in WO2021199039A1 or WO2022023761 A2, each of which is incorporated by reference herein.

[0224] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain I of WT GCase, such as WT human GCase, and does not contain any mutations in Domain II or Domain III of WT GCase.

[0225] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain II of WT GCase, such as WT human GCase, and does not contain any mutations in Domain I or Domain III of WT GCase.

[0226] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain III of WT GCase, such as WT human GCase, and does not contain any mutations in Domain I or Domain II of WT GCase.

[0227] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain I and Domain III of WT GCase, such as WT human GCase, and does not contain any mutations in Domain II of WT GCase.

[0228] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain II and Domain III of WT GCase, such as WT human GCase, and does not contain any mutations in Domain I of WT GCase.

[0229] In some aspects, the GCase polypeptide or fragment thereof comprises one or more substitutions in Domain I and Domain II of WT GCase, such as WT human GCase, and does not contain any mutations in Domain III of WT GCase.

[0230] In some aspects, the GCase polypeptide or fragment thereof comprises no more than 10 amino acid substitutions in Domain I of WT GCase, such as WT human GCase, no more than 10 amino acid substitutions in Domain II of WT GCase, such as WT human GCase, and no more than 5, 4, 3, 2, or 1 substitution in Domain III of WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase, such as WT human GCase.

[0231] In some aspects, the GCase polypeptide or fragment thereof comprises no more than 5 amino acid substitutions in Domain I of WT GCase, such as WT human GCase, no more than 10 amino acid substitutions in Domain II of WT GCase, such as WT human GCase, and no more than 5, 4, 3, 2, or 1 substitution in Domain III of WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase, such as WT human GCase.

[0232] In some aspects, the GCase polypeptide or fragment thereof comprises no more than 2 amino acid substitutions in Domain I of WT GCase, such as WT human GCase, no more than 10 amino acid substitutions in Domain II of WT GCase, such as WT human GCase, and no more than 5, 4, 3, 2, or 1 substitution in Domain III of WT GCase, such as WT human GCase. In someaspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase, such as WT human GCase.

[0233] In some aspects, the GCase polypeptide or fragment thereof comprises no more than 1 amino acid substitution in Domain I of WT GCase, such as WT human GCase, no more than 7 amino acid substitutions in Domain II of WT GCase, such as WT human GCase, and no more than 5, 4, 3, 2, or 1 amino acid substitutions in Domain III of WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase, such as WT human GCase.

[0234] In some aspects, the GCase polypeptide or fragment thereof comprises no more than 20 amino acid substitutions as compared to a corresponding region of WT GCase, such as WT human GCase. In some aspects, the Gcase polypeptide or fragment thereof comprises no more than 15 amino acid substitutions as compared to a corresponding region of WT GCase, such as WT human GCase. In some aspects, the Gcase polypeptide or fragment thereof comprises no more than 10 amino acid substitutions as compared to a corresponding region of WT GCase, such as WT human GCase. In some aspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase.

[0235] In some aspects, the Gcase polypeptide or fragment thereof comprises 5-20 amino acid substitutions as compared to a corresponding region of WT GCase such as WT human GCase. In some aspects, the Gcase polypeptide or fragment thereof comprises 5-15 amino acid substitutions as compared to a corresponding region of WT GCase such as WT human GCase. In some aspects, the Gcase polypeptide or fragment thereof comprises 5-10 amino acid substitutions as compared to a corresponding region of WT GCase such as WT human GCase. In some aspects, the GCase polypeptide or fragment thereof comprises a V78I substitution as compared to WT GCase. In some aspects, the GCase polypeptide of fragment thereof comprises a W351C and / or A380C substitution as compared to WT GCase.

[0236] In some aspects, the human GCase polypeptide or fragment thereof comprises one or more substitutions relative to SEQ ID NO:241 selected from the group consisting of V78I, N102E, L103E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E, H374W, or any combination thereof. In some aspects, the human GCase polypeptide or fragment thereof comprises one or more substitutions relative to SEQ ID NO:241 comprising one or more of V78I, N102E, L103E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E, H374W, or any combination thereof. In some aspects, the human GCase polypeptide or fragment thereof comprises no more than 5 mutations relative to SEQ ID NO:241 selected from the group consisting of V78I, N102E, L103E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E, and H374W. In some aspects, the human GCase polypeptide or fragment thereof comprises no more than 4 mutations relative to SEQ ID NO:241 selected from the group consisting of V78I, N102E, L103E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E, and H374W. In some aspects, the human GCase polypeptide or fragment thereof comprises no more than 3 mutations relative to SEQ ID NO:241 selected from the group consisting of V78I, N102E, L103E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E, and H374W.

[0237] In some aspects, the human GCase polypeptide or fragment thereof does not comprise any of the following mutations relative to SEQ ID NO: 241 : L34P; K224N / G, T369E and N370D.

[0238] In some aspects, the GCase polypeptide or fragment thereof comprises the substitutions corresponding to W312C, A341C, and H495R.

[0239] In some aspects, the GCase polypeptide or fragment thereof comprises the substitution corresponding to V78I.

[0240] In some aspects, the GCase polypeptide or fragment thereof comprises the N102E mutation.

[0241] In some aspects, the GCase polypeptide or fragment thereof comprises the I130T mutation.

[0242] In some aspects, the GCase polypeptide or fragment thereof comprises the A168S mutation.

[0243] In some aspects, the GCase polypeptide or fragment thereof comprises the VI 9 IM mutation.

[0244] In some aspects, the GCase polypeptide or fragment thereof comprises the R21 IN mutation.

[0245] In some aspects, the GCase polypeptide or fragment thereof comprises the Q226T mutation.

[0246] In some aspects, the GCase polypeptide or fragment thereof comprises the L241I mutation.

[0247] In some aspects, the GCase polypeptide or fragment thereof comprises the S242P mutation.

[0248] In some aspects, the GCase polypeptide or fragment thereof comprises the V343T mutation.

[0249] In some aspects, GCase polypeptide or fragment thereof comprises the M361E mutation.

[0250] In some aspects, the GCase polypeptide or fragment thereof comprises the H374W mutation.

[0251] In some aspects, the GCase polypeptide or fragment thereof comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of any one of SEQ ID NOs:242, 245, 247, and 267-279.

[0252] In some aspects, the GCase polypeptide or fragment thereof comprises the amino acid sequence of any one of SEQ ID NOs: 242, 245, 247, and 267-279.

[0253] In some aspects, the GCase polypeptide or fragment thereof comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:246.

[0254] In some aspects, the GCase polypeptide or fragment thereof comprises the amino acid sequence of SEQ ID NO:246.

[0255] In some aspects, the GCase polypeptide or fragment thereof does not comprise the amino acid sequence of SEQ ID NO: 246.

[0256] In some aspects, the GCase polypeptide or fragment thereof is capable of hydrolyzing glucosylceramide.

[0257] In some aspects, the GCase polypeptide or fragment thereof has increased protein expression in CHO cells as compared to wildtype human GCase.

[0258] In some aspects, the GCase polypeptide or fragment thereof has greater than 50, 60, 70, 80, or 90% monomer purity after one step of ProA purification.

[0259] In some aspects, the GCase polypeptide or fragment thereof has greater stability in human serum than wildtype human GCase or Cerezyme.

[0260] In some aspects, the GCase polypeptide or fragment thereof has a melting temp (Tm) greater than 60° C.

[0261] In some aspects, the GCase polypeptide or fragment thereof has an aggregation temperature (Tagg) greater than 65° C.II. Antigen-Binding Domains and Compositions Comprising Antigen-Binding Domains

[0262] In some aspects, compositions provided herein may comprise an antigen-binding domain.

[0263] In some aspects, compositions provided herein may comprise a fusion protein or complex. In some aspects, the fusion protein or complex comprises an antigen-binding domain.

[0264] In some aspects, compositions provided herein may comprise an antibody or an antibody fragment thereof.

[0265] In some aspects, compositions provided herein may comprise an antigen-binding fragment. Antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab’, Fab’-SH, F(ab’)2, Fv, and scFv fragments, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0266] Diabodies are antibody fragments with two antigen-binding sites that can be bivalent and / or bispecific. See, for example, EP404097; WO 1993 / 01161; Hudson et al. Nat. Med. 9: 129- 134 (2003). Triabodies and tetrabodies are also described in Hudson et al. Nat. Med. 9:129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some aspects, a single-domain antibody is a human single-domain antibody (see, e.g., U.S. Patent No. 6,248,516).

[0267] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0268] As provided herein, a composition comprising an antigen-binding domain provided herein can be chimeric. Certain chimeric antibodies are described, e.g., in U.S. Patent No.4,816,567. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody.

[0269] As provided herein, a composition comprising an antigen-binding domain provided herein can be humanized. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some aspects, a humanized antibody is substantially non-immunogenic in humans. In some aspects, a humanized antibody has substantially the same affinity for a target as an antibody from another species from which the humanized antibody is derived. See, e.g., U.S. Pat. No. 5,530,101; 5,693,761; 5,693,762; and 5,585,089. In some aspects, amino acids of an antibody variable domain that can be modified without diminishing the native affinity of the antigen-binding domain while reducing its immunogenicity are identified. See, e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619. Generally, a humanized antibody comprises one or more variable domains in which CDRs (or portions thereof) are derived from a non-human antibody, and framework regions (FRs) (or portions thereof) are derived from human antibody sequences. A humanized antibody can comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0270] Humanized antibodies and methods of making them are reviewed, for example, in Almagro et al. Front. Biosci. 13: 161 9-1633 (2008), and are further described, e.g., in U.S. Patent Nos. 5,821,337; 7,527,791; 6,982,321; and 7087409. Human framework regions that can be used for humanization include but are not limited to: framework regions selected using the “best- fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Set. USA 89:4285 (1992); and Presta et al., J. Immunol. 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson Front. Biosci. 13 : 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al. J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al. J. Biol. Chem. 271 :22611-22618 (1996)).

[0271] As provided herein, a composition comprising an antigen-binding domain provided herein can be human. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk et al. Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg Curr. Opin. Immunol. 20:450-459 (2008).

[0272] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. One can engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci in anticipation that such mice would produce human antibodies in the absence of mouse antibodies. Large human Ig fragments can preserve the large variable gene diversity as well as the proper regulation of antibody production and expression. By exploiting the mouse machinery for antibody diversification and selection and the lack of immunological tolerance to human proteins, the reproduced human antibody repertoire in these mouse strains can yield high affinity fully human antibodies against any antigen of interest, including human antigens. Using the hybridoma technology, antigen-specific human MAbs with the desired specificity can be produced and selected. Certain exemplary methods are described in U.S. Pat. No. 5,545,807, EP546073, and EP546073. See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology. Human variable regions from intact antibodies generated by such animals can be further modified, e.g., by combining with a different human constant region.

[0273] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol. 133:3001 (1984) and Boemer et al. J. Immunol. 147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al. Proc. Natl. Acad. Sci. USA, 1 03:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (Trioma technology) is also described in Vollmers et al. Histology and Histopathology 20(3):927-937 (2005) and Vollmers et al. Methods and Findings in Experimental and Clinical Pharmacology 27(3): 185-91 (2005). Human antibodies can also be generated byisolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0274] In some aspects, an antibody is a human antibody isolated by in vitro methods and / or screening combinatorial libraries for antibodies with the desired activity or activities. Suitable examples include but are not limited to phage display (CAT, Morphosys, Dyax, Biosite / Medarex, Xoma, Symphogen, Alexion (formerly Proliferon), Affimed) ribosome display (CAT), yeast display (Adimab), and the like. In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. Ann. Rev. Immunol. 12: 433-455 (1994). For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. See also Sidhu et al. J. Mol. Biol. 338(2): 299-310, 2004; Lee et al. J. Mol. Biol. 340(5): 1073-1093, 2004; Fellouse Proc. Natl. Acad. Sci. USA 101(34): 12467- 12472 (2004); and Lee et al. J. Immunol. Methods 284(2): 1 19-132 (2004). Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also selfantigens without any immunization as described by Griffiths et al. EMBO J. 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers comprising random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom et al. J. Mol. Biol., 227: 381-388, 1992. Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2007 / 0292936 and 2009 / 0002360. Antibodies isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.III. Fc Domains and Regions

[0275] A composition provided herein can comprise an Fc domain or region or fragment thereof. In some aspects, an Fc domain or region is of IgG class, the IgM class, or the IgA class. In some aspects, an Fc domain or region or fragment thereof is an IgG Fc domain or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG Fc domainor fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgGl Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG2 Fc domain or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a human IgG4 Fc domain or fragment thereof.

[0276] In some aspects, an Fc domain or region or fragment thereof is a wild-type human IgG Fc domain or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a wild-type human IgGl Fc domain or region or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a wild-type human IgG2 Fc domain or fragment thereof. In some aspects, an Fc domain or region or fragment thereof is a wild-type human IgG4 Fc domain or fragment thereof.

[0277] In some aspects provided herein, a composition provided herein comprises a modified Fc domain or region or fragment thereof. In some aspects, the modified Fc domain or region or fragment thereof is a modified IgGl Fc comprising one or more modifications. For example, in some aspects, the IgGl modified Fc comprises one or more amino acid substitutions (e.g., relative to a wild-type Fc domain of the same isotype).

[0278] In some aspects, the modified Fc domain or fragment thereof is a modified IgG2 Fc domain. In some aspects, the modified Fc domain or fragment thereof is a modified IgG2 Fc domain comprising one or more modifications relative to a wild-type IgG2. In some aspects, the modified Fc domain or fragment thereof is a modified IgG4 Fc domain relative to a wild-type IgG4. In some aspects, the modified Fc domain or fragment thereof is a modified IgG4 Fc domain comprising one or more modifications relative to a wild-type IgG4.

[0279] In some aspects, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) Eur J Immunol 23:403-411), D265A (Shields et al. (2001) A. J. Biol. Chem. 276, 6591-6604), L234A, L235A (Hutchins et al. (1995) Proc Natl Acad Sci USA, 92: 11980- 11984; Alegre et al., (1994) Transplantation 57: 1537-1543. 31; Xu et al., (2000) Cell Immunol, 200: 16-26), G237A (Alegre et al. (1994) Transplantation 57: 1537-1543. 31; Xu et al. (2000) Cell Immunol, 200: 16-26), C226S, C229S, E233P, L234V, L234F, L235E (McEarchern et al., (2007) Blood, 109: 1185-1192), P331S (Sazinsky et al., (2008) Proc Natl Acad Sci USA 2008, 105:20167-20172), K322A (Hezareh et al. (2001) J Virol. 75(24) 12161-12168), S267E, L328F, A330L, M252Y, S254T, E430G, and / or T256E, where the amino acid position is according to the EU numbering convention. In some aspects, the Fc domain or region or fragment thereof comprises the amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EUnumbering. In some aspects, the Fc domain or region or fragment thereof comprises N325S and L328F mutations according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises P329G or P329S according to EU numbering. In some aspects, the Fc domain or region or fragment thereof comprises K322A according to EU numbering.

[0280] In some aspects, the Fc domain comprises S228P according to EU numbering. In some aspects, the Fc domain comprises L235E according to EU numbering. In some aspects, the Fc domain comprises S228P and L235E according to EU numbering. In some aspects, the modified Fc domain or fragment thereof is a modified IgG4 and comprises S228P and L235E according to EU numbering.

[0281] Any suitable Fc domain or fragment thereof is contemplated in the constructs described herein, and exemplary Fc domains are provided in Table 1, below.Table 1: Exemplary Fc Domains

[0282] In some aspects provided herein, a composition provided herein comprises one or more mutations to promote heterodimerization of Fc domains. In some aspects, a Fc region of a complex provided herein is formed by Fc domains that contain amino acid mutations, substitutions, additions, or deletions to promote heterodimerization in which different polypeptides comprising different Fc domains can dimerize to yield a heterodimer configuration. In some aspects, a bispecific of the present disclosure comprises a first Fc sequence comprising a first CH3 region, and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first and second CH3 regions are different and are such that the heterodimeric interaction between said first and second CH3 regions is stronger than each of the homodimeric interactions of said first and second CH3 regions.

[0283] Methods to promote heterodimerization of Fc domains include amino acid deletions, additions, or substitutions of the amino acid sequence of the Fc domain, such as by including a set of “knob-into-hole” deletions, additions, or substitutions or including amino acid deletions, additions, or substitutions to effect electrostatic steering of the Fc to favor attractive interactions among different polypeptide chains. Methods for promoting heterodimerization of complementary Fc polypeptides have been previously described in, for example, Ridgway et al, 1996, Protein Eng, 9:617-621; Merchant et al, 1998, Nature Biotechnol, 16:677-681; Moore et al, 2011, MAbs, 3:546-557; Von Kreudenstein et al, 2013, 5:646-654; Gunasekaran et al, 2010, J Biol Chem, 285: 19637-19464; Leaver-Fay et al, 2016, Structure, 24:641-651; Ha et al, 2016, Frontiers in Immunology, 7: 1; Davis et al, 2010, Protein Eng Des Sei, 23: 195-202; PCT Pub.Nos. W01996 / 027011; WO 1998 / 050431; W02006 / 028936; W02009 / 089004;WO201 1 / 143545; WO2014 / 067011; WO2012 / 058768; WO2018 / 027025; US Pub. Nos. US2014 / 0363426; US2015 / 0307628; US2018 / 0016354; US2015 / 0239991; US2017 / 0058054;U.S. Pat. Nos. 5,731,168; 7,183,076; 9,701,759; 9,605,084; 9,650,446; 8,216,805; 8,765,412; and 8,258,268.

[0284] In some aspects, complementary Fc polypeptides of an Fc heterodimer include a mutation to alter charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc domains support favorable attractive interactions, thereby promoting desired Fc heterodimer formation; whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation (Guneskaran et al, 2010, J Biol Chem, 285:19637-19646). When co-expressed in a cell, association between the polypeptide chains is possible but the chains do not substantially self-associate due to charge repulsion.

[0285] “Knob-hole” or “knob-into-hole” configurations are complementary Fc polypeptides of an Fc heterodimer that promote heterodimerization of two Fc polypeptides. “Knob-into-hole” technology is described in U.S. Pat. Nos. 5,731,168; 7,695,936; 8,216,805; 8,765,412; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g., tyrosine or tryptophan).Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.

[0286] In some aspects, a composition provided herein comprises a Fc dimer comprising a “knob” mutation in one Fc domain and a “hole” mutation in the other Fc domain. In some aspects, the “knob” mutation comprises the amino acid substitution T366W according to EU numbering. In some aspects, the “hole” mutation comprises the amino acids substitutions T366S, L368A, and Y407V according to EU numbering. In some aspects, the “knob” mutation comprises the amino acid substitution T366W in one of the two subunits of the Fc dimer, and the “hole” mutation comprises the amino acid substitutions T366S, L368A and Y407V in the other subunit of the Fc dimer. In some aspects, the subunit of the Fc dimer comprising the “knob” mutation additionally comprises the amino acid substitution S354C, and the subunit of the Fc dimer comprising the “hole” mutation additionally comprises the amino acid substitution Y349C.Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc dimer, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). Thus, in such configurations, a first Fc polypeptide comprises amino acid modifications to form the “knob” and a second Fc polypeptide comprises amino acid modifications to form the “hole” thus forming an Fc heterodimer comprising complementary Fc polypeptides.

[0287] Exemplary paired amino acid modifications of complementary Fc polypeptides of an Fc heterodimeric configuration are set forth below in Table 2 (EU numbering).Table 2: Exemplary paired Fc modifications for heterodimeric Fc domainsIV. Compositions that Bind to Blood-Brain Barrier Receptors or Proteins

[0288] Provided herein are compositions that specifically bind to human receptors or proteins of the blood-brain barrier. Such compositions are capable of crossing the blood-brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the composition across the BBB. Exemplary human receptors or proteins of the blood-brain barrier are transferrin receptor (TfR) and CD98 heavy chain (CD98hc).

