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

Abstract

The present disclosure is generally directed GCase polypeptides as well as fusion proteins and complexes comprising a GCase polypeptide and an antigen-binding domain that specifically binds to blood-brain barrier (BBB) target such as human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc). Such fusion proteins and complexes can transport the GCase polypeptide across 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 Appl. No. 63 / 734,577, filed December 16, 2024; U.S. Provisional Appl. No. 63 / 771,955, filed March 14, 2025; U.S. Provisional Appl. No. 63 / 801,552, filed May 7, 2025; U.S. Provisional Appl. No. 63 / 855,633, filed August 1, 2025; U.S. Provisional Appl. No. 63 / 884,871, filed September 19, 2025; U.S. Provisional Appl. No. 63 / 911,865, filed November 5, 2025; and U.S. Provisional Appl. No. 63 / 934,878, filed December 9, 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_036PC07_Sequencelisting_ST26.xml; Size: 547,310 bytes; and Date of Creation: December 4, 2025) is herein incorporated by reference in its entirety.FIELD OF THE PRESENT DISCLOSURE

[0003] The present disclosure relates to variant P-Glucocerebrosidase (variant GCase) polypeptides and fusion proteins and complexes for transporting variant GCase polypeptides across the blood-brain barrier (BBB), uses (e.g., therapeutic uses) of such variant GCase polypeptides, fusion proteins, or complexes, and nucleic acids encoding such variant GCase polypeptides.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 havehigher GCase substrate levels than idiopathic PD patients. In vitro data show that accumulation of the substrate GlcSph is linked with higher alpha-synuclein aggregation. In addition, compound GBA1 mutations can cause a lysosomal storage disorder called Gaucher Disease.

[0006] 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 variant glucocerebrosidase (GCase) polypeptides comprising one or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the one or more amino acid substitutions comprises: V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241.

[0008] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343S with reference to numbering of SEQ ID NO: 241.

[0009] In some aspects, the variant GCase polypeptide comprises one or more amino acid substiutions comprises V343Q with reference to numbering of SEQ ID NO: 241.

[0010] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343E with reference to numbering of SEQ ID NO: 241.

[0011] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343M with reference to numbering of SEQ ID NO: 241.

[0012] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343R with reference to numbering of SEQ ID NO: 241.

[0013] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343L with reference to numbering of SEQ ID NO: 241.

[0014] In some aspects, the variant GCase polypeptide comprises one or more amino acid substitutions comprises V343K with reference to numbering of SEQ ID NO: 241.

[0015] In some aspects, the wild-type GCase polypeptide is a mammalian GCase. In some aspects, the wild-type Gcase ia a human GCase.

[0016] In some aspects, the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

[0017] In some aspects, the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0018] In some aspects, the GCase polypeptide further comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond.

[0019] In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L286C and A318C with reference to SEQ ID NO: 241. In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L240C and G250C with reference to SEQ ID NO: 241. In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise T43C and S488C with reference to SEQ ID NO: 241. In some aspects, the variant Gcase polypeptide does not comprise T43C and / or S488C with reference to SEQ ID NO: 241. In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise W312C and A341C with reference to SEQ ID NO: 241.

[0020] In some aspects, the variant GCase polypeptide does not comprise W312C and / or A341C with reference to SEQ ID NO: 241.

[0021] In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise G83C and L383C with reference to SEQ ID NO: 241.

[0022] In some aspects, the variant GCase polypeptide does not comprise G83C and / or L383C with reference to SEQ ID NO: 241.

[0023] In some aspects, the variant GCase polypeptide further comprises a T379I substitution with reference to SEQ ID NO: 241.

[0024] In some aspects, the variant GCase polypeptide further comprises a A338H substitution with reference to SEQ ID NO: 241.

[0025] In some aspects, the variant GCase polypeptide comprises V343S, L286C, and A318C substitutions with reference to SEQ ID NO: 241.

[0026] In some aspects, the variant GCase polypeptide comprises V343S, L286C, A318C, and T379I substitutions with reference to SEQ ID NO: 241.

[0027] In one aspect, the disclosure provides a variant GCase polypeptide comprising two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L286C and A318C with reference to the numbering of SEQ ID NO: 241.

[0028] In some aspects, the wild-type GCase polypeptide is a mammalian GCase. In some aspects, the wild-type Gcase ia a human GCase.

[0029] In some aspects, the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

[0030] In some aspects, the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0031] In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L286C and A318C with reference to SEQ ID NO: 241.

[0032] In one aspect, the disclosure provides a variant GCase polypeptide comprising two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L240C and G250C with reference to SEQ ID NO: 241.

[0033] In some aspects, the wild-type GCase polypeptide is a mammalian GCase. In some aspects, the wild-type Gcase ia a human GCase.

[0034] In some aspects, the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

[0035] In some aspects, the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0036] In some aspects, the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L240C and G250C with reference to SEQ ID NO: 241.

[0037] In some aspects, the variant GCase polypeptide further comprises V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241

[0038] In some aspects, the variant GCase polypeptide further comprises V343A or V343T with reference to SEQ ID NO: 241.

[0039] In one aspect, the disclosure provides a variant GCase polypeptide comprising three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343T, W312C, and A341C with reference to SEQ ID NO: 241.

[0040] In one aspect, the disclosure provides a variant GCase polypeptide comprising three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343M, W312C, and A341C with reference to SEQ ID NO: 241.

[0041] In some asepcts, the wild-type GCase polypeptide is a mammalian GCase. In some aspects, the wild-type Gcase ia a human GCase.

[0042] In some aspects, the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

[0043] In some aspects, the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0044] In some aspects, the variant GCase polypeptide does not comprise an amino acid change at any of positions Glu235, Glu340, and Cys342 with reference to SEQ ID NO: 241.

[0045] In some aspects, the variant GCase polypeptide comprises at least 96% amino acid sequence identity to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises at least 97% amino acid sequence identity to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises at least 98% amino acid sequence identity to SEQ ID NO :241. In some aspects, the variant GCase polypeptide comprises at least 99% amino acid sequence identity to SEQ ID NO: 241.

[0046] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 453, 454, or 455.

[0047] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 395, 396, 288, or 246.

[0048] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 399.

[0049] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 395.

[0050] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 392.

[0051] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 379, 245, 377, 376, or 374.

[0052] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 245.

[0053] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 385, 388, or 387.

[0054] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 389 or 390.