[0289] In some aspects, the compositions provided herein comprise a TfR antigen-binding domain. In some aspects, the compositions provided herein comprise a CD98hc antigen-binding domain.A. Antigen-Binding Domains That Bind to TfR

[0290] Provided herein are antigen-binding domains that specifically bind to human TfR. Such anti-TfR antigen-binding domains are capable of crossing the blood-brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the BBB. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and are capable of being internalized in BBB epithelial cells.

[0291] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 13 or 14 below (i.e. the three VH CDRs of the antibody listed in Table 3 or 13 and the three VL CDRs of the same antibody listed in Table 4 or 14). In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 below. In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by Kabat numbering. In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six Chothia CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15.

[0292] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human TfR can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732- 745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of Antibody Variable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and comprise VH and VL CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by the method in MacCallum RM et al.

[0293] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human TfR can be determined according to the AbM numbering scheme, which refers to AbM hypervariable regions, which represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antigen-binding domains that specifically bind to human TfR and comprise VH and VL CDRs of an antibody listed in Tables 3, 4, 5, 13, 14, or 15 as determined by the AbM numbering scheme.

[0294] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six IMGT CDRs of an antibody listed in listed in Tables 3, 4, 5, 13, 14, or 15 according to the IMGT numbering system as described in Lefranc M-P, (1999) TheImmunologist 7: 132-136 and Lefranc M-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL- CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0295] In some aspects, an antigen-binding domain that specifically binds to human TfR provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone. See, for example, Rader C et al., (1998) PNAS 95: 8910- 8915, which is incorporated herein by reference in its entirety, describing the humanization of the mouse anti-avP3 antibody by identifying a complementing light chain or heavy chain, respectively, from a human light chain or heavy chain library, resulting in humanized antibody variants having affinities as high or higher than the affinity of the original antibody. See also Clackson T et al., (1991) Nature 352: 624-628, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VL domain (or VH domain) and screening a library for the complementary variable domains. The screen produced 14 new partners for a specific VH domain and 13 new partners for a specific VL domain, which were strong binders, as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45: 572-577, which is incorporated herein by reference in its entirety, describing methods of producing antibodies that bind a specific antigen by using a specific VH domain and screening a library (e.g., human VL library) for complementary VL domains; the selected VL domains in turn could be used to guide selection of additional complementary (e.g., human) VH domains.

[0296] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Table 5 or 15.

[0297] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VL of antibody listed in Table 5 or 15.

[0298] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH and the VL of an antibody listed in Table 5 or 15 (i.e., the VH of the antibody listed in Table 5 or 15 and the VL of the same antibody listed in Table 5 or 15.

[0299] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VH amino acid sequence of an antibody listed in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibodyin Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs). In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody in Table 5 or 15.

[0300] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 5 or 15, and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in listed in Tables 3, 4, 13, or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0301] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0302] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0303] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody inTable 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0304] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13 or 14 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0305] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 5 or 15 and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody in Tables 3, 4, 13 or 14. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3, 4, 13, or 14 (e.g., the non- identical amino acids in the VH and / or VL are outside of the CDRs).

[0306] In some aspects, provided herein is an antigen-binding domain that competitively inhibits binding to TfR of as an antibody comprising a VH amino acid sequence of an antibody in Table 5 or 15 and a VL amino acid sequence of the same antibody in Table 5 or 15.

[0307] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises a VH and a VL on a single polypeptide chain (e.g., a VH and VL in Table 5 or 15).

[0308] In some aspects, the antigen-binding domain comprises an scFv. In some aspects, an scFv comprises a VH and a VL of an antibody listed in Table 5 or 15. The scFv can comprise a VH that is N-terminal to a VL or a VL that is N-terminal to a VH. The scFv can comprise a linker, e.g., between a VH and a VL. Accordingly, the scFv can be in the orientation VH-linker- VL or VL-linker-VH. Such a linker can be about 5 to about 25 amino acids in length. Such a linker can be about 5 to about 20 amino acids in length. Such a linker can be about 10 to about 25 amino acids in length. Such a linker can be about 10 to about 20 amino acids in length. Such a linker can be, e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker can comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 6). Sucha linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0309] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab). The Fab comprises one constant domain, one VH, and one VL. In some aspects, the antigen-binding domain comprises a Fab comprising a constant domain, a VH of an antibody listed in Table 5 or 15 and a VL of the same antibody in Table 5 or 15. In some aspects, the antigen-binding domain that comprises a Fab comprises the CDRs of the antibody in Tables 3, 4, 13 or 14 (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0310] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the antigen-binding fragment of a heavy chain only antibody (e.g., a VHH or nanobody). In some aspects, an antigen-binding domain comprises a VHH comprising the VH of an antibody listed in Table 5 or 15. In other aspects, the antigen-binding domain comprises a VHH comprises the heavy chain CDRs of an antibody listed in Table 3 or 13.

[0311] In some aspects, an antigen-binding domain that specifically binds to human TfR is a murine antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a chimeric antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a humanized antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human TfR is a human antigenbinding domain.

[0312] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus monkey TfR.

[0313] In certain embodiments, an antibody that binds to TfR has a dissociation constant (KD) of about 0.01 nM to about 10,000 nM, less than about 20 pM, less than about 15 pM, less than about 12 pM, less than about 10 pM, less than about 7.5 pM, less than about 5 pM, less than about 2.5 pM, less than about 1 pM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0314] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 8, 15, 25, 43, 55, and 61, respectively.

[0315] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 102 and 158, respectively.

[0316] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 28, 392, 58, and 62, respectively.

[0317] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 117 and 400, respectively.

[0318] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 30, 46, 58, and 62, respectively.

[0319] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 118 and 170, respectively.

[0320] In some aspects, the anti-TfR antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 10, 22, 28, 395, 58, and 62, respectively.

[0321] In some aspects, the anti-TfR antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 117 and 403, respectively.

[0322] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of from about 0.01 nM to about 50 nM, such as about 0.01 nM to about 1 nM, about 0.01 nM to about 5 nM, about 0.01 nM to about 25 nM, about 1 nM to about 5 nM, about 1 nM to about 25 nM, about 1 nM to about 50 nM, about 5 nM to about 25 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, about 10 nM to about 50 nM, about 25 nM to about 50 nM, and values and ranges there between.

[0323] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity from about 51 nM to about 750 nM, such as about 51 nM to about 100 nM, about 51 nM to about 250 nM, about 51 nM to about 500 nM, about 100 nM to about 250 nM, about 100 nM to about 500 nM, about 100 nM to about 750 nM, about 250 nM to about 500 nM, about 250 nM to about 750 nM, about 500 nM to about 750 nM, and values and ranges there between.

[0324] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity from about 751 nM to about 10,000 nM, such as about 751 nM to about 2,500 nM, about 751 nM to about 5,000 nM, about 2,500 nM to about 5,000 nM, about 2,500 nM to about 10,000 nM, about 5,000 nM to about 10,000 nM, and values and ranges there between.

[0325] In some aspects, an antigen-binding domain provided herein bind to human TfR with “low” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity between 500 nM and 10 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 2 pM and 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 1 pM and 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 2 pM and 8 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 750 nM and 2 pM.

[0326] In some aspects, the antigen-binding domain binds human TfR with an affinity between about 750 nM and about 10,000 nM (or about 10 pM), such as about 751 nM to about 10,000 nM, such as about 751 nM to about 2,500 nM, about 751 nM to about 5,000 nM, about 2,500 nM to about 5,000 nM, about 2,500 nM to about 10,000 nM, about 5,000 nM to about 10,000 nM, and values and ranges there between. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 2 pM and about 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 1 pM and about 5 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 2 pM and about 8 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 750 nM and about 2 pM.

[0327] In some aspects, an antigen-binding domain provided herein bind to human TfR with “medium” affinity. In some aspects, an antigen-binding domain provided herein bind to human TfR with “medium” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity between 10 nM and 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 50 nM and 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 100 nM and 250 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 10 nM and 100 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 250 nM and 500 nM.

[0328] In some aspects, the antigen-binding domain binds human TfR with an affinity between 51 nM and 750 nM, such as about 51 nM to about 100 nM, about 51 nM to about 250 nM, about 51 nM to about 500 nM, about 100 nM to about 250 nM, about 100 nM to about 500 nM, about 100 nM to about 750 nM, about 250 nM to about 500 nM, about 250 nM to about 750 nM, about 500 nM to about 750 nM, and values and ranges therebetween. In some aspects, the antigenbinding domain binds human TfR with an affinity between about 50 nM and about 500 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 100 nM and about 250 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 10 nM and about 100 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 250 nM and about 500 nM.

[0329] In some aspects, an antigen-binding domain provided herein bind to human TfR with “high” affinity. In some aspects, the antigen-binding domain binds human TfR with an affinity less than 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.01 nM and 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.1 nM and 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between 0.01 nM and 1 nM.

[0330] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds with an affinity of about 0.01 nM to about 50 nM (e.g., about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 25 nM, or about 15 nM to about 25 nM). In some embodiments, the antigen-binding domain binds human TfR with an affinity less than about 50 uM. In some aspects, the antigen-binding domain binds human TfR with an affinity less than about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM about about 50 pM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM and about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.1 nM and about 10 nM. In some aspects, the antigen-binding domain binds human TfR with an affinity between about 0.01 nM and about 1 nM.

[0331] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of no more than 250 nM (e.g., 10 pM to 250 nM, 5 pM to 250 nM, 1 nM to 250 nM or 3 nM to 250 nM), an affinity of no more than 200 nM (e.g., 10 pM to 200 nM, 5 pM to 200 nM, 1 nM to 200 nM or 3 nM to 200 nM), or an affinity of no more than 150 nM(e.g., 10 pM to 150 nM, 5 pM to 150 nM, 1 nM to 150 nM or 3 nM to 150 nM), optionally wherein the affinity is measured using surface plasmon resonance.

[0332] In some aspects, an antigen-binding domain provided herein specifically binds to human TfR with an affinity of less than about 250 nM (e.g., about 10 pM to about 250 nM, about 5 pM to about 250 nM, about 1 nM to about 250 nM or about 3 nM to about 250 nM), an affinity of less than about 200 nM (e.g., about 10 pM to about 200 nM, about 5 pM to about 200 nM, about 1 nM to about 200 nM or about 3 nM to about 200 nM). In some aspects, an antigenbinding domain provided herein specifically binds to human TfR with an affinity of less than about 150 nM (e.g., about 10 pM to about 150 nM, about 5 pM to about 150 nM, about 1 nM to about 150 nM or about 3 nM to about 150 nM).

[0333] In some aspects, affinity is measured using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to human TfR as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0334] In some aspects, the affinity between the antigen-binding domain and TfR is measured using surface plasmon resonance, such as a BIACORE™ system. In some aspects, the affinity between the antigen-binding domain and TfR is measured using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to human TfR as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0335] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, or about 5 nM to about 15 nM), of about 51 nM to about 1250 nM (such as about 100 nM to about 1250 nM or about 150 nM to about 1250 nM), or of about 1251 nM to about 10,000 nM (such as about 1250 nM to about 5000 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the BIACORE™.

[0336] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to (i) human TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM, about 10 nM to about 50 nM, about 10 nM to about 25 nM, or about 15 nM to about 25 nM) and cynomolgus TfR with an affinity of about 0.01 nM to about 50 nM (such as about 1 nM to about 50 nM, about 5 nM to about 50 nM,about 5 nM to about 30 nM, about 5 nM to about 25 nM, or about 5 nM to about 15 nM); (ii) human TfR with an affinity of about 51 nM to about 750 nM (such as about 100 nM to about 750 nM or about 100 nM to about 700 nM) and cynomolgus TfR wih an affinity of about 51 nM to about 1250 nM (such as about 100 nM to about 1250 nM or about 150 nM to about 1250 nM); or (iii) human TfR with an affinity of about 751 nM to about 10,000 nM (such as about 1000 nM to about 5000 nM) and cynomolgus TfR with an affinity of about 1251 nM to about 10,000 nM (such as about 1250 nM to about 5000 nM), optionally wherein the affinity is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using BIACORE™.

[0337] In some aspects, binding of an antigen-binding domain provided herein to human TfR is measured using surface plasmon resonance. In some aspects, affinity is measured using the Carterra LSA platform

[0338] In some aspects, an antigen-binding domain provided herein that binds to human TfR with moderate to high affinity (e.g., 0.01 nM to 750 nM) results in more rapid brain uptake and clearance. This improved property can be particularly useful, e.g., for complexing with enzymes or proteins that tend to have a faster clearance in the periphery.

[0339] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR reduces cell surface expression by more than 40%, 60%, or 80% relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0340] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR does not significantly increase cell surface expression of TfR on HCMED / D3 cells relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0341] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR does not significantly increase cell surface expression of TfR on HCMED / D3 cells relative to cell surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0342] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR accumulates at least 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50-fold more than an isotype control after peripheral injection. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR at least 5-fold more than binding to an irrelevantprotein and / or specifically binds to cynomolgus TfR at least 5-fold more than binding to an irrelevant protein.

[0343] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR accumulates at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold, or at least about 50-fold more than an isotype control after peripheral injection. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR at least 5-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 5-fold more than binding to an irrelevant protein.

[0344] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR specifically binds to human TfR at least 10-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 10-fold more than binding to an irrelevant protein. In some embodiments, an antigen-binding domain provided herein that specifically binds to human TfR specifically binds human TfR at least 20-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 20-fold more than binding to an irrelevant protein. In some embodiments, an antigen-binding domain provided herein that specifically binds to human TfR specifically binds human TfR at least 30-fold more than binding to an irrelevant protein and / or specifically binds to cynomolgus TfR at least 30-fold more than binding to an irrelevant protein.

[0345] Also provided herein are antigen-binding domains that bind to the same epitope of TfR as a TfR antigen-binding domain provided herein. Also provided herein are antigen-binding domains that competitively inhibit binding to TfR of TfR antigen-binding domain provided herein.

[0346] As provided herein, antigen-binding domains provided herein are capable of crossing the BBB. In some aspects, antigen-binding domains provided herein are internalized in bloodbrain barrier epithelial cells greater than 5 fold, or 10-fold, as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0347] As provided herein, antigen-binding domains provided herein are capable of crossing the BBB. In some aspects, antigen-binding domains provided herein are internalized in bloodbrain barrier epithelial cells greater than about 5-fold, about 10-fold, about 20-fold, or about 30-fold as compared to internalization by an isotype control. The blood-brain barrier endothelial cells can be, e.g., HCMEC / D3 cells.

[0348] In some aspects, antigen-binding domains provided herein do not reduce cell-surface expression of TfR on HCMEC / D3 cells by more than 20%, 40%, or 60% relative to cell-surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS. In some aspects, compositions provided herein do not significantly increase cell-surface expression of TfR on HCMEC / D3 cells relative to cell-surface expression of TfR on HCMEC / D3 cells treated with an isotype control. Cell surface expression can be measured, e.g., using Western blot or FACS.

[0349] In some aspects, compositions provided herein accumulate at least 4-fold, 5 -fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold or 50-fold or more than an isotype control in vessel-depleted mouse brain.

[0350] In some aspects, compositions provided herein accumulate at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 40-fold or at least about 50-fold or more than an isotype control in vessel-depleted mouse brain.

[0351] In some aspects, compositions provided herein bind human TfR with an equilibrium dissociation constant (KD) of about 6.7 nM to about 340 nM and binds cynomolgus monkey TfR with a KD of about 18 nM to about 870 nM.Table 3. Heavy Chain CDR Sequences of Anti-TfR AntibodiesTable 4: Light Chain CDR Sequences of Anti-TfR AntibodiesTable 5: VH and VL Sequences of Anti-TfR AntibodiesB. Agents Comprising Anti-TfR Antigen-Binding Domains

[0352] Provided herein are agents comprising an antigen-binding domain that specifically binds to human TfR.Fusion Proteins or Complexes Comprising Anti-TfR Antigen-Binding Domains

[0353] In some aspects, a fusion protein or complex provided herein comprises: an antigenbinding domain that specifically binds to human TfR and a heterologous protein, such as a GCase protein. In some aspects, a fusion protein or complex provided herein comprises: (i) an antigenbinding domain that specifically binds to human TfR and (ii) a GCase polypeptide or fragment thereof or fragment thereof. In some aspects, the GCase protein is a protein or polypeptide or fragment thereof useful in protein replacement therapy (PRT). In some aspects, the GCase polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some aspects, the GCase polypeptide or protein is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof.

[0354] Exemplary formats for a fusion protein or complex as disclosed herein are depicted in Figure 1.

[0355] In some aspects, the GCase polypeptide or fragment thereof in the fusion protein or complex is N-terminal to the antigen-binding domain that specifically binds to human TfR.

[0356] In some aspects, the GCase polypeptide or fragment thereof in the fusion protein or complex is C-terminal to the antigen-binding domain that specifically binds to human TfR.

[0357] In some aspects, the GCase fusion polypeptide or fragment thereof and the antigenbinding domain that specifically binds to human TfR are directly connected via a peptide bond. In some aspects, the GCase fusion polypeptide or fragment thereof and the antigen-binding domain that specifically binds to human TfR are connected via a linker, e.g., a peptide linker.

[0358] In some aspects, the peptide linker comprises the amino acid sequence corresponding to SEQ ID NO: 285 (GAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAGS).

[0359] In some aspects, the fusion protein or complex comprises an antigen-binding domain that specifically binds to human TfR, a GCase polypeptide or fragment thereof, and an Fc portion. In some aspects, the antigen-binding domain that specifically binds to human TfR and the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0360] In some aspects, the antigen-binding domain that specifically binds to human TfR and the GCase protein are both linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the antigen-binding domain that specifically binds to human TfR is linked to the N-terminus of the Fc portion and the GCase polypeptide or fragment thereof is linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the GCase polypeptide or fragment thereof is linked to the N-terminus of the Fc portion of the fusion protein or complex and the antigen-binding domain that specifically binds to human TfR is linked to the C-terminus of the Fc portion.

[0361] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, or VHH, or Fab antigen-binding domain that specifically binds to human TfR and (ii) a GCase polypeptide or fragment thereof, wherein the fusion protein or complex comprises two copies of the GCase polypeptide or fragment thereof. In some aspects, the fusion protein or complex comprises an Fc domain. The Fc may be a heterodimeric Fc, comprising a first Fc polypeptide with a knob mutation and a second Fc polypeptide with a hole mutation (a “knobhole” or “knob-into-hole” or “knob-hole Fc”, as described herein). In some aspects, the single scFv, Fab or VHH antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain (i.e., to one of the two heavy chains of the Fc domain) while the two copies of the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc. An example of this 2+1 format is shown in (vi) of Fig. 1.

[0362] In some aspects, the single scFv, Fab or VHH antigen-binding domain that specifically binds to human TfR is linked to the N-terminus of the Fc domain (i.e., to one of the two heavy chains of the Fc domain) while the two copies of the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc domain. An example of this 2+1 format is shown as (v) in Fig. 1.

[0363] In some aspects, disclosed herein is a fusion protein or complex comprising (i) an antibody that specifically binds to human TfR, wherein the antibody comprises two heavy chains and two light chains; and (ii) two copies of a GCase polypeptide or fragment thereof linked to the C-terminus of the two antibody heavy chains. An example of this 2+1 format is shown as (vii) in Fig. 1.

[0364] In some aspects, the fusion protein or complex comprises (i) two scFv, Fab, or VHH antigen-binding domains that specifically bind to human TfR linked to the C-terminus of the heavy chain and (ii) two copies of the fusion proteins or fusion protein variants (or complexes or complex variants) linked to the N-terminus of the Fc domain. An example of this 2+2 format is shown as (viii) in Fig. 1. In some aspects, the fusion protein or complex comprises (i) two scFv, Fab, or VHH antigen-binding domains that specifically bind to human TfR linked to the N- terminus of the heavy chain and (ii) two copies of the fusion proteins or fusion protein variants (or complexes or complex variants) linked to the C-terminus of the Fc domain.

[0365] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) an Fc domain, and (iii) a single copy of the GCase protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the C-terminus of the Fc domain and the GCase polypeptide or fragment thereof is linked to N-terminus of the Fc domain. In some aspects, the Fc is a single chain, engineered monovalent Fc domain. An example of this single chain 1+1 format of a fusion protein or complex is shown as (i) in Fig. 1.

[0366] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) a Fc domain, and (iii) a single copy of the GCase polypeptide or fragment thereof, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human TfR is linked to the N-terminus of the Fc domain and the GCase polypeptide or fragment thereof linked to C-terminus of the Fc domain. In some aspects, the Fc is a single chain, engineered monovalent Fc. An example of this1+1 format of a fusion protein or complex is shown as (ii) in Fig. 1. Such monovalent formats can have improved properties for purification and manufacturing.

[0367] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) a Fc domain, and (iii) a single copy of the GCase polypeptide or fragment thereof, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and the GCase polypeptide or fragment thereof are both linked to the N-terminus of the Fc domain. An example of this 1+1 format of a fusion protein or complex is shown as (iv) in Fig. 1. Such N-terminal monozyme formats can result in improved serum PK (longer half-life).

[0368] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR, (ii) a Fc domain, and (iii) a single copy of the GCase polypeptide or fragment thereof, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and the GCase polypeptide or fragment thereof are both linked to the C-terminus of the Fc domain.C. Antigen-Binding Domains That Bind to CD98hc

[0369] Provided herein are antigen-binding domains that specifically bind to human CD98hc.

[0370] Such antigen-binding domains can be capable of crossing the blood-brain barrier (BBB) and capable of transporting other agents (e.g., therapeutically active agents) associated with the antigen-binding domain across the BBB. Accordingly, in some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc that are capable of being internalized in BBB epithelial cells.

[0371] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 6A, 6B, 7A, or 7B (i.e. the three VH CDRs of the antibody listed in Table 6A or 6B and the three VL CDRs of the same antibody listed in Table 7A or 7B). In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six CDRs of an antibody listed in Tables 6A, 6B, 7A, or 7B as determined by Kabat numbering. In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six Chothia CDRs of an antibody listed in Tables 6A, 6B, 7A, or 7B

[0372] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence and Structure Analysis of AntibodyVariable Domains,” in Antibody Engineering, Kontermann and Diibel, eds., Chapter 31, pp. 422- 439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Table 6A, 6B, 7A, or 7B as determined by the method in MacCallum RM et al.

[0373] In some aspects, the CDRs of an antigen-binding domain that specifically binds to human CD98hc can be determined according to the AbM numbering scheme (as described above). In some aspects, provided herein are antigen-binding domains that specifically bind to human CD98hc and comprise VH and VL CDRs of an antibody listed in Tables 6A, 6B, 7A, or 7B as determined by the AbM numbering scheme.

[0374] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the six IMGT CDRs of an antibody listed in Tables 6A, 6B, 7A, or 7B according to the IMGT numbering system as described above.

[0375] In some aspects, an antigen-binding domain that specifically binds to human CD98hc provided herein is described by its VL domain alone, or its VH domain alone, or by its 3 VL CDRs alone, or its 3 VH CDRs alone.

[0376] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH of an antibody listed in Table 8A or 8B.

[0377] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VL of antibody listed in Table 8A or 8B.

[0378] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VH and the VL of an antibody listed in Table 8A or 8B (i.e., the VH of the antibody listed in Table 8A or 8B and the VL of the same antibody listed in Table 8A or 8B.

[0379] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 80% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 80% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs). In some aspects, an antigenbinding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 85% identical to a VH amino acid sequence of an antibody inTable 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 85% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B.

[0380] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 90% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 90% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0381] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 95% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 95% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0382] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 96% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 96% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0383] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 97% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 97% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0384] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 98% identical to a VHamino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 98% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0385] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises (i) a VH comprising an amino acid sequence that is at least 99% identical to a VH amino acid sequence of an antibody in Table 8A or 8B and (ii) a VL comprising an amino acid sequence that is at least 99% identical to the VL amino acid sequence of the same antibody in Table 8A or 8B. In some aspects, the antigen-binding domain that specifically binds to human CD98hc also comprises the CDRs of the antibody in Tables 6A, 6B, 7A, or 7B (e.g., the nonidentical amino acids in the VH and / or VL are outside of the CDRs).

[0386] In some aspects, provided herein is an antigen-binding domain that competitively inhibits binding to CD98hc of as an antibody comprising a VH amino acid sequence of an antibody in Table 8A or 8B and a VL amino acid sequence of the same antibody in Table 8A or 8B

[0387] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH and a VL on a single polypeptide chain (e.g., a VH and VL in Table 8A or 8B). In some aspects, the antigen-binding domain comprises an scFv. The scFv can comprise a VH that is N-terminal to a VL or a VL that is N-terminal to a VH. The scFv can comprise a linker, e.g., between a VH and a VL. Accordingly, the scFv can be in the orientation VH-linker-VL or VL-linker-VH. Such a linker can be about 5 to about 25 amino acids in length. Such a linker can be about 5 to about 20 amino acids in length. Such a linker can be about 10 to about 25 amino acids in length. Such a linker can be about 10 to about 20 amino acids in length. Such a linker can be, e.g., a glycine linker, a glycine-rich linker, or a glycine-serine linker. Such a linker can comprise the amino acid sequence of GGSEGKSSGSGSESKSTGGS (SEQ ID NO: 6). Such a linker can comprise the amino acid sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 7).

[0388] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises a VH on a first polypeptide and a VL on a second polypeptide (e.g., a Fab).

[0389] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the antigen-binding fragment of a heavy chain only antibody (e.g., a VHH or nanobody).

[0390] In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a murine antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a chimeric antigen-binding domain. In some aspects, an antigenbinding domain that specifically binds to human CD98hc is a humanized antigen-binding domain. In some aspects, an antigen-binding domain that specifically binds to human CD98hc is a human antigen-binding domain.

[0391] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc also binds to cynomolgus monkey CD98hc.

[0392] In certain aspects, an antibody that binds to CD98hc has a dissociation constant (KD) of about 10 nM to about 1500 nM, about 100 nM to about 500 nM, about 500 nM to about 10 pM, or less than about 20 pM, less than about 15 pM, less than about 12 pM, less than about 10 pM, less than about 7.5 pM, less than about 5 pM, less than about 2.5 pM, less than about 1 pM, less than about 10 pM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10'8M or less, e.g., from 10'8M to 10'13M, e.g., from 10'9M to 10'13M).

[0393] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 291, 193, 197, 198, and 204, respectively.

[0394] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 210 and 216, respectively.

[0395] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 190, 193, 320, 198, and 204, respectively.

[0396] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 210 and 340, respectively.

[0397] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and alight chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 292, 193, 197, 198, and 204, respectively.

[0398] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 332 and 216, respectively.

[0399] In some aspects, the anti-CD98hc antigen-binding domain comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 184, 297, 193, 197, 198, and 204, respectively.

[0400] In some aspects, the anti-CD98hc antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 337 and 216, respectively.

[0401] In some aspects, an antigen-binding domain provided herein binds to human CD98hc with an affinity of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1100 nM, about 1200 nM, about 1300 nM, about 1400 nM or about 1500 nM.

[0402] In some aspects, an antigen-binding domain provided herein bind to human CD98hc with “low” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 500 nM and 10 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 2 pM and 5 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 1 pM and 5 pM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 2 pM and 8 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 750 nM and 2 pM.

[0403] In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 500 nM and about 10 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 2 pM and about 5 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 1 pM and about 5 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 2 pM and about 8 pM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 750 nM and about 2 pM.

[0404] In some aspects, an antigen-binding domain provided herein bind to human CD98hc with “medium” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 10 nM and 500 nM. In some aspects, the antigen-binding domain bindshuman CD98hc with an affinity between 50 nM and 500 nM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 100 nM and 250 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 10 nM and 100 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 250 nM and 500 nM.

[0405] In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 100 nM and 500 nM, such as about 100 nM to about 499 nM, about 100 nM to about 250 nM, about 250 nM to about 500 nM, and values and ranges therebetween. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 50 nM and about 500 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 100 nM and about 250 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 250 nM and about 500 nM.

[0406] In some aspects, an antigen-binding domain provided herein bind to human CD98hc with “high” affinity. In some aspects, the antigen-binding domain binds human CD98hc with an affinity less than 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 0.01 nM and 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 0.1 nM and 10 nM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between 0.01 nM and 1 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between 0.01 nM and 10 nM.

[0407] In some aspects, the antigen-binding domain binds human CD98hc with an affinity less than about 100 nM, such as less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 0.01 nM and about 100 nM, such as about 0.1 nM to about 10 nM, about 0.1 nM to about 25 nM, about 1 nM to about 25 nM, about 1 nM to about 50 nM, about 25 nM to about 50 nM, about 25 nM to about 100 nM, about 50 nM to about 100 nM, and values and ranges therebetween. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 0.1 nM and about 10 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 0.01 nM and about 10 nM.

[0408] In some aspects, an antigen-binding domain provided herein that binds to human CD98hc with a lower affinity (e.g., an affinity of up to 1 pM) can have improved transport into the brain parenchyma, e.g., as a result of the fact that they would not remain associated with blood vessels for as long a time (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1). In some aspects, an antigen-binding domain provided herein that binds to human CD98hc with a lower affinity (e.g., an affinity of up to 1 pM) can have an improved safety profile (e.g., as compared to an antigen-binding domain with a higher affinity such as the parental WH1).

[0409] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc binds to human CD98hc is measured using surface plasmon resonance. Surface plasmon resonance can be measured, e.g., using the Carterra LSA platform. In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR binds to human TfR as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0410] Also provided herein are antigen-binding domains that bind to the same epitope of CD98hc as a CD98hc antigen-binding domain provided herein. Also provided herein are antigenbinding domains that competitively inhibit binding to CD98hc of CD98hc antigen-binding domain provided herein.Table 6A: Heavy Chain CDR Sequences of Anti-CD98hc AntibodiesTable 6B: Heavy Chain CDR Sequences of Anti-CD98hc AntibodiesTable 7A: Light Chain CDR Sequences of Anti-CD98hc AntibodiesTable 7B: Light Chain CDR Sequences of Anti-CD98hc AntibodiesTable 8A: VH and VL Sequences of Anti-CD98hc AntibodiesTable 8B: VH and VL Sequences of Anti-CD98hc AntibodiesIllD. Agents Comprising Anti-CD98hc Antigen-Binding Domains

[0411] Provided herein are agents comprising an antigen-binding domain that specifically binds to human CD98hc.Fusion Proteins or Complexes Comprising CD98hc binding domains

[0412] In some aspects, a fusion protein or complex provided herein comprises: an antigenbinding domain that specifically binds to human CD98hc and a heterologous protein, such as a GCase protein. In some aspects, a fusion protein or complex provided herein comprises: (i) an antigen-binding domain that specifically binds to human CD98hc and (ii) a heterologous protein or polypeptide or fragment thereof. In some aspects, the heterologous protein is a protein or polypeptide or fragment thereof useful in protein replacement therapy (PRT), such as a GCase protein. In some aspects, the heterologous polypeptide is an enzyme (e.g., an enzyme for use in enzyme replacement therapy (ERT)) or a catalytically active fragment thereof. In some aspects, the heterologous polypeptide or protein is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof.

[0413] Exemplary formats for a fusion protein or complex as disclosed herein are depicted in Figure 1.

[0414] In some aspects, the heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof) in the fusion protein or complex is N-terminal to the antigen-binding domain that specifically binds to human CD98hc.

[0415] In some aspects, the heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof) in the fusion protein or complex is C-terminal to the antigen-binding domain that specifically binds to human CD98hc.

[0416] In some aspects, in a fusion protein, complex, or polypeptide provided herein, the GCase and the antigen-binding domain that specifically binds to human CD98hc are directly connected via a peptide bond. In some aspects, in a fusion protein, complex, or polypeptide provided herein, the GCase and the antigen-binding domain that specifically binds to human CD98hc are connected via a linker, e.g., a peptide linker.

[0417] In some aspects, the fusion protein or complex comprises an antigen-binding domain that specifically binds to human CD98hc, a heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof), and an Fc portion. In some aspects, the antigen-binding domain that specifically binds to human CD98hc and the heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof) are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0418] In some aspects, the antigen-binding domain that specifically binds to human CD98hc and the heterologous protein (e.g., a GCase polypeptide or fragment thereof) are both linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the antigenbinding domain that specifically binds to human CD98hc is linked to the N-terminus of the Fc portion, and the heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof) is linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the heterologous protein or polypeptide (e.g., a GCase polypeptide or fragment thereof) is linked to the N-terminus of the Fc portion of the fusion protein or complex and the antigenbinding domain that specifically binds to human CD98hc is linked to the C-terminus of the Fc portion.

[0419] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, or VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and (ii) a heterologous protein or polypeptide, wherein the fusion protein or complex comprises two copies of the heterologous protein or polypeptide. In some aspects, the fusion protein or complex comprises an Fc domain. The Fc may be a heterodimeric Fc, comprising a first Fc polypeptide with a knob mutation and a second Fc polypeptide with a hole mutation (a “knob-hole”, “knob- into-hole”, “knob-hole Fc”, as described herein). In some aspects, the single scFv, Fab or VHH antigen-binding domain that binds to human CD98hc is linked to the C-terminus of the Fc domain (i.e., to one of the two heavy chains of the Fc domain) while the two copies of the heterologous protein or polypeptide are linked to the N-terminus of the Fc. An example of this 2+1 format is shown in (vi) of Fig. 1.

[0420] In some aspects, the single scFv, Fab or VHH antigen-binding domain that specifically binds to human CD98hc is linked to the N-terminus of the Fc domain (i.e., to one of the two heavy chains of the Fc domain) while the two copies of the heterologous protein or polypeptide are linked to the C-terminus of the Fc domain. An example of this 2+1 format is shown as (v) in Fig. 1.

[0421] In some aspects, disclosed herein is a fusion protein or complex comprising (i) an antibody that specifically binds to human CD98hc, wherein the antibody comprises two heavy chains and two light chains; and (ii) two copies of a heterologous protein or polypeptide linked to the C-terminus of the two antibody heavy chains. An example of this 2+1 format is shown as (vii) in Fig. 1.

[0422] In some aspects, the fusion protein or complex comprises (i) two scFv, Fab, or VHH antigen-binding domains that specifically bind to human CD98hc linked to the C-terminus of the heavy chain and (ii) two copies of the fusion proteins or fusion protein variants (or complexes or complex variants) linked to the N-terminus of the Fc domain. An example of this format is shown as (viii) in Fig. 1. In some aspects, the fusion protein or complex comprises (i) two scFv, Fab, or VHH antigen-binding domains that specifically bind to human CD98hc linked to the N- terminus of the heavy chain and (ii) two copies of the fusion proteins or fusion protein variants (or complexes or complex variants) linked to the C-terminus of the Fc domain.

[0423] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc, (ii) an Fc domain, and (iii) a single copy of the heterologous protein or peptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human CD98hc is linked to the C-terminus of the Fc domain and the heterologous protein or polypeptide is linked to N-terminus of the Fc domain. In some aspects, the Fc is a single chain, engineered monovalent Fc domain. An example of this single chain 1+1 format of a fusion protein or complex is shown as (i) in Fig. 1.

[0424] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc, (ii) a Fc domain, and (iii) a single copy of the heterologous protein or polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that binds to human CD98hc is linked to the N- terminus of the Fc domain and the heterologous protein or polypeptide linked to C-terminus of the Fc domain. In some aspects, the Fc is a single chain, engineered monovalent Fc. An example of this 1+1 format of a fusion protein or complex is shown as (ii) in Fig. 1.

[0425] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc, (ii) a Fc domain, and (iii) a single copy of the heterologous protein or polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and the heterologous protein or polypeptide are both linked to the N-terminus of the Fc domain. An example of this 1+1 format of a fusion protein or complex is shown as (iv) in Fig. 1.

[0426] In some aspects, disclosed herein is a fusion protein or complex comprising (i) a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc, (ii) a Fc domain, and (iii) a single copy of the heterologous protein or polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and the heterologous protein or polypeptide are both linked to the C-terminus of the Fc domain.V. Compositions that Comprise a GCase Polypeptide or Fragment Thereof and Antigen- Binding Domain that Binds to Blood-Brain Barrier Receptors or Proteins

[0427] Provided herein are compositions that specifically bind to human receptors or proteins of the blood-brain barrier.

[0428] In some aspects, the composition comprises any of the GCase polypeptides or fragments thereof described herein.

[0429] In some aspects, the composition comprises a GCase polypeptide or fragment thereof comprising at least one substitution. In some aspects, the at least one substitution is selected from the group consisting of V78I, N102E, L103E, H30T, A168S, V191M, R211N, Q226T, L241I, S242P, W312C, A341C, V343T, M361E, H374W, H495R, and any combination thereof.

[0430] In some aspects, the GCase protein comprises the substitution corresponding to V78I.

[0431] In some aspects, the GCase protein comprises the substitutions corresponding to W312C, A341C, and H495R.

[0432] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 241-247.

[0433] In some aspects, the GCase protein comprises the amino acid sequence corresponding to any one of SEQ ID NOs: 241-247.

[0434] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 241.

[0435] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 241.

[0436] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 242.

[0437] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 242.

[0438] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 243.

[0439] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 243.

[0440] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 245.

[0441] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 245.

[0442] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 246.

[0443] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 246.

[0444] In some aspects, the GCase protein does not comprise the amino acid sequence corresponding to SEQ ID NO: 246.

[0445] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 247.

[0446] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 247.

[0447] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 267-279.

[0448] In some aspects, the GCase protein comprises the amino acid sequence corresponding to any one of SEQ ID NOs: 267-279.

[0449] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 267.

[0450] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 267.

[0451] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 268.

[0452] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 268.

[0453] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 269.

[0454] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 269.

[0455] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 270.

[0456] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 270.

[0457] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 271.

[0458] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 271.

[0459] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 272.

[0460] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 272.

[0461] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 273.

[0462] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 273.

[0463] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 274.

[0464] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 274.

[0465] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 275.

[0466] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 275.

[0467] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 276.

[0468] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 276.

[0469] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 277.

[0470] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 277.

[0471] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 278.

[0472] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 278.

[0473] In some aspects, the GCase protein comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 279.

[0474] In some aspects, the GCase protein comprises the amino acid sequence corresponding to SEQ ID NO: 279.

[0475] In some aspects, the composition comprises any of the fusion proteins or complexes disclosed herein.

[0476] In some aspects, the composition comprises a fusion protein or complex comprising (i) an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) the GCase polypeptide or fragment thereof.

[0477] In some aspects, the antigen-binding domain is an anti-TfR antigen-binding domain.

[0478] In some aspects, the GCase polypeptide or fragment thereof in the fusion protein or complex is N-terminal to the anti-TfR antigen-binding domain.

[0479] In some aspects, the GCase polypeptide or fragment thereof in the fusion protein or complex is C-terminal to the anti-TfR antigen-binding domain.

[0480] In some aspects, the GCase fusion polypeptide or fragment thereof and the anti-TfR antigen-binding domain are directly connected via a peptide bond.

[0481] In some aspects, the GCase fusion polypeptide or fragment thereof and the anti-TfR antigen-binding domain are connected via a linker. In some aspects, the linker is a peptide linker.

[0482] In some aspects, the fusion protein or complex further comprises iii) an Fc portion.