[0055] In some aspects, the variant GCase polypeptide is capable of hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell-free enzyme assay. In some aspects, the variant GCasepolypeptide is at least as efficient as a polypeptide of SEQ ID NO: 241 in hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell-free enzyme assay. In some aspects, the variant GCase polypeptide is at least ten times as efficient as a polypeptide of SEQ ID NO: 241 in hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell-free enzyme assay.

[0056] In some aspects, the variant GCase polypeptide is capable of converting 4- Methylumbelliferyl-P-D-glucopyranosiduronic acid (4-MUG) into 4-Methylumbelliferone (4- MU). In some aspects, the variant GCase polypeptide is at least as efficient as a polypeptide of SEQ ID NO: 241 in converting 4-MUG into 4-MU. In some aspects, the variant GCase polypeptide is at least two times as efficient as a polypeptide of SEQ ID NO:241 in converting 4- MUG into 4-MU. In some aspects, the variant GCase polypeptide is at least five times as efficient as a polypeptide of SEQ ID NO:241 in converting 4-MUG into 4-MU. In some aspects, the variant GCase polypeptide is at least ten times as efficient as a polypeptide of SEQ ID NO: 241 in converting 4-MUG into 4-MU.

[0057] In some aspects, the variant GCase polypeptide is expressed at a level equivalent to or greater than the expression level of a polypeptide of SEQ ID NO: 241 in ExpiCHO cells.

[0058] In some aspects, the GCase polypeptide has a melting temperature equivalent to or greater than the melting temperature of a polypeptide of SEQ ID NO: 241.

[0059] In some aspects, the GCase polypeptide has a half-life equivalent to or longer than the half-life of a polypeptide of SEQ ID NO: 241 in PBS pH 7.4 at 37 °C.

[0060] In one aspect, the disclosure provides a fusion protein comprising an Fc domain and a variant GCase polypeptide described herein.

[0061] In some aspects, the variant GCase polypeptide is N-terminal to the Fc domain.

[0062] In some aspects, the fusion protein further comprising a linker between the Fc domain and the variant GCase polypeptide. In some aspects, the Fc domain is N-terminal to the variant GCase polypeptide. In some aspects, the Fc domain comprises a CH2 and a CH3 domain.

[0063] In one aspect, the disclosure provides a complex comprising a variant GCase polypeptide described herein and an antigen-binding domain that specifically binds to a blood brain barrier (BBB) target.

[0064] In some aspects, the BBB target is human transferrin receptor (TfR).

[0065] In some aspects, 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, VL CDR2, and VL 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;(xxxvi) SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively;(xxxvii) SEQ ID NOs: 10, 22, 30, 50, 58, and 62, respectively;(xxxviii) SEQ ID NOs: 10, 22, 461, 50, 58, and 62, respectively;(xxxix) SEQ ID NOs: 10, 22, 28, 463, 58, and 62, respectively;(xl) SEQ ID NOs: 10, 22, 28, 464, 58, and 62, respectively;(xli) SEQ ID NOs: 10, 22, 28, 465, 58, and 62, respectively;(xlii) SEQ ID NOs: 10, 22, 28, 466, 58, and 62, respectively;(xliii) SEQ ID NOs: 10, 22, 462, 50, 58, and 62, respectively;(xliv) SEQ ID NOs: 8, 14, 25, 41, 55, and 61, respectively;(xlv) SEQ ID NOs: 8, 15, 25, 467, 55, and 61, respectively;(xlvi) SEQ ID NOs: 8, 15, 25, 468, 55, and 61, respectively; or(xlviii) SEQ ID NOs: 8, 14, 25, 469, 55, and 61, respectively.

[0066] In some aspects, the antigen-binding domain that specifically binds to human TfR comprises a VH and a VL comprising 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) SEQ ID NOs: 77 and 131, respectively;(xxiv) SEQ ID NOs: 80 and 140, respectively;(xxv) SEQ ID NOs: 81 and 141, respectively;(xxvi) SEQ ID NOs: 82 and 131, respectively;(xxvii) SEQ ID NOs: 83 and 142, respectively;(xxviii) SEQ ID NOs: 77 and 143, respectively;(xxix) SEQ ID NOs: 75 and 131, respectively;(xxx) SEQ ID NOs: 75 and 144, respectively;(xxxi) SEQ ID NOs: 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) SEQ ID NOs: 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) SEQ ID NOs: 88 and 131, respectively;(xli) SEQ ID NOs: 75 and 149, respectively;(xlii) SEQ ID NOs: 89 and 150, respectively;(xliii) SEQ ID NOs: 90 and 151, respectively;(xliv) SEQ ID NOs: 77 and 152, respectively;(xlv) SEQ ID NOs: 79 and 153, respectively;(xlvi) SEQ ID NOs: 77 and 139, respectively;(xlvii) SEQ ID NOs: 91 and 131, respectively;(xlviii) SEQ ID NOs: 92 and 131, respectively;(xlix) SEQ ID NOs: 79 and 154, respectively;(1) SEQ ID NOs: 93 and 155, respectively;(li) SEQ ID NOs: 80 and 131, respectively;(lii) SEQ ID NOs: 94 and 131, respectively;(liii) SEQ ID NOs: 95 and 131, respectively;(liv) SEQ ID NOs: 66 and 156, respectively;(Iv) SEQ ID NOs: 97 and 138, respectively;(Ivi) SEQ ID NOs: 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;(cii) SEQ ID NOs: 117 and 178, respectively;(ciii) SEQ ID NOs: 118 and 174, respectively;(civ) SEQ ID NOs: 470 and 174, respectively;(cv) SEQ ID NOs: 117 and 471, respectively;(cvi) SEQ ID NOs: 117 and 472, respectively;(cvii) SEQ ID NOs: 117 and 473, respectively;(cviii) SEQ ID NOs: 117 and 474, respectively;(cix) SEQ ID NOs: 475 and 174, respectively;(ex) SEQ ID NOs: 101 and 154, respectively;(cxi) SEQ ID NOs: 102 and 476, respectively;(cxii) SEQ ID NOs: 102 and 477, respectively; or(cxiii) SEQ ID NOs: 101 and 478, respectively.

[0067] In some aspects, the BBB target is human CD98 heavy chain (CD98hc).