[0483] In some aspects, the anti-TfR antigen-binding domain and the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0484] In some aspects, the anti-TfR antigen-binding domain and the GCase protein are both linked to the C-terminus of the Fc portion of the fusion protein or complex.

[0485] In some aspects, the anti-TfR antigen-binding domain is linked to the N-terminus of the Fc portion and the GCase polypeptide or fragment thereof is linked to the C-terminus of the Fc portion of the fusion protein or complex.

[0486] In some aspects, the GCase polypeptide or fragment thereof is linked to the N-terminus of the Fc portion of the fusion protein or complex and the anti-TfR antigen-binding domain is linked to the C-terminus of the Fc portion.

[0487] In some aspects, the anti-TfR antigen-binding domain is a single scFv, or a VHH, or a Fab.

[0488] In some aspects, the fusion protein or complex comprises two copies of the GCase polypeptide or fragment thereof.

[0489] In some aspects, the fusion protein or complex comprises an Fc domain.

[0490] In some aspects, the single scFv, Fab or VHH is linked to the C-terminus of the Fc domain while the two copies of the GCase polypeptide or fragment thereof are linked to the N- terminus of the Fc domain.

[0491] In some aspects, the single scFv, Fab or VHH is linked to the N-terminus of the Fc domain. In some aspects, the two copies of the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc domain.

[0492] In some aspects, the anti-TfR antigen binding domain is an anti-TfR antibody. In some aspects, the antibody comprises two heavy chains and two light chains.

[0493] In some aspects, two copies of a GCase polypeptide or fragment thereof are linked to the C-terminus of the two antibody heavy chains.

[0494] In some aspects, the anti-TfR antigen binding domain comprises two scFv, Fab, or VHH domains linked to the C-terminus of the heavy chain.

[0495] In some aspects, two copies of the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc domain.

[0496] In some aspects, the anti-TfR antigen binding domain comprises two scFv, Fab, or VHH antigen-binding domains that specifically bind to human TfR linked to the N-terminus of the heavy chain.

[0497] In some aspects, two copies of the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc domain.

[0498] In some aspects, the anti-TfR antigen binding domain comprises a single scFv, VHH, or Fab antigen-binding domain.

[0499] In some aspects, the fusion protein or complex further comprises (ii) an Fc domain, and (iii) a single copy of the GCase protein or peptide.

[0500] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the C-terminus of the Fc domain and the GCase polypeptide or fragment thereof is linked to N- terminus of the Fc domain.

[0501] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the N-terminus of the Fc domain and the GCase polypeptide or fragment thereof linked to C- terminus of the Fc domain.

[0502] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the GCase polypeptide or fragment thereof are both linked to the N-terminus of the Fc domain.

[0503] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the GCase polypeptide or fragment thereof are both linked to the C-terminus of the Fc domain.

[0504] In some aspects, the Fc is a single chain, engineered monovalent Fc domain.

[0505] In some aspects, the fusion protein or complex comprises any of the anti-TfR antigenbinding domains disclosed herein.

[0506] In some aspects, the antigen-binding domain is an anti-CD98hc antigen-binding domain.

[0507] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the anti- CD98hc antigen-binding domain.

[0508] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the anti- CD98hc antigen-binding domain.

[0509] In some aspects, the GCase polypeptide or fragment thereof and the anti-CD98hc antigen-binding domain are directly connected via a peptide bond.

[0510] In some aspects, the GCase polypeptide or fragment thereof and the anti-CD98hc antigen-binding domain are connected via a linker.

[0511] In some aspects, the linker is a peptide linker.

[0512] In some aspects, the fusion protein or complex further comprises (iii) a Fc portion.

[0513] In some aspects, the anti-CD98hc antigen-binding domain and the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0514] In some aspects, the anti-CD98hc antigen-binding domain and the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc portion of the fusion protein or complex.

[0515] In some aspects, the anti-CD98hc antigen-binding domain is linked to the N-terminus of the Fc portion and the GCase polypeptide or fragment thereof is linked to the C-terminus of the Fc portion.

[0516] In some aspects, the GCase polypeptide or fragment thereof is linked to the N-terminus of the Fc portion of the fusion protein or complex and the anti-CD98hc antigen-binding domain is linked to the C-terminus of the Fc portion.

[0517] In some aspects, the anti-CD98hc antigen-binding domain is a single scFv, or VHH, or Fab antigen-binding domain.

[0518] In some aspects, the fusion protein or complex comprises two copies of the GCase polypeptide or fragment thereof.

[0519] In some aspects, the fusion protein or complex further comprises an Fc domain.

[0520] In some aspects, the Fc domain is a heterodimeric Fc, comprising a first Fc polypeptide with a knob mutation and a second Fc polypeptide with a hole mutation.

[0521] In some aspects, the single scFv, Fab or VHH antigen-binding domain is linked to the C-terminus of the Fc domain. In some aspects, the two copies of the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc domain.

[0522] In some aspects, the single scFv, Fab or VHH antigen-binding domain is linked to the N-terminus of the Fc domain. In some aspects, the two copies of the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc domain.

[0523] In some aspects, the anti-CD98hc antigen-binding domain comprises an antibody that specifically binds to human CD98hc. In some aspects, the antibody comprises two heavy chains and two light chains.

[0524] In some aspects, the fusion protein or complex comprises two copies of a GCase polypeptide or fragment thereof linked to the C-terminus of the two antibody heavy chains.

[0525] In some aspects, the anti-CD98hc antigen-binding domain comprises two scFv, Fab, or VHH antigen-binding domains linked to the C-terminus of the heavy chain. In some aspects, two copies of the GCase polypeptide or fragment thereof are linked to the N-terminus of the Fc domain.

[0526] In some aspects, the anti-CD98hc antigen-binding domain comprises two scFv, Fab, or VHH antigen-binding domains linked to the N-terminus of the heavy chain. In some aspects, two copies of the GCase polypeptide or fragment thereof are linked to the C-terminus of the Fc domain.

[0527] In some aspects, the anti-CD98hc antigen-binding domain comprises a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc. In some aspects, the single scFv, VHH, or Fab antigen-binding domain that binds to human CD98hc is linked to the C-terminus of the Fc domain.

[0528] In some aspects, a single copy of the GCase protein or peptide is linked to N-terminus of the Fc domain.

[0529] In some aspects, the anti-CD98hc antigen-binding domain comprises a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc. In some aspects, the single scFv, VHH, or Fab antigen-binding domain that binds to human CD98hc is linked to the N-terminus of the Fc domain.

[0530] In some aspects, a single copy of the GCase polypeptide or fragment thereof is linked to C-terminus of the Fc domain.

[0531] In some aspects, the anti-CD98hc antigen-binding domain comprises a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc. In some aspects, the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and a single copy of the GCase polypeptide or fragment thereof are both linked to the N-terminus of the Fc domain.

[0532] In some aspects, the anti-CD98hc antigen-binding domain comprises a single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc. In some aspects, the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human CD98hc and a single copy of the GCase polypeptide or fragment thereof are both linked to the C-terminus of the Fc domain.

[0533] In some aspects, the Fc domain is a single chain, engineered monovalent Fc domain.

[0534] In some aspects, the fusion protein or complex comprises any of the anti-CD98hc antigen-binding domains disclosed herein.

[0535] In some aspects, the fusion protein or complex comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 248-266, or 280-289.

[0536] In some aspects, the fusion protein or complex comprises the amino acid sequence corresponding to any one of SEQ ID NOs: 248-266, or 280-289.

[0537] In some aspects, the fusion protein or complex comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs: 248-266, 280-289, or 373-390.

[0538] In some aspects, the fusion protein or complex comprises the amino acid sequence corresponding to any one of SEQ ID NOs: 248-266, 280-289, or 373-390.

[0539] In some aspects, the fusion protein or complex comprises any one of the amino acid sequences disclosed in Table 21, Table 22, Table 23, or Table 24.

[0540] In some aspects, the fusion protein or complex comprises any one of the amino acid sequences disclosed in Table 21, Table 22, Table 23, Table 24, or Table 26.

[0541] In some aspects, provided herein are complexes comprising (i) an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) a GCase polypeptide of fragment thereof.

[0542] In some aspects, the complex comprises a first polypeptide comprising the GCase polypeptide or fragment there and a first Fc domain; a second polypeptide comprising a VH of the antigen-binding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigen-binding domain.

[0543] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the first Fc domain.

[0544] In some aspects, the first polypeptide further comprises an antibody hinge between the GCase polypeptide or fragment thereof and the first Fc domain.

[0545] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the second Fc domain.

[0546] In some aspects, the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation.

[0547] In some aspects, the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

[0548] In some aspects, the second polypeptide is an antibody heavy chain.

[0549] In some aspects, the third polypeptide further comprises a CL domain. In some aspects, the third polypeptide is an antibody light chain.

[0550] In some aspects, the first Fc domain comprises a CH2 and a CH3. In some aspects, the second Fc domain comprises a CH2 and a CH3.

[0551] In some aspects, the complex comprises a first polypeptide comprising an antibody heavy chain and a GCase polypeptide or fragment thereof and a second polypeptide comprising an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise the antigen-binding domain.

[0552] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the antibody heavy chain.

[0553] In some aspects, the complex further comprises a third polypeptide comprising a Fc domain.

[0554] In some aspects, the antibody heavy chain comprises a knob mutation and the Fc domain comprises a hole mutation.

[0555] In some aspects, the antibody heavy chain comprises a hole mutation and the Fc domain comprises a knob mutation.

[0556] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0557] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are contained in a single polypeptide.

[0558] In some aspects, the GCase polypeptide or fragment thereof and the antigen-binding domain are directly connected via a peptide bond.

[0559] In some aspects, the GCase polypeptide or fragment thereof and the antigen-binding domain are connected via a linker.

[0560] In some aspects, the linker is a peptide linker.

[0561] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the antigen-binding domain.

[0562] In some aspects, the GCase polypeptide or fragment thereof is C-terminal to the antigen-binding domain.

[0563] In some aspects, the complex further comprises an Fc domain.

[0564] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0565] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are both N-terminal to the Fc domain.

[0566] In some aspects, the antigen-binding domain and the GCase polypeptide or fragment thereof are both C-terminal to the Fc domain.

[0567] In some aspects, the antigen-binding domain is N-terminal to the Fc domain and the GCase polypeptide or fragment thereof is C-terminal to the Fc domain or fragment thereof.

[0568] In some aspects, the GCase polypeptide or fragment thereof is N-terminal to the Fc domain or fragment thereof and the antigen-binding domain is C-terminal to the Fc domain or fragment thereof.

[0569] In some aspects, the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of: L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, H184F, H184L, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P,H262N, H262Y, D263N, E272L, E272Q, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, H313N, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, W351C, F355A, L356F, K360A, K360N, M361E, S364A, T369E, N370D, L372N, H374W, V376T, A380C, A380T, N386D, R395K, H404K, 1406 A, I406T, I407C, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, D482C, I483V, D484C, H490K, S494C, H495R, S503C, R534C, R534H, R534N, and combinations thereof as compared to SEQ ID NO:241.

[0570] In some aspects, the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of V78I, N102E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E; H374W, and combinations thereof as compared to SEQ ID NO:241.

[0571] In some aspects, the GCase polypeptide or fragment thereof does not comprise any of the following mutations L34P, K224G, K224N, T369E and N370D relative to SEQ ID NO: 241.

[0572] In some aspects, the GCase polypeptide or fragment thereof comprises the mutations W351C and A380C relative to SEQ ID NO: 241.

[0573] In some aspects, the GCase polypeptide or fragment thereof comprises the sequence of SEQ ID NO:265.

[0574] In some aspects, the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:241 when expressed under the same conditions.

[0575] In some aspects, the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:243 when expressed under the same conditions.

[0576] In some aspects, the GCase polypeptide or fragment thereof has increased protein expression in CHO cells as compared to wildtype human GCase.

[0577] In some aspects, the complex comprises any of the GCase polypeptides or fragments thereof disclosed herein.

[0578] In some aspects, the antigen binding domain of the complex is a Fab.

[0579] In some aspects, the antigen-binding domain of the complex is an scFv.

[0580] In some aspects, the antigen-binding domain of the complex is a VHH.VI. Polynucleotides and Methods of Making the Same

[0581] In some aspects, provided herein is a nucleotide sequence encoding an antigen-binding domain that specifically binds to a human BBB protein or receptor and a GCase polypeptide or fragment thereof. In some aspects, provided herein are polynucleotides encoding an antigen-binding domain that specifically binds to a human BBB protein or receptor and encoding a GCase polypeptide or fragment thereof. In some aspects, the human BBB protein or receptor is TfR. In some aspects, the human BBB protein is CD98hc.

[0582] Provided herein are polynucleotides comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, that are optimized, e.g., by codon / RNA optimization, replacement with heterologous signal sequences, and / or elimination of mRNA instability elements. Methods to generate optimized nucleic acids for recombinant expression by introducing codon changes (e.g., a codon change that encodes the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in the mRNA can be carried out by adapting the optimization methods described in, e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, accordingly.

[0583] A polynucleotide comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3’ and 5’ ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells producing the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids comprising, e.g., the sequence encoding the light chain and / or heavy chain of an antigen-binding domain, antibody, or antigen-binding fragment thereof. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning, for example, to generate an antigen-binding domain that specifically bind to human TfR, fusion protein or complex, antibody, or antigen-binding fragment thereof described herein, or a domain thereof described herein.

[0584] Polynucleotides provided herein can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and DNA can be double-stranded or single-stranded. If single stranded, DNA can be the coding strand or non-coding (anti-sense) strand. In some aspects, the polynucleotide is a cDNA or a DNA lacking one more endogenous introns. In some aspects, a polynucleotide is a non-naturally occurring polynucleotide. In some aspects, a polynucleotide is recombinantly produced. In some aspects, the polynucleotides are isolated. In some aspects, the polynucleotides are substantially pure.

[0585] In some aspects, polynucleotides provided herein are in the form of RNA. In some aspects, polynucleotides provided herein are in the form of RNA encoding a fusion protein orcomplex provided herein. In some aspects, a polynucleotide provided herein is a synthetic messenger RNA (mRNA). In some aspects, the synthetic mRNA has at least one nucleoside modification. In some aspects, the at least one nucleoside modification is selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl- 1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l-methyl-pseudoisocytidine, 2- aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2- methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-m ethylinosine, 6-methoxy-guanosine, 1- methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl- 8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl- 6-thio-guanosine.

[0586] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, ora domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell or in an E. coli cell. A vector for the production of the compositions described herein, or a domain thereof described herein, can be produced, e.g., by recombinant DNA technology using techniques well known in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are replicable vectors comprising a nucleotide sequence encoding the compositions described herein, or a domain thereof described herein, operably linked to a promoter. Such vectors can, for example, include the nucleotide sequence encoding the constant region of an antigen-binding domain, antibody or antigen-binding fragment thereof (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and variable domains of the antigen-binding domain, antibody or antigen-binding fragment thereof can be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains. In some aspects, the vector is gene therapy vector (e.g., an AAV or lentiviral vector).

[0587] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a domain thereof described herein and a GCase polypeptide or fragment thereof. An expression system can be included on a vector. An expression system can also be integrated into a host cell chromosome. In some aspects, an expression system is a cell free expression system. In some aspects, an expressions system comprises a host cell comprising a polynucleotide and / or vector provided herein.

[0588] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing the compositions described herein and a GCase polypeptide or fragment thereof. In some aspects, for the expression of double-chained antigenbinding proteins, vectors encoding both the heavy and light chains, individually, can be coexpressed in the host cell for expression of the entire immunoglobulin. In some aspects, a host cell contains two different vectors, a first vector comprising a polynucleotide encoding a heavy chain of an antigen-binding protein described herein, and a second vector comprising a polynucleotide encoding a light chain of the an antigen-binding protein. In some aspects, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain.In some aspects, provided herein is a population of host cells comprising such first host cell and such second host cell.

[0589] In some aspects, provided herein are methods for producing the compositions described herein, or a domain thereof and a GCase polypeptide or fragment thereof described herein in a host cell.

[0590] An expression vector can be transferred to a cell (e.g., host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the compositions described herein, or a domain thereof described herein.

[0591] A variety of host-expression vector systems can be utilized to the compositions described herein, or a domain thereof described herein (see, e.g., U.S. Patent No. 5,807,715). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells which can, when transformed or transfected with the appropriate nucleotide coding sequences, express the compositions described herein, or a domain thereof described herein in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH3T3, HEK-293T, HepG2, SP210, Rl. l, B-W, L-M, BSC1, BSC40, YB / 20 and BMTIO cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). In some aspects, cells for expressing the compositions described herein, or a domain thereof described herein are CHO cells, for example CHO cells from the CHO GS System™ (Lonza). In some aspects, cells for expressing the compositions described herein, or a domain thereof described herein as described herein are human cells, e.g., human cell lines. In some aspects, a mammalian expression vector ispOptiVEC™ or pcDNA3.3. In some aspects, bacterial cells, such as Escherichia coli, or eukaryotic cells (e.g., mammalian cells) are used for the expression of the compositions described herein, or a domain thereof described herein. For example, mammalian cells such as Chinese hamster ovary (CHO) cells in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-105; and Cockett MI et al., (1990) Biotechnology 8: 662-667). In some aspects, the compositions described herein, or a domain thereof described herein is produced by CHO cells or NSO cells.

[0592] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, Rl.l, B-W, L-M, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells.

[0593] Once the compositions described herein, or a domain thereof described herein has been produced by recombinant expression, it can be purified by any method known in the art for purification, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Further, the compositions described herein, or a domain thereof described herein can be fused to heterologous polypeptide sequences to facilitate purification.

[0594] In some aspects, the compositions described herein are isolated or purified. Generally, an isolated or purified composition described herein is one that is substantially free of other proteins. For example, in some aspects, a preparation of the compositions described herein is substantially free of cellular material and / or chemical precursors.A. Polynucleotides Encoding Agents Comprising a GCase Polypeptide or Fragment Thereof and Antigen Binding Domains That Bind TfR and Methods of Making the Same

[0595] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a composition comprising an antigen binding domain that specifically binds to human TfR, as described herein and a GCase polypeptide or fragment thereof.

[0596] In some aspects, provided herein are polynucleotides comprising a first nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, as described herein and a second nucleotide sequence encoding a GCase polypeptide or fragment thereof. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0597] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antigen-binding domain that specifically bind to human TfR, a fusion protein or complex, described herein or a domain thereof described herein.

[0598] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human TfR provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human TfR provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human TfR provided herein and a nucleic acid molecule encoding the light chain of an antigenbinding domain that specifically binds to human TfR provided herein.

[0599] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the GCase polypeptide or fragment thereof as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding a domain of the GCase polypeptide or fragment thereof as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding one or more domains of the GCase polypeptide or fragment thereof as provided herein.

[0600] In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a complex provided herein, e.g., a complex with a format as shown in FIG. 1 (ii), (vi), or (vii), wherein the complex comprises an antigen-binding domain that binds to TfR. In some aspects, the polynucleotides in the combination or composition encode an Fc region comprising a knob mutation and a hole mutation.

[0601] In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a complex provided herein, e.g., a complex with a format as shown in FIG. 1 (iii), (iv), or (v), wherein the complex comprises an antigen-binding domain that binds to TfR. In some aspects, the polynucleotides in the combination or composition encode an Fc region comprising a knob mutation and a hole mutation.

[0602] In some aspects, combinations or compositions of polynucleotides are provided herein. In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, wherein the first, second, and third polynucleotides encode a complex provided herein, e.g., wherein the first polynucleotide encodes a first heavy chain (optionally wherein the first heavy chain comprises a knob mutation), the second polynucleotide encodes a second heavy chain (optionally wherein the second heavy chain comprises a hole mutation), and the third polynucleotide encodes a light chain, wherein the complex comprises two copies of the light chain, and wherein a GCase polypeptide is linked to the first and / or second heavy chain, e.g., to produce a complex with a 2+2 format as shown in FIG. 1 (vii), wherein the complex comprises an antigen-binding domain that binds to TfR.

[0603] In some aspects, disclosed herein are polynucleotides encoding a fusion protein or complex disclosed herein comprising a GCase polypeptide or fragment thereof. In some aspects, provided herein is a polynucleotide that encodes a GCase polypeptide or fragment thereof, an Fc domain, a linker, and an antigen-binding domain that specifically binds to human TfR provided herein. In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a GCase polypeptide or fragment thereof, an Fc domain, a linker, and a heavy chain of an antigen-binding domain that bind to human TfR provided herein, and wherein the second polynucleotide encodes a light chain of the antigen-binding domain that bind to human TfR provided herein. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0604] In certain aspects, provided herein are vectors e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding an antigen-binding domain that specifically bind to human TfR, fusion protein or complex described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell.

[0605] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a domain thereof described herein and a GCase polypeptide or fragment thereof.

[0606] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing an antigen-binding domain that specifically bind to human TfR, fusion protein or complex, antibody, or antigen-binding fragment thereof described herein, or a domain thereof described herein and a GCase polypeptide or fragment thereof.

[0607] In some aspects, provided herein are methods for producing an antigen-binding domain that specifically binds to human TfR described herein, or a domain thereof and a GCase polypeptide or fragment thereof described herein in a host cell.

[0608] In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human TfR, a linker, an Fc domain, and a GCase polypeptide or fragment thereof.

[0609] In some aspects, a composition that binds to human TfR as described herein is isolated or purified (e.g., is one that is substantially free of other proteins, and / or is substantially free of cellular material and / or chemical precursors).B. Polynucleotides Encoding Agents Comprising a GCase polypeptide or Fragment Thereof and Antigen Binding Domains That Bind CD98hc and Methods of Making the Same

[0610] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding a composition comprising an antigen binding domain that specifically binds to human CD98hc described herein or a domain thereof described herein, and GCase polypeptide or fragment thereof.

[0611] In some aspects, provided herein are polynucleotides comprising a first nucleotide sequence encoding an antigen-binding domain that specifically bind to human CD98hc, as described herein and a second nucleotide sequence encoding a GCase polypeptide or fragment thereof. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0612] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding compositions that specifically bind to human CD98hc described herein or a domain thereof described herein.

[0613] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hcprovided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the heavy chain of an antigen-binding domain that specifically binds to human CD98hc provided herein and a nucleic acid molecule encoding the light chain of an antigen-binding domain that specifically binds to human CD98hc provided herein.

[0614] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the GCase polypeptide or fragment thereof as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding a domain of a GCase polypeptide or fragment thereof as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding one or more domains of a GCase polypeptide or fragment thereof as provided herein.

[0615] In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides encode a complex provided herein, e.g., a complex with a format as shown in FIG. 1 (ii), (vi), or (vii), wherein the complex comprises an antigen-binding domain that binds to CD98hc. In some aspects, the polynucleotides in the combination or composition encode an Fc region comprising a knob mutation and a hole mutation.

[0616] In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, and a third polynucleotide, wherein the first, second, and third polynucleotides encode a complex provided herein, e.g., a complex with a format as shown in FIG. 1 (iii), (iv), or (v), wherein the complex comprises an antigen-binding domain that binds to CD98hc. In some aspects, the polynucleotides in the combination or composition encode an Fc region comprising a knob mutation and a hole mutation.

[0617] In some aspects, combinations or compositions of polynucleotides are provided herein. In some aspects, a combination or composition comprises a first polynucleotide, a second polynucleotide, a third polynucleotide, wherein the first, second, and third polynucleotides encode a complex provided herein, e.g., wherein the first polynucleotide encodes a first heavy chain (optionally wherein the first heavy chain comprises a knob mutation), the second polynucleotide encodes a second heavy chain (optionally wherein the second heavy chain comprises a hole mutation), and the third polynucleotide encodes a light chain, wherein the complex comprises two copies of the light chain, and wherein a GCase polypeptide is linked tothe first and / or second heavy chain, e.g., to produce a complex with a 2+2 format as shown in FIG. 1 (vii), wherein the complex comprises an antigen-binding domain that binds to CD98hc.

[0618] In some aspects, disclosed herein are polynucleotides encoding a fusion protein or complex disclosed herein comprising a GCase polypeptide or fragment thereof. In some aspects, provided herein is a polynucleotide that encodes a GCase polypeptide or fragment thereof, an Fc domain, a linker, and an antigen-binding domain that specifically binds to human CD98hc provided herein. In some aspects, a combination or composition comprises a first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a GCase polypeptide or fragment thereof, an Fc domain, a linker, and a heavy chain of an antigen-binding domain that bind to human CD98hc provided herein, and wherein the second polynucleotide encodes a light chain of the antigen-binding domain that bind to human CD98hc provided herein. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0619] In certain aspects, provided herein are vectors e.g., expression vectors) comprising polynucleotides comprising the compositions that bind CD98hc described herein, or a domain thereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell.

[0620] In certain aspects, provided herein are expression systems comprising polynucleotides comprising nucleotide sequences encoding the compositions that bind CD98hc described herein, or a domain thereof described herein and a GCase polypeptide or fragment thereof.

[0621] Accordingly, also provided herein are cells, e.g., host cells, comprising polynucleotides and / or vectors for recombinantly expressing the compositions that bind CD98hc described herein, or a domain thereof described herein and a GCase polypeptide or fragment thereof.

[0622] In some aspects, provided herein are methods for producing a composition that binds to human CD98hc as described herein, or a domain thereof, and a GCase polypeptide or fragment thereof described herein in a host cell.

[0623] In some aspects, provided herein are methods for producing a single chain antigenbinding domain that specifically binds to human CD98hc, a linker, an Fc domain, and a GCase polypeptide or fragment thereof.

[0624] A variety of host-expression vector systems and host cells are described herein.

[0625] In some aspects, a composition that binds to human CD98hc as described herein is isolated or purified (e.g., is one that is substantially free of other proteins, and / or is substantially free of cellular material and / or chemical precursors).VII. Pharmaceutical Compositions

[0626] Provided herein are pharmaceutical compositions comprising the GCase polypeptide or fragment thereof and compositions that bind to human TfR or a human CD98hc as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer (Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Also provided herein are pharmaceutical compositions comprising a GCase polypeptide or fragment thereof and compositions that bind to human CD98hc as described herein and a pharmaceutically acceptable carrier, excipient or stabilizer. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can comprise antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.

[0627] In some aspects, a pharmaceutical composition comprises a GCase polypeptide or fragment thereof and compositions that bind to human TfR as described herein and a pharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some aspects, a pharmaceutical composition comprises a GCase polypeptide or fragment thereof and compositions that bind to human CD98hc as described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions described herein are, in some aspects, for use as a medicament. The compositions to be used for in vivo administration can be sterile. This is readily accomplished by filtration through, e.g., sterile filtration membranes.

[0628] In some aspects, provided herein are pharmaceutical compositions comprising a polynucleotide encoding a GCase polypeptide or fragment thereof and a composition that binds to human TfR as described herein. Also provided herein are pharmaceutical compositions comprising a polynucleotide encoding a GCase polypeptide or fragment thereof and a composition that binds to human CD98hc as described herein. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is a synthetic mRNA. In some aspects, the polynucleotide is a modified mRNA. In some aspects, the pharmaceutical composition comprising a polynucleotide further comprises a lipid-based transfection reagent.

[0629] A pharmaceutical composition described herein can be used to exert a biological effect(s) in vivo or in vitro. For example, a pharmaceutical composition described herein can be used to cross a blood-brain barrier, e.g., in a subject.

[0630] In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, and CNS inflammation. In some aspects, a pharmaceutical composition provided herein is used to treat diseases or conditions such as Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, and traumatic brain injury. In some aspects, a pharmaceutical composition provided herein is used to treat frontotemporal dementia.

[0631] In some aspects, a pharmaceutical composition provided herein is formulated for intravenous administration. In some aspects, a pharmaceutical composition provided herein is formulated for subcutaneous administration.VIII. Methods of Using Agents Comprising a GCase polypeptide or fragment thereof and Antigen-Binding Domains that Bind to Blood-Brain Barrier Receptors or Proteins

[0632] Compositions provided herein comprise (i) an anti-TfR or anti-CD98hc antigen-binding domain and (ii) a GCase polypeptide or fragment thereof that can advantageously be transported across a blood-brain barrier.

[0633] Accordingly, provided herein are methods of administering or transporting compositions comprising (i) an antigen-binding protein that specifically binds to human TfR or human CD98hc and (ii) a GCase polypeptide or fragment thereof across the blood-brain barrier of a subject comprising administering to the subject such compositions.

[0634] In view of the ability of compositions provided herein to specifically bind to human TfR or human CD98hc and be transported across a blood-brain barrier, these compositions can be used to treat a neurological disease or disorder. In some aspects, a method of treating a neurological disease or disorder in a subject comprises administering to the subject a composition comprising (i) an antigen-binding protein that specifically binds to human TfR or human CD98hc and (ii) a GCase polypeptide or fragment thereof.

[0635] The neurological disease or disorder can be, for example, a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, orCNS inflammation. The neurological disease or disorder can be, for example, a neurodegenerative disease (such as Lewy body disease, postpoliomyelitis syndrome, Shy- Draeger syndrome, olivopontocerebellar atrophy, Parkinson’s disease, Gaucher disease, multiple system atrophy, striatonigral degeneration, spinocerebellar ataxia, spinal muscular atrophy), a tauopathy (such as Alzheimer disease and supranuclear palsy), a prion disease (such as bovine spongiform encephalopathy, scrapie, Creutz-feldt-Jakob syndrome, kuru, Gerstmann-Straussler- Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, a nervous system heterodegenerative disorders (such as Canavan disease, Huntington’s disease, neuronal ceroid-lipofuscinosis, Alexander’s disease, Tourette’s syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden- Spatz syndrome, lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (such as Pick’s disease, and spinocerebellar ataxia), cancer of the CNS and / or brain (such as glioblastoma or brain metastases resulting from cancer elsewhere in the body), Alzheimer’s disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), limbic-predominant age-related TDP-43 encephalopathy (LATE), cystic fibrosis, Angelman’s syndrome, Liddle syndrome, Parkinson’s disease, Pick’s disease, Paget’s disease, cancer, or traumatic brain injury. In some aspects, the neurological disease or disorder is dementia. In some aspects, the neurological disease or disorder is frontotemporal dementia. In some aspects, the neurological disease or disorder is Alzheimer’s disease. In some aspects, the neurological disease or disorder is Parkinson’s disease. In some aspects, the neurological disease or disorder is frontal temporal epilepsy. In some aspects, the neurological disease or disorder is autism. In some aspects, the neurological disease or disorder is lissencephaly.

[0636] In some aspects, provided herein is a method of treating a lysosomal storage disease with a fusion protein or complex disclosed herein. In some aspects, the lysosomal storage disease is selected from Gaucher disease, Ceroid lipofuscinosis (Batten disease), Mucopolysaccharidosis (MPS) Type I, MPS Type II and MPS Type III ),

[0637] In some aspects, provided herein is a method of transporting a GCase polypeptide or fragment thereof across the blood-brain barrier of a subject comprising administering to the subject any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotide or combination of polynucleotides disclosed herein.

[0638] In some aspects, provided herein is a method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject any of the GCase polypeptides or fragments thereof disclosed herein, any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein.

[0639] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease.

[0640] In some aspects, provided herein is a method of treating a CNS disease or disorder in a subject comprising administering to the subject any of the GCase polypeptides or fragments thereof disclosed herein, any of the complexes disclosed herein, any of the compositions disclosed herein, or any of the polynucleotides or combination of polynucleotides disclosed herein.

[0641] In some aspects, the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia.

[0642] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0643] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure. All citations throughout the disclosure are hereby expressly incorporated by reference.EXAMPLESExample 1: Engineering of Antigen-Binding Domains against Human Transferrin Receptor

[0644] Mammalian expression of human, cynomolgus macaque (cyno) and mouse variants of Transferrin Receptor antigens (SEQ ID NOs:218-224) was performed by cloning synthetic genes based on cDNA into mammalian expression vectors, followed by transient transfection and expression in Expi293 cells and purified by Ni-NTA agarose (QIAGEN 30230) using the manufacturer’s protocol. See Example 1 of PCT / US2023 / 071239, published as WO 2024 / 026472, filed July 28, 2023.Table 9: Avi-His tagged variants of TfR

[0645] Plasmid constructs each expressing full length human TfR and mouse TfR (SEQ ID NOs: 1 and 225, respectively) were used to generate CHO cells stably expressing human TfR and mouse TfR. The resulting CHO cells stably expressing human TfR and mouse TfR were analyzed for cell surface expression by flow cytometry. See Example 2 of PCT / US2023 / 071239, published as WO 2024 / 026472.Table 10. Full-length TfR sequences

[0646] Mouse lines were generated at Taconic Biosciences GmbH (Germany) to humanize the extracellular domains of TfR. CRISPR was used to replace the mouse ECD with the human version, while retaining the mouse intracellular and transmembrane portions under the control of the mouse promotor. Brain slices from these mice were evaluated by IHC and / or Western Blot to confirm expression and localization of the human ECD in vivo (data not shown). See Example 3 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0647] Hybridomas to obtain antibodies against TfR were generated by immunizing BALB / c mice or Sprague Dawley rats with purified extracellular domain polypeptides of human / cyno and / or mouse TfR. Lymph nodes were harvested from the mice or rats for hybridoma cell line generation. Sera from the animals were analyzed for reactivity to TfR and lymphocytes from animals whose sera demonstrated strong binding were used to generate hybridoma libraries. See Example 4 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0648] A total of 1536 anti-TfR hybridoma supernatants were screened via FACS for their ability to differentially bind CHO cells overexpressing human or mouse TfR compared to CHO parental cells. A total of 189 mouse hybridoma and 115 rat hybridoma clones displayed greater than 3-fold difference in binding (as determined by MFI) to CHO cells stably overexpressing human TfR compared to isotype control. See Example 5 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0649] Hybridoma culture supernatants from 319 hybridomas obtained as described above were screened for their ability to bind Avi-His-tagged huTfR ECD, huTfR apical domain, and muTfR ECD ) as compared to binding to an irrelevant Avi-His-tagged control protein. From this hybridoma supernatant screen, a total of 112 anti-TfR hybridoma clones were identified that displayed greater than 5-fold difference in binding to recombinant huTfR Avi-His over background, and 86 of these anti-TfR hybridoma clones also bound to the huTfR apical domain. See Example 6 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0650] Anti-TfR hybridoma antibodies were purified and screened for their ability to internalize into a blood-brain barrier endothelial cell line (hCMEC / D3). Internalization into endothelial cells at the blood-brain barrier is the first stage of transcytosis across the BBB and into the brain. To identify anti-TfR antibodies with internalization ability, HCMEC / D3 cells were seeded and treated with anti-TfR antibodies. A human IgG isotype and an anti-TfR antibody with known internalization ability were included in the assay as negative and positive controls, respectively. Antibodies (TfRl-32) were found to have a fold change of internalization over anirrelevant mouse IgG antibodies in a range from 1-2 fold to 714.7 fold. See Example 7 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0651] Anti-TfR antibodies from hybridomas were then cloned and out of 72 apical domain positive anti-TfR hybridoma clones, a total 32 unique sequences were identified. Out of 24 apical domain positive anti-TfR hybridoma clones from rats, an additional 21 unique sequences were identified. See Example 8 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0652] Example anti-TfR antibody sequences are shown in Tables 3, 4, 5, 13, 14, and 15.

[0653] Anti-TfR antibodies were then reformatted into a 2+1 bispecific antibody format, wherein a “2+1 bispecific antibody format” or “2+1 bispecific antibody” refers to a trivalent, bispecific antibody format comprising (i) a single antigen-binding domain that binds to human TfR or CD98hc and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains; and wherein the single antigen-binding domain that binds to human TfR or CD98hc is linked to the C-terminus of one of the two antibody heavy chains. Antibodies were selected for reformatting based on various criteria: 1) the antibodies covered a broad range of affinities based on ELISA and FACS binding assays, 2) the antibodies covered a broad range of internalization activity, 3) no outstanding high-risk modification sites were identified in the CDRs, and 4) the antibodies were phylogenetically diverse from each other within the hybridoma sequences obtained. An IgG isotype antibody with no target specificity (“inert isotype control antibody”) with knob-hole mutations in the constant domains of the heavy chains was used for formatting into a 2+1 bispecific antibody format. An example structure of a 2+1 bispecific antibody format comprised the following components: 1) Isotype control hlgGl wildtype antibody with a knob ((T366W) mutation and a hole mutation (T366S L368A Y407V) in the constant regions, 2) a (G4S)x3 linker (SEQ ID NO: 179 - GGGGSGGGGSGGGGS) between the “hole side” of the hlgGl antibody and a scFv, 3) a VH sequence of the scFv, 4) a 20 amino acid linker sequence between the VH and VL of the scFv, and 5) a VL sequence of the scFv. See Example 9 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0654] Once converted into a 2+1 bispecific antibody format, anti-TfR hybridoma antibodies were confirmed to retain TfR binding via ELISA and FACs. In total, 28 out of 32 reformatted antibodies retained binding affinity against TfR in the 2+1 bispecific antibody format. Importantly, only 6 of the 32 antibodies (TfR6, TfR9, TfR12, TfR15, TfR19, TfR27) showed strong cyno cross-reactivity. See Example 10 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0655] Certain 2+1 anti-TfR bispecific antibodies were tested for their ability to be internalized into the hCMEC / D3 cell line. The 2+1 anti-TfR bispecific antibody formats showed a wide range of internalization capability with many showing significantly higher internalization than an anti- TfR control antibody. See Example 11 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0656] The scFv portion of certain anti-TfR antibodies were then chosen for their crossreactivity, function, functional stability and sequence diversity for humanization. Structure-based antibody modeling was applied in the process of humanizing anti-TfR mouse monoclonal antibodies (mAbs) utilizing the BioMOE module of MOE (Molecular Operating Environment, Chemical Computing Group, Montreal, Canada). After the BioMOE module of MOE was used to visualize and classify potential residues for back mutation, the back potential back mutations were classified based on amino acid type difference, interaction potential with CDR residues, impact potential for VL / VH pairing, and potential change in hydrophobic and charged surface area in and near the CDRs. Mutations that were near the CDRs or the VL / VH interface, have a significant charge difference or contain strong H-bond interactions were individually evaluated and the significantly disrupting mutations were reverted back to the original query residues. As a result, humanized sequences may contain up to five back mutations. See Example 12 of PCT / US2023 / 071239, published as WO 2024 / 026472. Certain humanized anti-TfR antibody sequences for TfR9, TfR12, and TfR15 are shown in Tables 3, 4, and 5 above.