[0068] In some aspects, the the antigen-binding domain that specifically binds to human CD98 comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VHCDR2, VH CDR3 and light chain variable region (VL) CDR1, VL CDR2, and VL 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;(ix) SEQ ID NOs: 184, 234, 193, 197, 198, and 204; respectively,(x) SEQ ID NOs: 184, 291, 193, 197, 198, and 204, respectively;(xi) SEQ ID NOs: 184, 292, 193, 197, 198, and 204, respectively;(xii) SEQ ID NOs: 184, 293, 193, 197, 198, and 204, respectively;(xiii) SEQ ID NOs: 184, 294, 193, 197, 198, and 204, respectively;(xiv) SEQ ID NOs: 184, 295, 193, 197, 198, and 204, respectively;(xv) SEQ ID NOs: 184, 296, 193, 197, 198, and 204, respectively;(xvi) SEQ ID NOs: 184, 297, 193, 197, 198, and 204, respectively;(xvii) SEQ ID NOs: 184, 190, 193, 318, 198, and 204, respectively;(xviii) SEQ ID NOs: 184, 190, 193, 319, 198, and 204, respectively;(xix) SEQ ID NOs: 184, 190, 193, 320, 198, and 204, respectively;(xx) SEQ ID NOs: 184, 190, 193, 321, 198, and 204, respectively;(xxi) SEQ ID NOs: 184, 190, 193, 322, 198, and 204, respectively;(xxii) SEQ ID NOs: 184, 190, 193, 323, 198, and 204, respectively;(xxiii) SEQ ID NOs: 184, 190, 193, 324, 198, and 204, respectively;(xxiv) SEQ ID NOs: 184, 293, 193, 320, 198, and 204, respectively;(xxv) SEQ ID NOs: 184, 298, 193, 197, 198, and 204, respectively;(xxvi) SEQ ID NOs: 184, 190, 299, 197, 198, and 18, respectively;(xxvii) SEQ ID NOs: 184, 190, 300, 197, 198, and 204, respectively;(xxviii) SEQ ID NOs: 184, 190, 301, 197, 198, and 204, respectively;(xxix) SEQ ID NOs: 184, 190, 302, 197, 198, and 204, respectively;(xxx) SEQ ID NOs: 184, 190, 303, 197, 198, and 204, respectively;(xxxi) SEQ ID NOs: 184, 190, 304, 197, 198, and 204, respectively;(xxxii) SEQ ID NOs: 184, 190, 305, 197, 198, and 204, respectively;(xxxiii) SEQ ID NOs: 184, 190, 306, 197, 198, and 204, respectively;(xxxiv) SEQ ID NOs: 184, 190, 307, 197, 198, and 204, respectively;(xxxv) SEQ ID NOs: 184, 190, 193, 325, 198, and 204, respectively;(xxxvi) SEQ ID NOs. 184, 190, 193, 326, 198, and 204, respectively;(xxxvii) SEQ ID NOs: 184, 190, 193, 327, 198, and 204, respectively;(xxxviii) SEQ ID NOs: 184, 190, 193, 328, 198, and 204, respectively;(xxxix) SEQ ID NOs: 184, 190, 193, 329, 198, and 204, respectively;(xl) SEQ ID NOs: 184, 190, 193, 330, 198, and 204, respectively;(xli) SEQ ID NOs: 184, 190, 193, 197, 198, and 308, respectively;(xlii) SEQ ID NOs: 184, 190, 193, 197, 198, and 309, respectively;(xliii) SEQ ID NOs: 184, 190, 193, 197, 198, and 310, respectively;(xliv) SEQ ID NOs: 184, 190, 193, 197, 198, and 311, respectively;(xlv) SEQ ID NOs: 184, 190, 193, 197, 198, and 312, respectively;(xlvi) SEQ ID NOs: 184, 190, 193, 197, 198, and 313, respectively;(xlvii) SEQ ID NOs: 184, 190, 193, 197, 198, and 314, respectively;(xlviii) SEQ ID NOs: 184, 190, 193, 197, 198, and 315, respectively;(xlix) SEQ ID NOs: 184, 190, 193, 197, 198, and 316, respectively;(1) SEQ ID NOs: 184, 190, 193, 197, 198, and 317, respectively;(li) SEQ ID NOs: 290, 190, 193, 197, 198, and 204, respectively; or(lii) SEQ ID NOs: 290, 190, 193, 197, 198, and 317, respectively.

[0069] In some aspects, the antigen-binding domain that specifically binds to human CD98 comprises a VH and a VL comprising 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;(ix) SEQ ID NOs: 237 and 216, respectively;(x) SEQ ID NOs: 331 and 216, respectively;(xi) SEQ ID NOs: 332 and 216, respectively;(xii) SEQ ID NOs: 333 and 216, respectively;(xiii) SEQ ID NOs: 334 and 216, respectively;(xiv) SEQ ID NOs: 335 and 216, respectively;(xv) SEQ ID NOs: 336 and 216, respectively;(xvi) SEQ ID NOs: 337 and 216, respectively;(xvii) SEQ ID NOs: 210 and 338, respectively;(xviii) SEQ ID NOs: 210 and 339, respectively;(xix) SEQ ID NOs: 210 and 340, respectively;(xx) SEQ ID NOs: 210 and 341, respectively;(xxi) SEQ ID NOs: 210 and 342, respectively;(xxii) SEQ ID NOs: 210 and 343, respectively;(xxiii) SEQ ID NOs: 210 and 344, respectively;(xxiv) SEQ ID NOs: 333 and 340, respectively;(xxv) SEQ ID NOs: 345 and 216, respectively;(xxvi) SEQ ID NOs: 346 and 216, respectively;(xxvii) SEQ ID NOs: 347 and 216, respectively;(xxviii) SEQ ID NOs: 348 and 216, respectively;(xxix) SEQ ID NOs: 349 and 216, respectively;(xxx) SEQ ID NOs: 350 and 216, respectively;(xxxi) SEQ ID NOs: 351 and 216, respectively;(xxxii) SEQ ID NOs: 352 and 216, respectively;(xxxiii) SEQ ID NOs: 353 and 216, respectively;(xxxiv) SEQ ID NOs: 354 and 216, respectively;(xxxv) SEQ ID NOs: 210 and 355, respectively;(xxxvi) SEQ ID NOs: 210 and 356, respectively;(xxxvii) SEQ ID NOs: 210 and 357, respectively;(xxxviii) SEQ ID NOs: 210 and 358, respectively;(xxxix) SEQ ID NOs: 210 and 359, respectively;(xl) SEQ ID NOs: 210 and 360, respectively;(xli) SEQ ID NOs: 210 and 361, respectively;(xlii) SEQ ID NOs: 210 and 362, respectively;(xliii) SEQ ID NOs: 210 and 363, respectively;(xliv) SEQ ID NOs: 210 and 364, respectively;(xlv) SEQ ID NOs: 210 and 365, respectively;(xlvi) SEQ ID NOs: 210 and 366, respectively;(xlvii) SEQ ID NOs: 210 and 367, respectively;(xlviii) SEQ ID NOs: 210 and 368, respectively;(xlix) SEQ ID NOs: 210 and 369, respectively;(1) SEQ ID NOs: 210 and 370, respectively;(li) SEQ ID NOs: 371 and 215, respectively; or(lii) SEQ ID NOs: 371 and 370, respectively.