[0657] To determine whether the antibodies will transport efficiently into the brain without affecting the expression level or localization of the target receptor, the effect of the 2+1 anti-TfR bispecific antibody formats on recycling of the transferrin receptor in HCMEC / D3 cells were tested by both FACS and Western blot methods. To define effects of the 2+1 anti-TfR bispecific antibody formats on TfR recycling, the Mean Fluorescence Intensity (MFI) for cells treated with each individual 2+1 anti-TfR bispecific antibody format was normalized against that obtained for human IgG isotype. Treatment of HCMEC / D3 cells with the 2+1 anti-TfR bispecific antibody formats resulted in 40-80% reduction in cell-surface expression of TfR via FACs. Antibodies named TfR9 and TfR12, were selected for further engineering and a panel of humanized antibodies were generated, some of which displayed no adverse effect on receptor recycling (i.e., TfR9.1B and TfR9.5B). In contrast, all the clones generated from TfR12 strongly impaired receptor recycling. A panel of humanized antibodies based on TfR15 were generated (see Example 15 of PCT / US2023 / 071239, published as WO 2024 / 026472), and another antibodyclone, TfR15.WH8, was identified that showed no detrimental effects on TfR recycling. See Example 13 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0658] To validate the FACS data, the total amount of TfR protein in the cell lysates obtained from HCMEC / D3 following antibody treatment was quantified by Western blot. Similar to the FACS data, treatment of HCMEC / D3 cells with 2+1 anti-TfR bispecific antibodies had either minor or no effects on the total amount of TfR protein viaWestem blog, with TfR9. IB in particular having no effect on total protein levels. In contrast, the total TfR protein in HCMEC / D3 was strongly reduced upon exposure to humanized antibodies generated from TfR12. See Example 13 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0659] Humanized TfR9.1B was selected for further affinity optimization. TfR9.1B scFv was subjected to overlap extension PCR mutagenesis to generate randomized VH and VL libraries, which were packaged into bacteriophages each expressing a unique VH / VL combination and subsequently screened for TfR binding ability. 46 of the 9. IB scFv variants were selected to clone into a 2+1 bispecific antibody format and were expressed and purified for further characterization. Clone TfR9.1B.39 was chosen as the template for an additional round of random mutagenesis to remove two potential isomerization sites (D34 in VL and D62 in VH). Using biotinylated human and cyno TfR Avi-His protein as bait for panning and ELISA for screening, seven mutants with both potential isomerization sites fixed were selected to clone 2+1 bispecific antibody vector and used for further characterization. See Example 14 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0660] Engineering of humanized TfR.15.WH8.1 variants was carried out to optimize target affinity. See Example 16 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0661] 2+2 bispecific antibody formats were also analyzed, wherein a “2+2 bispecific antibody format” or a “2+2 bispecific antibody” refers to a refers to a tetravalent, bispecific antibody format comprising (i) two antigen-binding domains that bind to human TfR or CD8hc and (ii) an antibody, wherein the antibody comprises two heavy chains and two light chains; wherein one antigen-binding domain that binds to human TfR or CD98hc is linked to the C-terminus of one of the two antibody heavy chains, and the other antigen-binding domain that binds to human TfR or CD98hc is linked to the C-terminus of the other of the two antibody heavy chains.

[0662] Binding kinetics of 2+2 TfR.15.WH8.1 variant bispecific antibodies (Isotype IgG with 2 anti-TfR scFvs) to Avi-His tagged human and cynomolgus TfR apical domains were evaluated. The results illustrated that these 2+2 anti-TfR bispecific antibodies exhibit a range of affinitiesfrom approximately 3 nM to >2.5 pM for TfR apical domain binding. In particular, the affinity of 2+2 anti-TfR bispecific antibodies for binding to human TfR Avi-His ranged from 6 nM to >2.5 pM; and affinity of anti-TfR antibodies of the present disclosure for binding to cynomolgus TfR Avi-His ranged from 3 nM to >2.5 pM. See Example 17 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0663] Binding kinetics of certain humanized 2+1 anti-TfR bispecific antibodies to human and cyno TfR Avi-His were evaluated. The equilibrium dissociation constants (KD) were calculated from the fitted association and dissociation rate constants (k-on and k-off) for anti-TfR Avi-His antibodies of the present disclosure. The values were combined, means and standard deviation calculated. Some antibodies displayed heterogeneous binding profiles that do not fit to a 1 :1 binding model, thus their rate constants could not be determined.Table 11. Equilibrium dissociation constants (KD) for anti-TfR 2+1 bispecific antibodiesN=number of determinations; NT=not tested; NA=not applicable; LB=low binding (below limit of measurement); NB=no binding detected

[0664] The results of these studies illustrated that these 2+1 anti-TfR bispecific antibodies exhibit a range of affinities from approximately 6 nM to 900 nM for TfR Avi-His. In particular, affinity of these 2+1 anti-TfR bispecific antibodies for binding to huTfR Avi-His ranged from 6.7 nM to 340 nM, and affinity of these anti-TfR antibodies for binding to cyno TfR Avi-His ranged from 18 nM to 870 nM. See Example 18 of PCT / US2023 / 071239, published as WO 2024 / 026472.Example 2: Further Studies of 2+1 Anti-TfR Bispecific Antibodies

[0665] To establish in vivo proof of concept for brain penetration of 2+1 anti-TfR bispecific antibodies, a heterozygous knock-in (KI) mouse was generated in which the ectodomain region of mouse TfR was replaced with that of human in one of the two alleles. This was because the 2+1 anti-TfR bispecific antibodies specifically bind to primate but not mouse TfR. A group of WT and hu-TfRmu / hu mice were dosed with 10 mg / kg of either isotype IgG or 2+1 anti-TfRbispecific antibodies at day 1 and 14 and blood samples were collected from antibody treated animals at different timepoints. By measuring antibody concentrations in the vessel depleted mice brains, levels of 2+1 anti-TfR bispecific antibodies were 4-5 fold higher than isotype IgG in hu-TfRmu / hu mice. In contrast, brain uptake of 2+1 anti-TfR bispecific antibodies in WT animals, which do not express target receptor, was similar to that observed for isotype IgG. These results indicated that the 2+1 anti-TfR bispecific antibodies have enhanced ability for brain entry mediated by the transferrin receptor. See Example 19 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0666] To determine if 2+1 anti-TfR bispecific antibodies dysregulate receptor recycling in target cells in vivo, the total amount of TfR protein was quantified in the vessel portion of brain tissues, collected from antibody treated animals. Using quantitative analysis, no significant differences in TfR band densities were observed between samples obtained from hu-TfR+ / - KI mice that were injected with anti-TfR antibodies (i.e., TfR-9.1B.39 and TfR-15.WH8) compared to those from isotype treated animals, which indicated that 2+1 anti-TfR bispecific antibodies do not alter TfR recycling at the blood-brain barrier (BBB) in vivo. See Example 20 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0667] Anti-TfR antibodies are known to induce cell death in reticulocyte cell population in vivo. To examine this, a group of WT and hu-TfR+ / - KI mice were injected with either 2+1 anti- TfR bispecific antibodies (TfR-9. IB.39 and TfR-15.WH8), a human IgGl isotype or the previously published anti -ms TfR antibody mTfRl (Boado et al, Biotechnol Bioeng. 2009 Mar 1; 102(4): 1251-1258), which is known to drive strong depletion of reticulocytes in mice. Injection of mTfRl antibody resulted in a complete loss of reticulocytes in WT animals, whereas this cell population was not altered in isotype treated animals, regardless of genotype. Similarly, no changes in reticulocytes numbers were observed in WT mice receiving anti-TfR 2+1 bispecific antibodies. In contrast, the hu-TfR+ / - KI mice treated with TfR-9. IB.39 and TfR-15.WH8 displayed ~ 80% to 90% reduction in reticulocytes numbers, respectively. See Example 21 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0668] To investigate the mechanism of reticulocyte depletion seen in 2+1 anti-TfR bispecific antibodies, the ability of these same antibodies was evaluated to elicit in vitro effector responses, such as complement dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Given that anti-TfR antibodies on wt huIgGl backbone have been shown to elicit ADCC responses, alternative Fc formats were tested, such as N325S / L328F (NSLF) andL234A / L235A / P331S (LALAPS), which have strongly decreased binding to activating FcgRs (in particular, FcgRIIIa which is the key driver of ADCC). Two of the 2+1 anti-TfR bispecific antibodies, which elicited ADCC signal (TfR9.1B.39 and TfR15.WH8) were re-expressed in a variety of Fc formats, including wildtype hlgGl, hlgGl NSLF and hlgGl LALAPS and tested in the ADCC assay. In this assay, both NSLF and LALAPS were able to reduce the ADCC signal significantly, with NSLF showing >90% reduction of signal at the highest concentration tested for both 2+1 anti-TfR bispecific antibodies tested. See Example 22 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0669] Binding kinetics of 2+1 anti-TfR bispecific antibodies to human TfR Avi-His were evaluated using a GatorBio BLI instrument (GatorBio, Palo Alto, CA). The equilibrium dissociation constants (KD) were calculated from the fitted association and dissociation rate constants (k-on and k-off) for 2+1 anti-TfR bispecific antibodies. The values were combined, means and standard deviation calculated, and graphs prepared using GraphPad Prism. Some antibodies, as indicated in Table 12, displayed heterogeneous binding profiles that do not fit to a 1 : 1 binding model, thus their rate constants could not be determined. The KD values are summarized in Table 12 below.Table 12. Equilibrium dissociation constants (KD) for anti-TfR 2+1 bispecific antibodiesN=number of determinations; NT=not tested; NA=not applicable; LB=low binding (below limit of measurement); NB=no binding detected.

[0670] These results illustrate that 2+1 anti-TfR bispecific antibodies exhibit a range of affinities from approximately 30 nM to 4 pM for TfR Avi-His. Affinity of 2+1 anti-TfR bispecific antibodies for binding to human TfR Avi-His ranged from 65 nM to 3.5 pM; affinity of 2+1 anti-TfR bispecific antibodies for binding to cyno TfR Avi-His ranged from 38 nM to 2.3 pM. See Example 23 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0671] In order to evaluate the pharmacokinetics and pharmacy odynamics of 2+1 anti-TfR bispecific antibodies, a 2+1 anti-TfR bispecific antibody was generated with: (i) TfR9.1B.39.38 scFv; and (ii) a monoclonal antibody specific for human MS4A4A (referred to as “TD1”) in a 2+1 (hole) bispecific antibody format (referred to as “TDl-TfR”). To confirm that TDl-TfR retains the function of the parental TD1 (anti -human MS4A4A) antibody, a soluble TREM2 (sTREM2) assay was conducted using human monocyte-derived macrophages. TDl-TfR showed activity by increasing sTREM2 levels in an in vitro assay, as compared to hlgGl and Iso-TfR. This data indicates that addition of the anti-TfR scFv to the TD1 antibody did not impair function of TD1. See Example 24 of PCT / US2023 / 071239, published as WO 2024 / 026472.Example 3: NHP Studies for Antigen-Binding Domains against Human Transferrin Receptor

[0672] A PK / PD study was conducted in naive male cynomolgus monkeys (n=12) following intravenous (iv) dosing of 20 mg / kg of each of Iso-TfR, TDl-TfR, TD1 and huIgGl (isotype control) on Day 1 and 29 (total of 2 administrations). At pre-dose and following dose administration on days 3, 8, 15, 29, and 31, blood samples (~2 mL) for hematology were collected from the femoral vein. Overall, no notable changes were observed in any hematology parameters, and no consistent reduction in reticulocytes was observed over the course of the study. See Examples 25 and 26 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0673] Cerebrospinal fluid (CSF) and serum were collected from the NHPs at various times following the first and second administrations and tested for antibody levels. An increased CSF Cmax was seen with TDl-TfR, when compared to TD1. An increased CSF Cmax was also seen with Iso-TfR when compared to isotype control. Further, TDl-TfR demonstrated faster serum clearance in NHPs as compared to either Iso-TfR or TD1 alone. See Example 27 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0674] Antibody levels were also assessed directly in multiple NHP brain regions (frontal cortex, entorhinal cortex and hippocampus) using vessel-depleted brain lysates. The TfR BBB targeting arm was able to increase brain uptake of (i) TDl-TfR compared to TD1 in all 3 brain regions tested (ranging from a 2- to 11 -fold increase) and (ii) Iso-TfR compared to isotypeparental antibody in all 3 brain regions tested (ranging from an 8 to 64 fold increase). See Example 28 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0675] CSF and serum from NHP test subjects and NHP brain lysates were further tested for downstream biomarkers of TD1 function, including sTREM2 and CSF1R. Serum levels of soluble TREM2 (sTREM2) were increased in NHPs treated with TDl-TfR as compared to TD1 alone, Iso-TfR or hlgGl following administration of the first and the second dose. CSF-1 levels were increased in CSF from NHPs treated with TDl-TfR as compared to TD1 alone, Iso-TfR or hlgGl. These data showed that the enhanced brain penetration also led to a stronger pharmacodynamic response. See Example 29 of PCT / US2023 / 071239, published as WO 2024 / 026472.

[0676] Anti-TfR antibodies have been shown to be able to degrade the receptor (Niewoehner et al, Neuron 2014 8:49-60), which is an undesirable side effect of a BBB-penetrating anti-TfR molecule. In order to test for this effect brain tissue samples were collected from the non-human primates dosed with 2+1 anti-TfR bispecific antibody and control antibodies and total TfR levels were quantified by an MSD method. Quantitative data analysis revealed a slight decline in TfR expression levels in the brain tissues of animals treated with 2+1 anti-TfR bispecific antibodies compared to those injected with either hlgGl Isotype control antibody or TD1 antibody alone that was not statistically significant. These results show that 2+1 anti-TfR bispecific antibodies disclosed herein do not have a significant impact on TfR expression in vivo. See Example 30 of PCT / US2023 / 071239, published as WO 2024 / 026472.Example 4: Generation of Affinity-Engineered Anti-TfR Antibodies

[0677] To generate affinity-engineered TfR antibodies, humanized anti-TfR antibodies TfR.9.1B.39.38, TfR.15.WH8.E24A, and TfR.15.WH8.1.42Q were selected for further affinity tuning. The generation of humanized antibodies TfR.9.1B.39.38, TfR.15. WH8.1.24 A and TfR.15.WH8.1.42Q was described in International Publication No. WO2024 / 026472. Amino acid substitutions at selected positions in the CDRs were performed to generate affinity variants. The variants were expressed in Expi293 culture, and either the supernatant or purified protein was used for screening.

[0678] The antibody sequences of the affinity-engineered anti-TfR antibodies (or "affinity variants") are provided in Tables 13-15 below.Table 13. Heavy Chain CDR Sequences of Anti-TfR AntibodiesTable 14. Light Chain CDR Sequences of Anti-TfR AntibodiesTable 15. VH and VL Sequences of Anti-TfR AntibodiesExample 5: Assessment of Affinity of Anti-TfR Binding Domains

[0679] To assess the affinity of anti-TfR antibodies, a Biacore T200 instrument was used to evaluate the binding kinetics (Global Life Sciences Solutions USA LLC, Marlborough, MA). The anti-TfR antibodies were formatted as Fabs with a monovalent Fc. Anti-TfR antibodies were prepared by dilution in running buffer HBS-EP+ (Teknova) with BSA (MP Biomedicals) and captured by an anti-Fab antibody (Cat. 28958325, Cytiva) immobilized on a CM4 Sensor S Series sensor chip surface (Cytiva) that was prepared according to the manufacturer’s recommendations. A fixed concentration of molecules was captured on flow cell. A concentration series of human or cyno apical TfR analyte was then injected over all four flow cells, followed by dissociation. Blank injections were performed and used for double-referencing. All surfaces were regenerated with 10 mM glycine pH 2.1 buffer (Cytiva) at the end of each injection cycle. Data were processed and analyzed using Biacore T200 BiaEvaluation software (Cytiva). Results for the various anti-TfR binding domains are shown in Table 16.Table 16. Affinities of anti-TfR antibodies in (Fab format)Example 6: Nonspecific Binding Assay for Anti-TfR Antibodies

[0680] To assess nonspecific binding of the anti-TfR antibodies with varying affinities ("affinity variants"), an ELISA assay was performed to assess binding to baculovirus particles (BVP) and double stranded DNA (dsDNA). BVP particles derived from insect cells contain phospholipid, carbohydrate, glycoproteins, extracellular matrix, nucleic acids, viral capsid, allowing for detection of electrostatic and hydrophobic interactions (Hotzel et al., mAbs, 2012). dsDNA is one of the agents used in ELISA assay to study polyreactivity in natural antibodyrepertoires during B-cell maturation. A majority of the antibodies expressed from early immature B cells showed ELISA binding to dsDNA, ssDNA, insulin, and LPS (Wardemann et al., 2003).

[0681] An ELISA plate was coated with either BVP particles or dsDNA, washed and incubated with blocking buffer, washed and incubated with the anti-TfR antibodies tested, followed by detection with anti-human IgG HRP antibody. A BVP score was calculated from the OD450 value of the sample wells to the background (no protein) wells.

[0682] Anti-TfR affinity variants in Table 17 were tested for nonspecific binding to BVP and dsDNA. In the BVP binding assay, the following concentrations of each antibody were tested: 1 pM, 0.33 pM, 0.11 pM or 0.037 pM. Control antibodies (isotype, negative and positive control) were also tested at these concentrations. In the dsDNA binding assay, antibodies were tested at 10 pg / mL.Table 17. Anti-TfR Antibodies Tested for Non-specific Binding to BVP and dsDNA

[0683] As shown in FIGs. 3 A and 3B, all anti-TfR affinity variants showed low BVP scores. As shown in FIGs. 4A and 4B, all anti-TfR affinity variants showed low dsDNA binding.Example 7: Assessment of Characteristics for Manufacturing of Anti-TfR Affinity Variants

[0684] The physiochemical and biochemical properties of a protein can impact protein stability and can pose potential risks during manufacturing. In this study, an assessment was conducted by testing selected anti-TfR antibodies in IgG format, in order to determine and identify thesecharacteristics. Several analytical techniques were utilized as shown in Table 18 below, demonstrating that the properties of those selected anti-TfR antibodies met the criteria for further development.Table 18: Overview of Characteristics for Selected Anti-TfR Affinity VariantsExample 8: In-vitro Cell Uptake of Anti-TfR Affinity Variants

[0685] Anti-human TfR affinity variant antibodies were assessed for extent of cell uptake using hCMEC / D3 cells in an acute 2-hour incubation assay. Anti-TfR affinity variants were fused to an N-terminus of a monovalent Fc (mvFc), with an isotype control scFv fused to the C-terminus of the mvFc ("anti-TfR Fab-mvFc-Iso-scFv") and were assessed by a 2-point titration at 500 nM and 100 nM concentrations. Anti-TfR antibodies H6-4, H3-7, 24A, L7-2, LIO-16, 24A.42Q, L10- 1, L-35, LI 0-8, and 42Q and an isotype control were tested. FIG. 5 shows the detection of the anti-TfR antibodies at 500 nM concentration. Cell uptake was observed for anti-TfR antibodies but not for the isotype control antibody (in an Iso Fab-mvFc - Iso scFv format). Generally, the extent of cell uptake correlated with the affinity of the antibodies (see FIG. 5). The cell uptake was proportionally weaker when incubated at the lower dose of 100 nM (data not shown).Example 9: Brain Uptake of Anti-TfR Affinity Variants

[0686] In vivo studies were conducted to determine the brain penetration of a panel of anti-TfR antibodies with varying affinities to human transferrin receptor (TfR) apical domain ranging from double digit nM to single digit uM (see Table 16). huTfR knockin (KI) mice were injected with a single dose of antibody in a monovalent format to determine the brain uptake at the 24-hour timepoint. Brain uptake was determined from both vessel-depleted and whole brain fractions to assess whether the higher affinity anti-TfR antibodies were retained in the brain vasculature.

[0687] To produce vessel depleted brain fractions, brain tissues were minced and homogenized in HBSS buffer (MilliporeSigma #55037C) containing 10 mM HEPES (#15630130, Gibco) using a hand-operated tissue grinder. Subsequently, samples were centrifuged to separate the vessel portion from the parenchymal portion, lysed in RIPA with protease inhibitors, and total protein concentration in the lysates was determined by BCA Assay (Pierce #23225). Lysates were subsequently frozen on dry ice and stored at -80°C until analysis.

[0688] Antibody concentrations in the brain were measured using an MSD (Meso Scale Discovery) assay. Antibody concentrations were normalized to the total protein concentration in the lysate and then graphed as either antibody concentration (ng antibody / mg total protein) or fold-change over the format matched isotype control antibody.

[0689] At 24-hour post i.v. injection, an increase in brain uptake in vessel depleted brain was observed for all anti-TfR antibodies as compared to the format-matched isotype control (FIGs. 6A and 6B). FIG. 6A shows brain uptake of various anti-TfR affinity variants (administered at 5 mg / kg) after 24 hrs in vessel-depleted brain. FIG. 6B shows the fold change over the matched isotype control in vessel-depleted brain.

[0690] In addition, whole brain antibody concentration was determined, and a ratio of vessel depleted to whole brain antibody concentration was calculated (Figure 7). The data indicates that some fraction of the higher affinity anti-TfR antibodies remained in the vessels at the 24 hour timepoint, while the lower affinity TfR-antibodies (such as H3-7) had equivalent antibody levels in vessel depleted or whole brain, indicating that there was not a significant retention in the blood vessels. A similar trend was also observed in another in vivo study with other anti-TfR affinity variants (data not shown).

[0691] Overall, this study showed that the anti-TfR antibodies can drive brain uptake even at a low dose.Example 10: Analysis of Blood Reticulocyte Levels

[0692] To assess the safety of higher affinity TfR antibodies, the population of reticulocytes in whole blood samples at 24 hours after administration was assessed. Whole blood samples from animals dosed with anti-TfR affinity variants or isotype control antibodies were drawn from the animals prior to perfusion and collected into K2EDTA tubes. A hematological analysis was performed (Idexx Laboratories (Fremont CA)). A fully automated diagnostic instrumentSYSMEX XT-V was used for routine hematology testing. The complete blood count (CBC) and reticulocyte count were measured by flow cytometry.

[0693] As shown in FIG. 8, no significant effect on blood reticulocyte levels was observed among groups in this experiment at the time point sampled. Although some variability is observed, there were no substantial decreases in reticulocyte levels or associated acute clinical symptoms.Example 11: Assessment of TfR Degradation by Anti-TfR Affinity Variants

[0694] High-affinity TfR binders have been reported to drive TfR to the lysosome and induce TfR degradation (Bien-Ly, N. et al., J. Exp. Med. 2014 Feb 10;211(2):233-44.), which is undesirable as it may impact the capacity of TfR to transport its natural ligand and any cargo attached to the anti-TfR antibody. To determine whether the anti-TfR affinity variants led to TfR degradation, the whole brain lysate from the in vivo study described above in Example 9 were prepared in RIPA buffer with protease and phosphatase inhibitors. Total protein concentration in the lysates were determined by BCA assay (Pierce #23225), and 40 pg of each sample were separated by SDS-PAGE. The gels were transferred to nitrocellulose membranes for immunoblot. An anti-TfR antibody detecting both human and mouse TfR (Thermo #13-6800) and mouse anti-GAPDH (Millipore MAB374) were incubated overnight. Images were captured using a Biorad ChemiDoc system and analyzed with Image Lab 6.1 software. Band intensities for TfR were normalized to levels of GAPDH for each sample and the isotype control mean.

[0695] FIG. 9 shows that TfR levels in animals treated with anti-TfR antibodies L-21, L-6, and L-35 were reduced in comparison to the isotype controls; however, this difference was not statistically significant. The TfR levels in anti-TfR antibodies 42Q, 24A, and 39.38 also did not decrease significantly. Overall, these results suggest that the anti-TfR antibodies tested did not significantly degrade TfR, with a subset of the anti-TfR antibodies slightly reducing TfR levels at the 24 hour timepoint and 5 mg / kg dose.Example 12: Engineering of Antigen-Binding Domains against Human CD98 Heavy Chain

[0696] Avi-His tagged variants of the extra-cellular domain (ECD) of human, cynomolgus macaque (cyno), and mouse CD98 heavy chain (CD98hc) were used were used to produce CHO cells stably expressing human CD98hc and mouse CD98hc, whose cell surface expression was confirmed by FACS. See Examples 1 and 2 of PCT / US2023 / 071238, published as WO 2024 / 026471, filed July 28, 2023.Table 19. Avi-His tagged variants of CD98hc ECDTable 20. Full Length CD98hc Sequences

[0697] Humanized mouse lines expressing human extracellular domains of CD98hc were generated (Taconic Biosciences GmbH (Germany)). CRISPR was used to replace the mouse ECD with the human version, while retaining the mouse intracellular and transmembraneportions under the control of the mouse promotor. See Example 3 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0698] To obtain antibodies against CD98hc, BALB / c mice or Sprague Dawley rats were immunized with purified ECD polypeptides of human, cyno and / or mouse CD98hc. Sera from the animals were analyzed for reactivity to CD98hc (human, mouse or cyno), and lymphocytes from animals whose sera demonstrated strong CD98hc binding were isolated and fused with myeloma fusion partners to produce hybridoma cell lines. See Example 4 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0699] From four hybridoma campaigns, 121 hybridoma clones displaying an MFI ratio greater than 2-fold for binding to CHO cells overexpressing human CD98hc, 120 out of the 121 anti- CD98hc hybridoma clones were found to bind to human and cyno CD98hc with OD450 values greater than 0.45. One hybridoma clone, CD98HC.02.020, bound to mouse CD98hc. See Examples 5 and 6 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0700] Anti-CD98hc antibodies were then tested for their ability to internalize into a bloodbrain barrier endothelial cell line (hCMEC / D3) (see Example 7 of PCT / US2023 / 071238, published as WO 2024 / 026471), and subsequently cloned (see Example 8 of PCT / US2023 / 071238, published as WO 2024 / 026471). Certain anti-CD98hc antibodies were formatted into a 2+ 1 bispecific antibody format. See Example 9 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0701] CD98hc binding retention of the 2+1 bispecific antibodies was tested and cross reactivity to mouse CD98hc and cyno CD98hc was also tested, along with ability to internalize into the hCMEC / D3 cell line. Examples 11-13 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0702] Binding kinetics of the 2+1 anti-CD98hc bispecific antibodies were tested and they were found to exhibit a range of affinities from approximately 3 nM to 200 nM for CD98hc Avi- His binding. In particular, affinity of 2+1 anti-CD98hc bispecific antibodies for binding to human CD98hc Avi-His ranged from 3.1 nM to 210 nM; affinity of anti-CD98hc antibodies of the present disclosure for binding to cyno CD98hc Avi-His ranged from 3.2 nM to 145 nM. See Example 15 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0703] The 2+ 1 anti-CD98hc bispecific antibodies were then tested for whether they affect any dysregulation of cell surface CD98hc expression and no reduction in cell-surface expression of CD98hc was found. However, certain antibodies were found to result in increased CD98hc cellsurface expression and those antibodies were excluded from further development. See Example 16 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0704] Studies in mice with the anti-CD98hc bispecific antibodies showed that after antibody administration, antibody concentrations in vessel depleted brains, levels of 2+1 anti-CD98hc bispecific antibodies were determined to be up to 2-fold higher than isotype IgG in HET huCD98hc KI mice and three of the 2+1 anti-CD98hc bispecific antibodies (CD98hc.04.062, CD98hc.04.063, CD98hc.04.064) tested showed a significant (5-17) fold change in brain / serum ratio at the 24 hour timepoint tested. See Example 17 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0705] CD98hc.04.064 was humanized and further optimized to remove liabilities and optimize affinity, resulting in a 4.064 humanized panel that bound human CD98hc at a range of about 18 nM to 35 nM and cynomolgus CD98hc at a range of about 340 nM to 1.5 pM. Examples 18-21 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0706] Certain exemplary anti-CD98hc antibody sequences are shown in Tables 6-8 above.

[0707] One member of the CD98hc.04.064 humanized panel (CD98hc.04.064. le) was formatted into a bi-specific antibody with an antibody specific for human MSA4A (TD1), in a 2+1 (hole) bispecific antibody format (TDl-CD98hc), which was intravenously injected into naive male cynomolgus monkeys, along with a TD1 control and human IgGl, where no notable changed were observed in hematology parameters, including no consistent reduction in reticulocytes. See Examples 25 and 26 of PCT / US2023 / 071238, published as WO 2024 / 026471.

[0708] The CD98hc BBB targeting arm was further seen to be able to increase brain uptake of the TDl-CD98hc compared to TD1 alone up to 3 -fold in the frontal cortex, and up to 4-fold in the entorhinal cortex, with no significant increase seen in the hippocampus, and was further able to increase serum levels of soluble TREM2. See Examples 27-29 of PCT / US2023 / 071238, published as WO 2024 / 026471.Example 13: Engineering of GCase Enzyme

[0709] In order to generate GCase variants that possess improved enzyme activity, better stability, pharmacokinetics, and / or manufacturability, a previously published GC...

Claims

WHAT IS CLAIMED IS:

1. A complex comprising (i) an antigen-binding domain that specifically binds to human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) a GCase polypeptide or fragment thereof.

2. The complex of claim 1, wherein the complex comprises a first polypeptide comprising the GCase polypeptide or fragment thereof and a first Fc domain; a second polypeptide comprising a VH of the antigen-binding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigen-binding domain.

3. The complex of claim 2, wherein the GCase polypeptide or fragment thereof is N- terminal to the first Fc domain.

4. The complex of claim 3, wherein the first polypeptide further comprises an antibody hinge between the GCase polypeptide or fragment thereof and the first Fc domain.

5. The complex of claim 2, wherein the GCase polypeptide or fragment thereof is C- terminal to the second Fc domain.

6. The complex of any one of claims 2-5, wherein the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation.

7. The complex of any one of claims 2-5, wherein the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

8. The complex of any one of claims 2-7, wherein the second polypeptide is an antibody heavy chain.

9. The complex of any one of claims 2-8, wherein the third polypeptide further comprises a CL domain, optionally wherein the third polypeptide is an antibody light chain.

10. The complex of any one of claims 2-9, wherein the first Fc domain comprises a CH2 and a CH3 and / or wherein the second Fc domain comprises a CH2 and a CH3.

11. The complex of claim 2, wherein the complex comprises a first polypeptide comprising an antibody heavy chain and a GCase polypeptide or fragment thereof and a secondpolypeptide comprising an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise the antigen-binding domain.

12. The complex of claim 11, wherein the GCase polypeptide or fragment thereof is C- terminal to the antibody heavy chain.

13. The complex of claim 11, wherein the complex further comprises a third polypeptide comprising a Fc domain.

14. The complex of claim 13, wherein the antibody heavy chain comprises a knob mutation and the Fc domain comprises a hole mutation.

15. The complex of claim 13, wherein the antibody heavy chain comprises a hole mutation and the Fc domain comprises a knob mutation.

16. The complex of any one of claims 13-15, wherein the Fc domain comprises a CH2 and a CH3.

17. The complex of claim 1, wherein the antigen-binding domain and the GCase polypeptide or fragment thereof are contained in a single polypeptide.

18. The complex of claim 17, wherein the GCase polypeptide or fragment thereof and the antigen-binding domain are directly connected via a peptide bond.

19. The complex of claim 17, wherein the GCase polypeptide or fragment thereof and the antigen-binding domain are connected via a linker.

20. The complex of claim 19, wherein the linker is a peptide linker.

21. The complex of any one of claims 17-20, wherein the GCase polypeptide or fragment thereof is N-terminal to the antigen-binding domain.

22. The complex of any one of claims 17-20, wherein the GCase polypeptide or fragment thereof is C-terminal to the antigen-binding domain.

23. The complex of any one of claims 17-22, further comprising an Fc domain.

24. The complex of claim 23, wherein the Fc domain comprises a CH2 and a CH3.

25. The complex of claim 23 or 24, wherein the antigen-binding domain and the GCase polypeptide or fragment thereof are both N-terminal to the Fc domain.

26. The complex of claim 23 or 24, wherein the antigen-binding domain and the GCase polypeptide or fragment thereof are both C-terminal to the Fc domain.

27. The complex of claim 23 or 24, wherein the antigen-binding domain is N-terminal to the Fc domain and the GCase polypeptide or fragment thereof is C-terminal to the Fc domain or fragment thereof.

28. The complex of claim 23 or 24, wherein the GCase polypeptide or fragment thereof is N- terminal to the Fc domain or fragment thereof and the antigen-binding domain is C- terminal to the Fc domain or fragment thereof.

29. The complex of any one of claims 1-28, wherein the GCase polypeptide or fragment comprises one or more mutations.

30. The complex of any one of claims 1-29, wherein the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of: L34P, T36Q, S38A, R47K, L51R, H60W, Q70H, V78I, L91I, A95K, N102D, N102E, L103E, L103N, L103R, G115E, A124G, I130T, D140G, D140N, Q143E, H145K, H145R, H162K, L165Q, Q166A, A168S, S181A, T183A, H184F, H184L, L185M, V191M, S196T, I204K, R211N, E222K, K224G, K224N, Q226T, L241I, S242P, H262N, H262Y, D263N, E272L, E272Q, T272S, H274K, H274R, N275D, L286S, K293Q, T297S, E300R, H313N, K321E, A322D, N333D, T334F, T334K, T334Y, M335F, V343T, K346H, W351C, F355A, L356F, K360A, K360N, M361E, S364A, T369E, N370D, L372N, H374W, V376T, A380C, A380T, N386D, R395K, H404K, I406A, I406T, I407C, K408R, T410E, L420I, L420M, S431A, V437S, Q440E, M450Q, H451N, S465D, K473W, A476D, L480I, D482C, I483V, D484C, H490K, S494C, H495R, S503C, R534C, R534H, R534N, and combinations thereof as compared to SEQ ID NO:241.

31. The complex of claim 30, wherein the GCase polypeptide or fragment thereof comprises a mutation selected from the group consisting of V78I, N102E, I130T, A168S, V191M, R211N, Q226T, L241I, S242P, V343T, M361E; H374W, and combinations thereof as compared to SEQ ID NO:241.

32. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof does not comprise any of the following mutations L34P, K224G, K224N, T369E and N370D relative to SEQ ID NO: 241 or wherein the GCase polypeptide or fragment thereof does not comprise the amino acid sequence of SEQ ID NO: 246.

33. The complex of any one of claims 1-32, wherein the GCase polypeptide or fragment thereof comprises the mutations W351C and A380C relative to SEQ ID NO: 241.

34. The complex of any one of claims 1-28, wherein the GCase polypeptide or fragment thereof comprises the sequence of SEQ ID NO:265.

35. The complex of any one of claims 1-34, wherein the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:241 when expressed under the same conditions.

36. The complex of any one of claims 1-35, wherein the GCase polypeptide or fragment thereof has greater monomeric purity than a GCase polypeptide of SEQ ID NO:243 when expressed under the same conditions.

37. The complex of any one of claims 1-36, wherein the GCase polypeptide or fragment thereof has increased protein expression in CHO cells as compared to wildtype human GCase.

38. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises one of more of the following mutations relative to SEQ ID NO:241 a. V78I b. N102E c. I130T d. A168S e. V191M f. R211N g. Q226T h. L241I i. S242P j. V343Tk. M36 IE; and l. H374W; wherein the human GCase polypeptide or fragment thereof does not comprise any of the following mutations relative to SEQ ID NO: 241 : L34P; K224N / G, T369E and N370D.

39. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the V78I mutation as compared to SEQ ID NO:241.

40. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the N102E mutation as compared to SEQ ID NO:241.

41. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the I130T mutation as compared to SEQ ID NO:241.

42. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the A168S mutation as compared to SEQ ID NO:241.

43. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the V191M mutation as compared to SEQ ID NO:241.

44. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the R21 IN mutation as compared to SEQ ID NO:241.

45. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the Q226T mutation as compared to SEQ ID NO:241.

46. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the L241I mutation as compared to SEQ ID NO:241.

47. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the S242P mutation as compared to SEQ ID NO:241.

48. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the V343T mutation as compared to SEQ ID NO:241.

49. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the M361E mutation as compared to SEQ ID NO:241.

50. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the H374W mutation as compared to SEQ ID NO:241.

51. The complex of any one of claims 1-31, wherein the Gcase polypeptide or fragment thereof comprises an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs:242, 245, 247, and 267-279.

52. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof comprises the amino acid sequence any one of SEQ ID NOs: 242, 245, 247, and 267-279.

53. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof does not comprise the amino acid sequence of SEQ ID NO: 246.

54. The complex of any one of claims 1-53, wherein the GCase polypeptide or fragment thereof is capable of hydrolyzing glucosylceramide.

55. The complex of any one of claims 1-54, wherein the GCase polypeptide or fragment thereof has greater than 60%, 70%, 80%, or 90% monomer purity after one step of Pro A purification.

56. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof has greater stability in human serum than wildtype human GCase or Cerezyme.

57. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof has a melting temp (Tm) greater than 60° C.

58. The complex of any one of claims 1-31, wherein the GCase polypeptide or fragment thereof has an aggregation temperature (Tagg) greater than 65° C.

59. The complex of any one of claims 1-58, wherein the antigen binding domain is a Fab.

60. The complex of any one of claims 1-58, wherein the antigen-binding domain is an scFv.

61. The complex of any one of claims 1-58, wherein the antigen-binding domain is a VHH.

62. The complex of any one of claims 1-61, wherein the antigen-binding domain that specifically binds to human TfR comprises heavy chain variable region (VH)complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:(i) SEQ ID NOs: 8, 11, 24; and 40, 55, and 61; respectively;(ii) SEQ ID NOs: 8, 11, 25; and 41, 55, and 61; respectively;(iii) SEQ ID NOs: 8, 12, 26; and 42, 55, and 61; respectively;(iv) SEQ ID NOs: 8, 12, 27; and 42, 55, and 61; respectively;(v) SEQ ID NOs: 8, 13, 25; and 42, 55, and 61; respectively;(vi) SEQ ID NOs: 8, 14, 25; and 42, 55, and 61; respectively;(vii) SEQ ID NOs: 8, 15, 25; and 43, 55, and 61; respectively;(viii) SEQ ID NOs: 8, 16, 25; and 42, 55, and 61; respectively;(ix) SEQ ID NOs: 8, 17, 25; and 44, 55, and 61; respectively;(x) SEQ ID NOs: 9, 18, 28; and 45, 56, and 62; respectively;(xi) SEQ ID NOs: 9, 19, 28; and 45, 56, and 62; respectively;(xii) SEQ ID NOs: 9, 20, 28; and 46, 57, and 62; respectively;(xiii) SEQ ID NOs: 9, 20, 28; and 46, 58, and 62; respectively;(xiv) SEQ ID NOs: 9, 20, 28; and 47, 59, and 62; respectively;(xv) SEQ ID NOs: 9, 21, 28; and 46, 57, and 62; respectively;(xvi) SEQ ID NOs: 9, 21, 28; and 47, 59, and 62; respectively;(xvii) SEQ ID NOs: 9, 22, 28; and 46, 57, and 62; respectively;(xviii) SEQ ID NOs: 9, 22, 28; and 46, 58, and 62; respectively;(xix) SEQ ID NOs: 9, 22, 28; and 47, 59, and 62; respectively;(xx) SEQ ID NOs: 10, 22, 28; and 46, 58, and 62; respectively;(xxi) SEQ ID NOs: 10, 22, 30; and 46, 58, and 62; respectively;(xxii) SEQ ID NOs: 10, 22, 31; and 46, 58, and 62; respectively;(xxiii) SEQ ID NOs: 10, 22, 32; and 46, 58, and 62; respectively;(xxiv) SEQ ID NOs: 10, 22, 33; and 46, 58, and 62; respectively;(xxv) SEQ ID NOs: 10, 22, 34; and 46, 58, and 62; respectively;(xxvi) SEQ ID NOs: 10, 22, 35; and 46, 58, and 62; respectively;(xxvii) SEQ ID NOs: 10, 22, 36; and 46, 58, and 62; respectively;(xxviii) SEQ ID NOs: 10, 22, 37; and 46, 58, and 62; respectively;(xxix) SEQ ID NOs: 10, 22, 38; and 46, 58, and 62; respectively;(xxx) SEQ ID NOs: 10, 22, 39; and 46, 58, and 62; respectively;(xxxi) SEQ ID NOs: 10, 22, 28; and 49, 58, and 62; respectively;(xxxii) SEQ ID NOs: 10, 22, 28; and 50, 58, and 62; respectively;(xxxiii) SEQ ID NOs: 10, 22, 28; and 51, 58, and 62; respectively;(xxxiv) SEQ ID NOs: 10, 22, 28; and 52, 58, and 62; respectively;(xxxv) SEQ ID NOs: 10, 22, 28; and 53, 58, and 62; respectively; or(xxxvi) SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively.