[0070] In some aspects, the complex comprises a first polypeptide comprising the variant GCase polypeptide and a first Fc domain; a second polypeptide comprising a VH of the antigenbinding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigenbinding domain.

[0071] In some aspects, the variant GCase polypeptide is N-terminal to the first Fc domain.

[0072] In some aspects, the first polypeptide further comprises an antibody hinge between the variant GCase polypeptide or fragment thereof and the first Fc domain.

[0073] In some aspects, the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation. In some aspects, the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

[0074] In some aspects, the second polypeptide chain is an antibody heavy chain.

[0075] In some aspects, the third polypeptide further comprises a CL domain, optionally wherein the third polypeptide is an antibody light chain.

[0076] In some aspects, the first Fc domain comprises a CH2 and a CH3 and / or wherein the second Fc domain comprises a CH2 and a CH3.

[0077] In some aspects, the complex comprises the amino acid sequence of SEQ ID NO: 510, 511, 514, or 517. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 437, 429, and 431. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 526, 429, and 431. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 526, 528, and 529. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 526, 429, and 430. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 437, 429, and 430. In some aspects, the complex comprises the amino acid sequences of SEQ ID NOs: 526, 429, and 428; or SEQ ID NOs: 437, 429, and 428.

[0078] In one aspect, the disclosure provides a viral vector comprising an expression cassette comprising a nucleotide sequence encoding a variant GCase polypeptide described herein.

[0079] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) vector comprising an expression cassette comprising a nucleotide sequence encoding a variant GCase polypeptide described herein.

[0080] In some aspects, the nucleotide encoding the variant GCase polypeptide is operably linked to a promoter.

[0081] In some aspects, the promoter induces expression of the variant GCase polypeptide in the central nervous system.

[0082] In some aspects, the promoter induces expression of the variant GCase polypeptide in macrophages.

[0083] In some aspects, the nucleotide encoding the variant GCase polypeptide is operably linked to an enhancer.

[0084] In some aspects, the nucleotide encoding the variant GCase polypeptide is codon optimized for expression in a target cell.

[0085] In one aspect, the disclosure provides a rAAV viral particle comprising an AAV capsid protein encapsidating an rAAV vector described herein.

[0086] In some aspects, the viral particle comprises an AAV9 capsid protein. In some aspects, the viral particle comprises an AAVrhlO capsid protein. In some aspects, the viral particle comprises an AAV2 capsid protein. In some aspects, the viral particle comprises an AAV8 capsid protein.

[0087] In some aspects, the capsid further comprises a binding moiety which targets human TfR.

[0088] In one aspect, the disclosure provides a polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding a variant GCase polypeptide described herein, a fusion protein described herein, or a complex described herein.

[0089] In one aspect, the disclosure provides a host cell comprising a polynucleotide or combination of polynucleotides described herein.

[0090] In one aspect, the disclosure provides a method of producing the variant GCase polypeptide described herein, the fusion protein described herein, or the complex described herein comprising culturing the host cell described herein.

[0091] In some aspects, a variant GCase polypeptide, fusion protein, or complex is produced by the method described herein.

[0092] In one aspect, the disclosure provides a host cell comprising the variant GCase polypeptide, fusion protein or complex described herein.

[0093] In one aspect, the disclosure provides a composition comprising the variant GCase polypeptide, fusion protein, or complex described herein or the polynucleotide or combination of polynucleotides described herein.

[0094] In one aspect, the disclosure provides a pharmaceutical composition comprising (a) the variant GCase polypeptide, fusion protein, or complex described herein or the polynucleotide or combination of polynucleotides described herein and (b) a pharmaceutically acceptable carrier, excipient or stabilizer.

[0095] In one aspect, the disclosure provides a pharmaceutical composition comprising a rAAV viral particle described herein and a pharmaceutically acceptable carrier, excipient, or stabilizer. In some aspects, the pharmaceutical composition comprises a dose of about 1011vg / kg to about 1014vg / kg.

[0096] In one aspect, the disclosure provides a method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides, or composition described herein.

[0097] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease.

[0098] In one aspect, the disclosure provides a method of reducing glucosyl sphingosine (GlcSph) accumulation in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition described herein.

[0099] In some aspects, the method reduces GlcSph accumulation in the liver. In some aspects, the method reduces GlcSph accumulation in the brain.

[0100] In one aspect, the disclosure provides a method of enhancing peripheral P- glucocerebrosidase (GCase) in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition described herein.

[0101] In one aspect, the disclosure provides a method of treating a CNS disease or disorder in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition described herein.

[0102] In some aspects, the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia. In some aspects, the CNS disorder is early Parkinson’s Disease and the subject has a GBA gene mutation.

[0103] In some aspects, the method reduces brain lipid accumulation in the subject. In some aspects, the method enhances peripheral Gcase activity in the subject. In some aspects, the method reduces liver glucosyl sphingosine (GlcSph) accumulation in the subject. In some aspects, the method reduces brain glucosylsphingosine (GlcSph) accumulation in the subject.

[0104] In some aspects, the administration is intravenous administration. In some aspects, the administration is subcutaneous administration.

[0105] In one aspect, the disclosure provides a variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition described herein for use in the methods decribed herein.

[0106] In one aspect, the disclosure provides a use of the GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition described herein in the methods described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1 (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 antigen-binding 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 antigen-binding 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 GCaselinked 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.

[0108] Figures 2A-2D (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 (monovalent-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).

[0109] Figure 3 (FIG. 3) shows the Vmax of GCase activity for wild type GCase, GC100, GC101, GC102, GC103, GC104, GC105, GC107, GC125, GC108, GC119, GC109, GC110, GC111, GC112, and GC3.

[0110] Figures 4A-4D (FIGs. 4A-4D) show hematology parameters of non-human primates treated with GCase-TfR constructs (G2, G3, and G4), including reticulocytes (FIG. 4A), red blood cell count (FIG. 4B), hemoglobin level (FIG. 4C), and normalized hematocrit (FIG. 4D), compared to excipient control (Gl).

[0111] Figures 5A-5B (FIGs. 5A-5B) show GCase activity in CSF collected from non-human primates treated with GCase-TfR constructs (G2, G3, and G4) compared to excipient control (Gl).