63. The complex of claim 62, wherein the antigen-binding domain that specifically binds to human TfR comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 64 and 129, respectively;(ii) SEQ ID NOs: 65 and 130, respectively;(iii) SEQ ID NOs: 66 and 131, respectively;(iv) SEQ ID NOs: 67 and 130, respectively;(v) SEQ ID NOs: 68 and 131, respectively;(vi) SEQ ID NOs: 69 and 130, respectively;(vii) SEQ ID NOs: 70 and 131, respectively;(viii) SEQ ID NOs: 71 and 130, respectively;(ix) SEQ ID NOs: 72 and 131, respectively;(x) SEQ ID NOs: 73 and 130, respectively;(xi) SEQ ID NOs: 74 and 131, respectively;(xii) SEQ ID NOs: 75 and 132, respectively;(xiii) SEQ ID NOs: 76 and 131, respectively;(xiv) SEQ ID NOs: 77 and 132, respectively;(xv) SEQ ID NOs: 77 and 133, respectively;(xvi) SEQ ID NOs: 78 and 134, respectively;(xvii) SEQ ID NOs: 77 and 135, respectively;(xviii) SEQ ID NOs: 75 and 136, respectively;(xix) SEQ ID NOs: 77 and 137, respectively;(xx) SEQ ID NOs: 77 and 138, respectively;(xxi) SEQ ID NOs: 79 and 131, respectively;(xxii) SEQ ID NOs: 77 and 139, respectively;(xxiii) SEQIDNOs: 77 and 131, respectively;(xxiv) SEQIDNOs: 80 and 140, respectively;(xxv) SEQIDNOs: 81 and 141, respectively;(xxvi) SEQIDNOs: 82 and 131, respectively;(xxvii) SEQ ID NOs: 83 and 142, respectively;(xxviii) SEQ ID NOs: 77 and 143, respectively;(xxix) SEQIDNOs: 75 and 131, respectively;(xxx) SEQIDNOs: 75 and 144, respectively;(xxxi) SEQIDNOs: 77 and 145, respectively;(xxxii) SEQ ID NOs: 84 and 131, respectively;(xxxiii) SEQ ID NOs: 75 and 146, respectively;(xxxiv) SEQ ID NOs: 85 and 131, respectively;(xxxv) SEQIDNOs: 86 and 138, respectively;(xxxvi) SEQ ID NOs: 79 and 139, respectively;(xxxvii) SEQ ID NOs: 77 and 147, respectively;(xxxviii) SEQ ID NOs: 75 and 148, respectively;(xxxix) SEQ ID NOs: 87 and 131, respectively;(xl) SEQIDNOs: 88 and 131, respectively;(xli) SEQIDNOs: 75 and 149, respectively;(xlii) SEQIDNOs: 89 and 150, respectively;(xliii) SEQIDNOs: 90 and 151, respectively;(xliv) SEQIDNOs: 77 and 152, respectively;(xlv) SEQIDNOs: 79 and 153, respectively;(xlvi) SEQIDNOs: 77 and 139, respectively;(xlvii) SEQIDNOs: 91 and 131, respectively;(xlviii) SEQIDNOs: 92 and 131, respectively;(xlix) SEQIDNOs: 79 and 154, respectively;(1) SEQIDNOs: 93 and 155, respectively;(li) SEQIDNOs: 80 and 131, respectively;(lii) SEQIDNOs: 94 and 131, respectively;(liii) SEQIDNOs: 95 and 131, respectively;(liv) SEQIDNOs: 66 and 156, respectively;(Iv) SEQIDNOs: 97 and 138, respectively;(Ivi) SEQIDNOs: 95 and 156, respectively;(Ivii) SEQ ID NOs: 98 and 157, respectively;(Iviii) SEQ ID NOs: 99 and 157, respectively;(lix) SEQ ID NOs: 100 and 157, respectively;(lx) SEQ ID NOs: 101 and 157, respectively;(Ixi) SEQ ID NOs: 102 and 158, respectively;(Ixii) SEQ ID NOs: 103 and 157, respectively;(Ixiii) SEQ ID NOs: 104 and 159, respectively;(Ixiv) SEQ ID NOs: 105 and 160, respectively;(Ixv) SEQ ID NOs: 106 and 161, respectively;(Ixvi) SEQ ID NOs: 107 and 162, respectively;(Ixvii) SEQ ID NOs: 106 and 163, respectively;(Ixviii) SEQ ID NOs: 108 and 164, respectively;(Ixix) SEQ ID NOs: 106 and 165, respectively;(Ixx) SEQ ID NOs: 108 and 166, respectively;(Ixxi) SEQ ID NOs: 109 and 165, respectively;(Ixxii) SEQ ID NOs: 110 and 167, respectively;(Ixxiii) SEQ ID NOs: 111 and 168, respectively;(Ixxiv) SEQ ID NOs: 112 and 160, respectively;(Ixxv) SEQ ID NOs: 113 and 169, respectively;(Ixxvi) SEQ ID NOs: 113 and 170, respectively;(Ixxvii) SEQ ID NOs: 113 and 171, respectively;(Ixxviii) SEQ ID NOs: 114 and 169, respectively;(Ixxix) SEQ ID NOs: 114 and 171, respectively;(Ixxx) SEQ ID NOs: 115 and 169, respectively;(Ixxxi) SEQ ID NOs: 115 and 170, respectively;(Ixxxii) SEQ ID NOs: 115 and 171, respectively;(Ixxxiii) SEQ ID NOs: 116 and 169, respectively;(Ixxxiv) SEQ ID NOs: 116 and 170, respectively;(Ixxxv) SEQ ID NOs: 116 and 171, respectively; (Ixxxvi) SEQ ID NOs: 117 and 170, respectively;(Ixxxvii) SEQ ID NOs: 118 and 170, respectively;(Ixxxviii) SEQ ID NOs: 119 and 170, respectively;(Ixxxix) SEQ ID NOs: 120 and 170, respectively;(xc) SEQ ID NOs: 121 and 170, respectively;(xci) SEQ ID NOs: 122 and 170, respectively;(xcii) SEQ ID NOs: 123 and 170, respectively;(xciii) SEQ ID NOs: 124 and 170, respectively;(xciv) SEQ ID NOs: 125 and 170, respectively;(xcv) SEQ ID NOs: 126 and 170, respectively;(xcvi) SEQ ID NOs: 127 and 170, respectively;(xcvii) SEQ ID NOs: 117 and 173, respectively;(xcviii) SEQ ID NOs: 117 and 174, respectively;(xcix) SEQ ID NOs: 117 and 175, respectively;(c) SEQ ID NOs: 117 and 176, respectively;(ci) SEQ ID NOs: 117 and 177, respectively; or(cii) SEQ ID NOs: 117 and 178, respectively.

64. The complex of any one of claims 1-61, wherein the antigen-binding domain that specifically binds to human TfR comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 sequences comprising the amino acid sequences of:(i) SEQ ID NOs: 10, 22, 30, 50, 58, and 62, respectively;(ii) SEQ ID NOs: 10, 22, 391, 50, 58, and 62, respectively;(iii) SEQ ID NOs: 10, 22, 28, 392, 58, and 62, respectively;(iv) SEQ ID NOs: 10, 22, 28, 393, 58, and 62, respectively;(v) SEQ ID NOs: 10, 22, 28, 394, 58, and 62, respectively;(vi) SEQ ID NOs: 10, 22, 28, 395, 58, and 62, respectively;(vii) SEQ ID NOs: 10, 22, 28, 50, 58, and 62, respectively;(viii) SEQ ID NOs: 8, 14, 25, 41, 55, and 61, respectively;(ix) SEQ ID NOs: 8, 15, 25, 396, 55, and 61, respectively;(x) SEQ ID NOs: 8, 15, 25, 397, 55, and 61, respectively; or(xi) SEQ ID NOs: 8, 14, 25, 398, 55, and 61, respectively.

65. The complex of claim 64, wherein the antigen-binding domain that specifically binds human TfR comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to the amino acid sequences of:(i) SEQ ID NOs: 118 and 174, respectively;(ii) SEQ ID NOs: 399 and 174, respectively;(iii) SEQ ID NOs: 117 and 400, respectively;(iv) SEQ ID NOs: 117 and 401, respectively;(v) SEQ ID NOs: 117 and 402, respectively;(vi) SEQ ID NOs: 117 and 403, respectively;(vii) SEQ ID NOs: 404 and 174, respectively;(viii) SEQ ID NOs: 101 and 154, respectively;(ix) SEQ ID NOs: 102 and 405, respectively;(x) SEQ ID NOs: 102 and 406, respectively; or(xi) SEQ ID NOs: 101 and 407, respectively.

66. The complex of any one of claims 1-61, wherein the antigen-binding domain that specifically binds to human CD98hc comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of:(i) SEQ ID NOs: 181, 185, 191, 194, 198, and 201; respectively;(ii) SEQ ID NOs: 182, 186, 191, 195, 199, and 202; respectively;(iii) SEQ ID NOs: 183, 187, 192, 196, 200, and 203; respectively;(iv) SEQ ID NOs: 9, 188, 192, 196, 200, and 203; respectively;(v) SEQ ID NOs: 184, 189, 193, 197, 198, and 204; respectively;(vi) SEQ ID NOs: 184, 190, 193, 197, 198, and 204; respectively;(vii) SEQ ID NOs: 184, 232, 193, 197, 198, and 204; respectively;(viii) SEQ ID NOs: 184, 233, 193, 197, 198, and 204; respectively; or(ix) SEQ ID NOs: 184, 234, 193, 197, 198, or 204; respectively.

67. The complex of claim 66, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of:(i) SEQ ID NOs: 205 and 211, respectively;(ii) SEQ ID NOs: 206 and 212, respectively;(iii) SEQ ID NOs: 207 and 213, respectively;(iv) SEQ ID NOs: 208 and 214, respectively;(v) SEQ ID NOs: 209 and 215, respectively;(vi) SEQ ID NOs: 210 and 216, respectively;(vii) SEQ ID NOs: 235 and 216, respectively;(viii) SEQ ID NOs: 236 and 216, respectively; or(ix) SEQ ID NOs: 237 and 216, respectively.

68. The complex of any one of claims 1-61, wherein the antigen-binding domain that specifically binds to human CD98hc comprises a heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and a light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 sequences comprising the amino acid sequences of: a. SEQ ID NOs: 184, 291, 193, 197, 198, and 204, respectively; b. SEQ ID NOs: 184, 292, 193, 197, 198, and 204, respectively; c. SEQ ID NOs: 184, 293, 193, 197, 198, and 204, respectively; d. SEQ ID NOs: 184, 294, 193, 197, 198, and 204, respectively; e. SEQ ID NOs: 184, 295, 193, 197, 198, and 204, respectively; f. SEQ ID NOs: 184, 296, 193, 197, 198, and 204, respectively; g. SEQ ID NOs: 184, 297, 193, 197, 198, and 204, respectively; h. SEQ ID NOs: 184, 190, 193, 318, 198, and 204, respectively; i. SEQ ID NOs: 184, 190, 193, 319, 198, and 204, respectively; j. SEQ ID NOs: 184, 190, 193, 320, 198, and 204, respectively; k. SEQ ID NOs: 184, 190, 193, 321, 198, and 204, respectively; l. SEQ ID NOs: 184, 190, 193, 322, 198, and 204, respectively; m. SEQ ID NOs: 184, 190, 193, 323, 198, and 204, respectively; n. SEQ ID NOs: 184, 190, 193, 324, 198, and 204, respectively; o. SEQ ID NOs: 184, 293, 193, 320, 198, and 204, respectively; p. SEQ ID NOs: 184, 298, 193, 197, 198, and 204, respectively; q. SEQ ID NOs: 184, 190, 299, 197, 198, and 18, respectively; r. SEQ ID NOs: 184, 190, 300, 197, 198, and 204, respectively; s. SEQ ID NOs: 184, 190, 301, 197, 198, and 204, respectively; t. SEQ ID NOs: 184, 190, 302, 197, 198, and 204, respectively; u. SEQ ID NOs: 184, 190, 303, 197, 198, and 204, respectively;v. SEQ ID NOs: 184, 190, 304, 197, 198, and 204, respectively; w. SEQ ID NOs: 184, 190, 305, 197, 198, and 204, respectively; x. SEQ ID NOs: 184, 190, 306, 197, 198, and 204, respectively; y. SEQ ID NOs: 184, 190, 307, 197, 198, and 204, respectively; z. SEQ ID NOs: 184, 190, 193, 325, 198, and 204, respectively; aa. SEQ ID NOs: 184, 190, 193, 326, 198, and 204, respectively; bb. SEQ ID NOs: 184, 190, 193, 327, 198, and 204, respectively; cc. SEQ ID NOs: 184, 190, 193, 328, 198, and 204, respectively; dd. SEQ ID NOs: 184, 190, 193, 329, 198, and 204, respectively; ee. SEQ ID NOs: 184, 190, 193, 330, 198, and 204, respectively; ff. SEQ ID NOs: 184, 190, 193, 197, 198, and 308, respectively; gg. SEQ ID NOs: 184, 190, 193, 197, 198, and 309, respectively; hh. SEQ ID NOs: 184, 190, 193, 197, 198, and 310, respectively; ii. SEQ ID NOs: 184, 190, 193, 197, 198, and 311, respectively; jj. SEQ ID NOs: 184, 190, 193, 197, 198, and 312, respectively; kk. SEQ ID NOs: 184, 190, 193, 197, 198, and 313, respectively;11. SEQ ID NOs: 184, 190, 193, 197, 198, and 314, respectively; mm. SEQ ID NOs: 184, 190, 193, 197, 198, and 315, respectively; nn. SEQ ID NOs: 184, 190, 193, 197, 198, and 316, respectively; oo. SEQ ID NOs: 184, 190, 193, 197, 198, and 317, respectively; pp. SEQ ID NOs: 290, 190, 193, 197, 198, and 204, respectively; or qq. SEQ ID NOs: 290, 190, 193, 197, 198, and 317, respectively.

69. The complex of claim 68, wherein the antigen-binding domain comprises a VH and a VL, wherein the VH and VL comprise amino acid sequences at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequences of:(i) SEQ ID NOs: 331 and 216, respectively;(ii) SEQ ID NOs: 332 and 216, respectively;(iii) SEQ ID NOs: 333 and 216, respectively;(iv) SEQ ID NOs: 334 and 216, respectively;(v) SEQ ID NOs: 335 and 216, respectively;(vi) SEQ ID NOs: 336 and 216, respectively;(vii) SEQ ID NOs: 337 and 216, respectively;(viii) SEQ ID NOs: 210 and 338, respectively;(ix) SEQ ID NOs: 210 and 339, respectively;(x) SEQ ID NOs: 210 and 340, respectively;(xi) SEQ ID NOs: 210 and 341, respectively;(xii) SEQ ID NOs: 210 and 342, respectively;(xiii) SEQ ID NOs: 210 and 343, respectively;(xiv) SEQ ID NOs: 210 and 344, respectively;(xv) SEQ ID NOs: 333 and 340, respectively;(xvi) SEQ ID NOs: 345 and 216, respectively;(xvii) SEQ ID NOs: 346 and 216, respectively;(xviii) SEQ ID NOs: 347 and 216, respectively;(xix) SEQ ID NOs: 348 and 216, respectively;(xx) SEQ ID NOs: 349 and 216, respectively;(xxi) SEQ ID NOs: 350 and 216, respectively;(xxii) SEQ ID NOs: 351 and 216, respectively;(xxiii) SEQ ID NOs: 352 and 216, respectively;(xxiv) SEQ ID NOs: 353 and 216, respectively;(xxv) SEQ ID NOs: 354 and 216, respectively;(xxvi) SEQ ID NOs: 210 and 355, respectively;(xxvii) SEQ ID NOs: 210 and 356, respectively;(xxviii) SEQ ID NOs: 210 and 357, respectively;(xxix) SEQ ID NOs: 210 and 358, respectively;(xxx) SEQ ID NOs: 210 and 359, respectively;(xxxi) SEQ ID NOs: 210 and 360, respectively;(xxxii) SEQ ID NOs: 210 and 361, respectively;(xxxiii) SEQ ID NOs: 210 and 362, respectively; (xxxiv) SEQ ID NOs: 210 and 363, respectively; (xxxv) SEQ ID NOs: 210 and 364, respectively; (xxxvi) SEQ ID NOs: 210 and 365, respectively; (xxxvii) SEQ ID NOs: 210 and 366, respectively; (xxxviii) SEQ ID NOs: 210 and 367, respectively; (xxxix) SEQ ID NOs: 210 and 368, respectively;(xl) SEQ ID NOs: 210 and 369, respectively;(xli) SEQ ID NOs: 210 and 370, respectively;(xlii) SEQ ID NOs: 371 and 215, respectively; or(xliii) (xliii) SEQ ID NOs: 371 and 370, respectively.

70. The complex of any one of claims 1-65, wherein the antigen-binding domain specifically binds to human TfR with an affinity of about 751 nM to about 10,000 nM.

71. The complex of any one of claims 1-65, wherein the antigen-binding domain specifically binds to human TfR with an affinity of about 51 nM to about 750 nM.

72. The complex of any one of claims 1-65, wherein the antigen-binding domain specifically binds to human TfR with an affinity of about 0.01 nM to about 50 nM.

73. The complex of any one of claims 1-61 and 66-69, wherein the antigen-binding domain specifically binds to human CD98hc with an affinity of about 500 nM to about 10,000 nM.

74. The complex of any one of claims 1-61 and 66-69, wherein the antigen-binding domain specifically binds to human CD98hc with an affinity of about 100 nM to about 499 nM.

75. The complex of any one of claims 1-61 and 66-69, wherein the antigen-binding domain specifically binds to human CD98hc with an affinity of less than about 100 nM.

76. The complex of any one of claims 70-75, wherein the affinity is measured using surface plasmon resonance.

77. A host cell comprising the complex of any one of claims 1-76.

78. A composition comprising the complex of any one of claims 1-76.

79. A pharmaceutical composition comprising (a) the complex of any one of claims 1-76 and (b) a pharmaceutically acceptable carrier, excipient or stabilizer.

80. A polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding the complex of any one of claims 1-76.

81. A method of transporting a GCase polypeptide or fragment thereof across the blood-brain barrier of a subject comprising administering to the subject the complex of any one of claims 1-76, the composition of claim 78 or 79, or the polynucleotide or combination of polynucleotides of claim 80.

82. A method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject the complex of any one of claims 1-76, the composition of claim 78 or 79, or the polynucleotide or combination of polynucleotides of claim 80.

83. The method of claim 82, wherein the lysosomal storage disease or disorder is Gaucher’s disease.

84. A method of treating a CNS disease or disorder in a subject comprising administering to the subject the complex of any one of claims 1-76, the composition of claim 78 or 79, or the polynucleotide or combination of polynucleotides of claim 80.

85. The method of claim 84, wherein the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia.

86. The complex of any one of claims 1-76, the composition of claim 78 or 79, or the polynucleotide or combination of polynucleotides of claim 80 for use in the method of any one of claims 81-85.

87. Use of the complex of any one of claims 1-76, the composition of claim 78 or 79, or the polynucleotide or combination of polynucleotides of claim 80 in the method of any one of claims 81-85.

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