[0112] Figure 6 (FIG. 6) shows the Vmax of GCase activity of GCase-TfR variants.

[0113] Figures 7A-7E (FIGs. 7A-7E) show pharmacokinetic (PK) and blood reticulocyte levels of mice treated with GCase-TfR constructs (G2, G3, and G4), including serum PK (FIG.7 A), serum GCase activity (FIG. 7B), brain PK (FIG. 7C), brain GCase activity (FIG. 7D), and blood reticulocyte levels (FIG. 7E) compared to excipient control (Gl).

[0114] Figure 8 (FIG. 8) shows the study design and treatment regimen a study in cynomolgus monkeys. The study included six groups with N = 3 female cynomolgus monkeys per group.(Group # may also referred to as G#, Test Article # or TA # herein.)

[0115] Figures 9A-9B (FIGs. 9A-9B) show the group mean concentration (FIG. 9A) and plasma GCase activity (FIG. 9B) of various GCase-TfR test articles at each timepoint.

[0116] Figures 10A-10B (FIGs. 10A-10B) show the group mean concentration (FIG. 10A) and CSF GCase activity (FIG. 10B) of test articles at each timepoint.

[0117] Figures 11A-11B (FIGs. 11A-11B) show the blood reticulocyte levels (FIG. 11 A) and red blood cell counts (FIG. 11B) for test articles at each timepoint.

[0118] Figures 12A-12B (FIGs. 12A-12B) show the hemoglobin levels (FIG. 12A) and hematocrit levels (FIG. 12B) for test articles at each timepoint.

[0119] Figure 13 (FIG. 13) shows the concentration of various GCase-TfR test articles in brain tissue lysate from frontal cortex, hippocampus, substantia nigra, and putamen of the non- human primates (NHPs) after sacrificing.

[0120] Figures 14A-14D (FIGs. 14A-14D) show GCase activity of various GCase-TfR test articles in the frontal cortex (FIG. 14A), hippocampus (FIG. 14B), substantia nigra (FIG. 14C), and putamen (FIG. 14D).

[0121] Figure 15 (FIG. 15) shows the ability of various GCase-TfR test articles to rescue GCase activity in GBA1 knock-out SH-SY5Y human neuroblastoma cells.

[0122] Figure 16 (FIG. 16) shows the experimental protocol for assessing the activity of GC109-mTfRl in Gba / -deficient mice and the brain glucosyl sphingosine (GlcSph) levels in the mice after administration of the test article or saline.

[0123] Figure 17A (FIG. 17A) shows liver Gcase activity measured using a fluorometric kinetic assay. Mean + standard deviation of n=4 mice per group. Figure 17B (FIG. 17B) shows brain GCase activity measured in the vessel-depleted fraction using a fluorometric kinetic assay.Mean + standard deviation of n=4 mice per group. Figure 17C (FIG. 17C) shows brain GlcSph measured by HPLC-MS / MS 7 days after a single dose with lOmg / kg of the test articles. Mean + standard deviation of n=4 mice per group. Sidak’s multiple comparisons test *p<0.05 **p<0.01.

[0124] Figure 18A (FIG. 18A) shows serum GCase-TfR concentration measured using an anti-Fc ELISA following dosing at Oh and 336h. Figure 18B (FIG. 18B) shows serum GCase activity measured using a fluorometric assay following dosing at Oh and 336h. Geometric mean and error, n=4 per group. Two undetectable data points were excluded from the saline group and three from the Cerezyme group.

[0125] Figures 19A-19C (FIGs. 19A-19C) show GCase enzyme activity measured using a fluorometric assay in tissue lysates prepared from liver (FIG. 19A), lung (FIG. 19B), or bone marrow (FIG. 19C) samples. Mean and standard deviation, n=4 per group.

[0126] Figure 20 (FIG. 20) shows liver GlcSph quantity measured by HPLC-MS / MS and normalized to tissue weight. Mean and standard deviation, n=4 per group.

[0127] Figure 21 (FIG. 21) shows absolute reticulocytes measured at different time points from terminal cardiac puncture samples. The dotted line represents the average of normal levels (saline and terminal time points).

[0128] Figure 22 (FIG. 22) shows brain GlcSph levels measured by HPLC-MS / MS at different time points following a single intravenous injection with GC103-mTfRl_21. Mean + standard deviation of n=4 per time point. The dotted line represents the average of Saline and d28-treated mice.

[0129] Figure 23 (FIG. 23) shows brain GCase activity measured in vessel-depleted lysates using a fluorogenic kinetic assay. Mean + standard deviation of n=4 mice per time point.

[0130] Figure 24 (FIG. 24) shows liver GlcSph levels determined by HPLC-MS / MS at different time points following injection with GC103-mTfRl_21. Mean + standard deviation of n=4 mice per time point. The dotted line represents the average of control mice.

[0131] Figure 25A (FIG. 25A) shows absolute reticulocyte counts determined 24h post 3rd and final dose or 7d post single dose. Mean + standard deviation. Figure 25B (FIG. 25B) shows GCase-TfR concentration as determined in vessel-depleted brain fraction 24h post final dose. Mean + standard deviation.

[0132] Figure 26A (FIG. 26A) shows GCase activity as determined in vessel-depleted brain fractions 24h post final dose. Mean + standard deviation. Figure 26B (FIG. 26B) shows brain GlcSph concentration determined 7 days post single dose or 24h post 3rd and final dose. Mean + standard deviation.

[0133] Figure 27A (FIG. 27A) shows absolute reticulocyte counts determined at various time points after injection. Mean + standard deviation. Figure 27B (FIG. 27B) shows brain GCase enzyme activity determined at various time points after injection. Mean + standard deviation. Figure 27C (FIG. 27C) shows brain GlcSph concentration determined at various time points after injection. Mean + standard deviation.

[0134] Figure 28A (FIG. 28A) shows brain Gcase-TfR concentration determined in vessel- depleted samples at the terminal time point. Mean + standard deviation n=6 / group. * Samples below lower limit of quantification are not graphed. Figure 28B (FIG. 28B) shows brain GCase activity determined in the vessel-depleted fraction using a kinetic fluorogenic enzymatic assay. Mean + standard deviation. *One sample was excluded because of mis-genotyping. Figure 28C (FIG. 28C) shows brain GlcSph concentration determined at the terminal time point. Mean + standard deviation n=6 / group. *One sample was excluded because of mis-genotyping. Figure 28D (FIG. 28D) shows absolute reticulocyte counts determined at terminal collection. Mean + standard deviation.

[0135] Figure 29 shows GCase activity in PBMC lysate measured prior to injection and 24h post second dose using a fluorescent kinetic assay. Mean + standard deviation.

[0136] Figure 30A (FIG. 30A) shows GlcCer and GlcSph concentrations determined by UPLC-MS / MS in liver lysates. Geom Mean + Geom SD. Figure 30B (FIG. 30B) shows GlcCer and GlcSph concentrations determined by UPLC-MS / MS in frontal cortex lysates. Geom Mean + Geom SD.DETAILED DESCRIPTION OF THE PRESENT DISCLOSURE

[0137] Recombinant GCase polypeptides have therapeutic potential 1 in treating conditions associated with deficiencies in GCase. The present disclosure relates to the identification of variant GCase polypeptides with improved functional properties, such as increased activity, expression, purity (e.g., monomer purity), stability (e.g., thermal stability), and / or half-life.

[0138] 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 therapeuticdirectly 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 variant GCase polypeptides that are capable of crossing the BBB as a result of binding to a BBB target such as transferrin receptor (TfR) or CD98 heavy chain (CD98hc).

[0139] In some aspects, a “1+1 format” comprising a BBB-binding domain refers to a format comprising (i) one antigen-binding domain that binds to a BBB target (e.g. human TfR or human CD98hc) and (ii) a variant GCase polypeptide. Such a “1+1” format can also comprise an Fc domain. Such a “1+1” format can also comprise an Fc domain. Exemplary 1+1 formats are shown in parts (i) to (iv) of Figure 1.

[0140] In some aspects, a “2+1 format” comprising a BBB-binding domain refers to a trivalent format comprising (i) a single antigen-binding domain that binds to a BBB target (e.g., human TfR or human CD98hc) and (ii) two copies of a variant GCase polypeptide. Such a “2+1” format can also comprise an Fc domain. In some aspects, a “2+1 format” comprising a BBB-binding domain refers to a trivalent format comprising (i) two antigen-binding domains that bind to a BBB target (e.g., human TfR or CD98hc) and (ii) a single copy of a variant GCase polypeptide. Such a “2+1” format can also comprise an Fc domain. Exemplary 2+1 formats are shown in parts (v) and (vi) of Figure 1.

[0141] In some aspects, a “2+2 format” comprising a BBB-binding domain refers to a tetraval ent format comprising (i) two antigen-binding domains that bind to a BBB target (e.g., human TfR or human CD98hc) and (ii) two copies of two copies of a variant GCase polypeptide. Such a “2+2” format can also comprise an Fc domain. Exemplary 2+2 formats are shown in parts (vii) and (viii) of Figure 1.

[0142] Certain 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 et al. 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

[0143] 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.

[0144] The terms “blood-brain barrier” or “BBB” refer to a network of brain capillary endothelial cells that are closely sealed by tight junctions.

[0145] A “central nervous system antigen” or “CNS antigen” is an antigen expressed in the CNS, including the brain.

[0146] A “brain antigen” is a CNS antigen expressed in the brain.

[0147] 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.

[0148] 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.

[0149] 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 lysosomal trafficking. 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.

[0150] “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.

[0151] 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.

[0152] 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.

[0153] 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 that results 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:MM DQ A RS AF SNLFGGEPLS YTRF SLARQ VDGDNSHVEMKL AVDEEENADNNTKANVT KPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPA ARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFK LSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHA NFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELS FFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSD WKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPG AAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSL HLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVE KLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEV AGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFR ATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLP ALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 1).

[0154] 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 CD98hc” refers to a polypeptide with the following amino acid sequence:MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPS ASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNEL EPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSP GWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAF QGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFDSLLQSAKKKSIRVILDLTPNYRGENSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDI ENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGST GEHTKSLVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGD EIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQ RSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASAS LPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (SEQ ID NO: 2).

[0155] 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.

[0156] 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.

[0157] 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-bindingdomain 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 about 0.01 nM to about 50 nM, about 51 nM to about 750 nM, about 751 nM to about 10,000 nM, less than about 20 pM, less than about 15 gM, less than about 12 gM, less than about 10 gM, less than about 7.5 gM , less than about 5 gM , less than about 2.5 gM, less than about 1 gM, 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). In certain embodiments, an anti-TfR antigen-binding domain binds to an epitope of TfR that is conserved among TfR from different species.

[0158] 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.

[0159] 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 about 10 nM to about 1500 nM, about 500 nM to about 10 pM, about 100 nM to about 500 nM, less than about 0.1 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). In certain aspects, an anti-CD98hc antigen-binding domain binds to an epitope of CD98hc that is conserved among CD98hc from different species.

[0160] 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 anFc 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.

[0161] 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.

[0162] The terms “VH” and “VH domain” are used interchangeably to refer to the heavy chain variable region of an antibody.

[0163] 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 (8), 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.

[0164] The terms “VL” and “VL domain” are used interchangeably to refer to the light chain variable region of an antibody.

[0165] 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 (1) based on the amino acid sequence of theconstant regions. Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0166] 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.

[0167] 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.

[0168] 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).

[0169] 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.

[0170] 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 antibodycan be derived from any animal species, such as rodents (e.g., mouse, rat, or hamster) and humans or can be artificially produced.

[0171] 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.

[0172] 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.

[0173] 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 disclosure include 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 twoheavy 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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).

[0179] 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.

[0180] 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.

[0181] In some aspects, the CDRs can be “contact” CDRs. The “contact” CDRs are based on an analysis of the available complex crystal structures.

[0182] 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

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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 replacednon-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.

[0188] 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.

[0189] “Framework” or “FR” residues are those variable-domain residues other than the CDR residues as herein defined.

[0190] 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 human immunoglobulin 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.

[0191] 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 inKabat 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.

[0192] 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.

[0193] 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.

[0194] 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.

[0195] 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 parent polypeptide, 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.

[0196] “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 animmunoreceptor 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.

[0197] “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 el al, (2013) Mabs 5(2):270-8.)

[0198] 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 orlower, 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.

[0199] 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 that comprises one or more prolines and can contain other amino acids so long as proline is the predominant species in the linker.

[0200] 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.

[0201] 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 orcompositions 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.

[0202] 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.

[0203] 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.

[0204] 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., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. 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.

[0205] “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.

[0206] 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 deliberatemutation. 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.

[0207] “ 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] 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 variant GCase polypeptide, an antigen-binding domain than specifically binds to a BBB target (e.g., CD98hc or TfR), an Fc region, and / or an Fc domain. Accordingly, non-limiting examples of a “complex” comprising an antigen-binding domain and a variant GCase polypeptide include (a) a fusion protein or complex comprising the antigen-binding domain and the variant GCase polypeptide in a single polypeptide chain (e.g., as shown in Figure 1 (i)), and (b) three proteins connected via noncovalent protein-protein interactions, wherein the first protein contains the variant 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 1 (iv)). Formats of other exemplary complexes are provided in Figures 1 and 2.

[0214] 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 variant GCase polypeptide can refer, e.g., to a fusion protein or complex comprising the variant GCase polypeptide can refer to a mixture or solution comprising such a variant GCase polypeptide, 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.”

[0215] As used herein the term “pharmaceutical composition” refers to a combination comprising an active agent (e.g. a variant GCase polypeptide, or fusion protein or complex disclosed herein) with at least one inert pharmaceutically acceptable agent (e.g., an excipient or a carrier).

[0216] A “recombinant adeno-associated virus vector” or “rAAV vector” refers to any vector that comprises or derives from components of an adeno-associated virus. The term rAAV vector can be used to designate a nucleic acid vector comprising at least one heterologous sequence (i.e., a nucleic acid sequence not of viral origin) flanked by at least one or at least two AAV inverted terminal repeat sequences (ITRs). rAAV vectors can be replicated and packaged into infectious viral particles when present in a host cell infected with a suitable helper virus or in a cell engineered to express suitable helper functions (e.g., adenovirus El, E2, E4, and / or VA genes) and that is expressing AAV rep and cap gene products (AAV rep and AAV cap proteins). In some aspects, a producer cell used to produce rAAV virus particles may express AAV rep, AAV cap, and / or the rAAV vector. An rAAV vector may be incorporated into a larger polynucleotide (such as a chromosome or another vector), wherein the incorporated rAAV vector is termed a pro-vector. The pro-vector can be rescued by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions to yield rAAV viral particles. In some aspects, the rAAV vector is suitable to infect mammalian cells (e.g., human cells). The term rAAV vector can be used to designate an AAV-type viral particle or virion comprising a payload. The rAAV vector can be derived from various serotypes, including combinations of serotypes (i.e., “pseudotyped” AAV) or from various genomes (e.g., single stranded or self- complementary). In addition, the rAAV vector can be replication defective and / or targeted. As used herein, the term “adeno-associated virus” (AAV), includes but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3 A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAVrh8, AAVrhlO, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, those AAV serotypes and clades disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and any other AAV. The terms “AAV genome,” “AAV capsid,” and “rAAV vector” can be used interchangeably. An rAAV vector may be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral particle, suchas an AAV viral particle. For example, an rAAV vector genome can be packaged into an AAV virus capsid to generate a recombinant AAV particle (rAAV particle).

[0217] As used herein, a “transgene” refers to a polynucleotide that is capable of being transcribed into RNA and then translated to express a protein product.

[0218] As used herein, an “AAV inverted terminal repeat” or "AAV ITR" sequence refers to a nucleotide sequence that is present at the termini of single stranded AAV genomes. Naturally occurring ITR sequences are approximately 145 bases each, and AAV plasmids typically include two ITR sequences. The outermost approximately 125 nucleotides of the ITR can be present in either of two orientations, leading to heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost approximately 125 nucleotides also contain shorter regions of self-complementarity, allowing intrastrand base-pairing to occur within the ITR.

[0219] As used herein, a “helper virus” in the context of AAV production refers to a virus that allows AAV to be replicated and packaged by a host cell. A helper virus provides helper functions, which allow for the replication of AAV. A plurality of helper viruses are known in the art, including adenoviruses, herpesviruses, and poxviruses. In some aspects, the helper virus is an adenovirus.

[0220] 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.

[0221] 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 to an “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.

[0222] 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 presenceof 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.

[0223] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0224] 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. |J-Glucocerebrosidase (GCase) Polypeptides

[0225] Human P-Glucocerebrosidase (GCase) is a globular protein of 497 amino acids (SEQ ID NO: 241), 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(11):5086-5095).

[0226] Enzyme replacement therapy (ERT) for Gaucher disease (caused by biallelic mutations in the GBA1 gene) has been available via periodic intravenous infusions of a recombinantly expressed GCase, of which imiglucerase, sold under the brand name 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 wild-type 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 for efficacious GCase ERT therapeutics with optimized stability and manufacturability that can cross the blood-brain barrier.

[0227] Certain aspects of the disclosure are related to variant GCase polypeptides. Certain GCase polypeptides are known in the art. For example, exemplary GCase polypeptides 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, which can have advantageous properties such as superior enzyme activity, half-life, stability, etc., are provided herein.

[0228] In some aspects, the variant GCase polypeptide 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%, or at least 99%, amino acid sequence identity to SEQ ID NO: 241.

[0229] In some aspects, a wild-type or naturally occurring variant GCase polypeptide comprises the amino acid sequence of NCBI Ref. Seq. NOs: NP_000148.2, NP_001005741.1, NP_001005742.1, NP_001165282.1, or NP_001165283.1. In some aspects, a wild-type or naturally occurring variant GCase polypeptide 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, a wild-type or naturally occurring variant GCase polypeptide 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.

[0230] In some aspects, the variant GCase polypeptide comprises at least one amino acid substitution as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises at least two substitutions as compared to SEQ ID NO :241. In some aspects, the variant GCase polypeptide comprises at least three substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises at least four substitutions as compared to SEQ ID NO: 241.

[0231] In some aspects, the GCase polypeptide comprises one to twenty amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the GCase polypeptide comprises one to nineteen amino acid substitutions as compared to SEQ ID NO :241. In some aspects, the variant GCase polypeptide comprises one to eighteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to seventeen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to sixteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to fifteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to fourteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to thirteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptidecomprises one to twelve amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to eleven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to ten amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to nine amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to eight amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to seven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to six amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to five amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to four amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises one to three amino acid substitutions as compared to SEQ ID NO: 241.

[0232] In some aspects, the variant GCase polypeptide comprises two to twenty amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to nineteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to eighteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to seventeen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to sixteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to fifteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to fourteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to thirteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to twelve amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to eleven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to ten amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to nine amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to eight amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptidecomprises two to seven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to six amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to five amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises two to four amino acid substitutions as compared to SEQ ID NO: 241.

[0233] In some aspects, the variant GCase polypeptide comprises three to twenty amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to nineteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to eighteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to seventeen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to sixteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to fifteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to fourteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to thirteen amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to twelve amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to eleven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to ten amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to nine amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to eight amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to seven amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to six amino acid substitutions as compared to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide comprises three to five amino acid substitutions as compared to SEQ ID NO: 241.

[0234] In some aspects, a variant GCase polypeptide provided herein comprises one or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the one or more amino acid substitutions comprises: V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptideprovided herein comprises one or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the one or more amino acid substitutions comprises: V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241, and wherein the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0235] In some aspects, a variant GCase polypeptide provided herein comprises a V343S amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343S amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0236] In some aspects, a variant GCase polypeptide provided herein comprises a V343Q amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343Q amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0237] In some aspects, a variant GCase polypeptide provided herein comprises a V343E amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343E amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0238] In some aspects, a variant GCase polypeptide provided herein comprises a V343M amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343M amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0239] In some aspects, a variant GCase polypeptide provided herein comprises a V343R amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343R amino acid substitution as compared to a wild-type GCase polypeptide withreference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0240] In some aspects, a variant GCase polypeptide provided herein comprises a V343L amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343L amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0241] In some aspects, a variant GCase polypeptide provided herein comprises a V343K amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343K amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0242] In some aspects, a variant GCase polypeptide provided herein comprises a V343T amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a V343T amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0243] In some aspects, a variant GCase polypeptide provided herein comprises a T379I amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a T379I amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0244] In some aspects, a variant GCase polypeptide provided herein comprises a A338H amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises a A338H amino acid substitution as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0245] In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, for example, L286C and A318C, L240C and G250C, T43C and S488C, W312C and A341C, or G83C and L383C with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, for example, L286C and A318C, L240C and G250C, T43C and S488C, W3 12C and A341C, or G83C and L383C with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0246] In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide selected from L286C and A318C, L240C and G250C, T43C and S488C, W312C and A341C, or G83C and L383C with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide selected from L286C and A318C, L240C and G250C, T43C and S488C, W3 12C and A341C, or G83C and L383C with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0247] In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide selected from L286C and A318C, L240C and G250C, T43C and S488C, W312C and A341C, or G83C and L383C with reference to SEQ ID NO: 241, and further comprises the amino acid substitution V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide selected from L286C and A318C, L240C and G250C, T43C and S488C, W312C and A341C, or G83C and L383C with reference to SEQ ID NO: 241, further comprises the amino acid substitution V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0248] In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L286C and A318C with reference to the numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L286C and A318C with reference to the numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0249] In some aspects, a variant GCase polypeptide provided herein comprises L286C and A318C amino acid substitutions as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises L286C and A318C amino acid substitutions as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0250] In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L240C and G250C with reference to the numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L240C and G250C with reference to the numbering of SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0251] In some aspects, a variant GCase polypeptide provided herein comprises L240C and G250C amino acid substitutions as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises L240C and G250C amino acid substitutions as compared to a wild-type GCase polypeptide with reference to numbering of SEQ ID NO: 241, and the variant GCase polypeptidecomprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0252] In some aspects, a variant GCase polypeptide provided herein comprises three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343T, W312C, and A341C with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343T, W312C, and A341C with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0253] In some aspects, a variant GCase polypeptide provided herein comprises three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise L286C, A318C, and V343S with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise L286C, A318C, and V343S with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0254] In some aspects, a variant GCase polypeptide provided herein comprises four or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the four or more amino acid substitutions comprise L286C, A318C, V343S, and T379I with reference to SEQ ID NO: 241. In some aspects, a variant GCase polypeptide provided herein comprises four or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the four or more amino acid substitutions comprise L286C, A318C, V343S, and T379I with reference to SEQ ID NO: 241, and the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

[0255] As provided herein, e.g., as demonstrated in the Examples Section, a variant GCase polypeptide can comprise any combination of amino acid substitutions provided herein.

[0256] In some aspects, the variant GCase polypeptide further comprises a combination of substitutions as disclosed in WO 2020 / 161483, WO 2024 / 163765, WO 2021 / 199039, and / or WO 2022 / 023761, each of which is incorporated by reference herein.

[0257] In some aspects, the variant GCase polypeptide does not comprise an amino acid change at positions Glu235 with reference to SEQ ID NO: 241. In some aspects, the variantGCase polypeptide does not comprise an amino acid change at positions Glu340 with reference to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide does not comprise an amino acid change at positions Cys342 with reference to SEQ ID NO: 241. In some aspects, the variant GCase polypeptide does not comprise an amino acid change at any of positions Glu235, Glu340, and Cys342 with reference to SEQ ID NO: 241.

[0258] In some aspects, the variant GCase polypeptide comprises one or more substitutions in Domain III of a wild-type GCase. In some aspects, the variant GCase polypeptide comprises one or more substitutions in Domain III of a wild-type GCase, and does not contain any mutations in Domain I and / or Domain II of a wild-type GCase.

[0259] In some aspects, the variant GCase polypeptide comprises two or more substitutions in Domain III of a wild-type GCase. In some aspects, the variant GCase polypeptide comprises two or more substitutions in Domain III of a wild-type GCase, and does not contain any mutations in Domain I or Domain II of a wild-type GCase.

[0260] In some aspects, the variant GCase polypeptide comprises three or more substitutions in Domain III of a wild-type GCase. In some aspects, the variant GCase polypeptide comprises three or more substitutions in Domain III of a wild-type GCase, and does not contain any mutations in Domain I or Domain II of a wild-type GCase.

[0261] In some aspects, the GCase polypeptide comprises one disulfide mutation (i.e., two amino acids changed to cysteine) capable of forming an intramolecular disulfide bond and one additional substitution in Domain III of a wild-type GCase. In some aspects, the GCase polypeptide comprises one disulfide mutation (i.e., two amino acids changed to cysteine) capable of forming an intramolecular disulfide bond and one additional substitution in Domain III of a wild-type GCase, and does not contain any mutations in Domain I or Domain II of a wild-type GCase.

[0262] In some aspects, the variant GCase polypeptide f comprises no more than 10 amino acid substitutions in Domain I of a wild-type GCase, no more than 10 amino acid substitutions in Domain II of a wild-type GCase, and no more than 10 substitutions in Domain III of a wild-type GCase.

[0263] In some aspects, the variant GCase polypeptide comprises no more than 5 amino acid substitutions in Domain I of a wild-type GCase, no more than 5 amino acid substitutions in Domain II of a wild-type GCase, and no more than 5 substitutions in Domain III of a wild-type GCase.

[0264] In some aspects, the variant GCase polypeptide comprises no more than 20 amino acid substitutions as compared to a corresponding region of a wild-type GCase. In some aspects, the GCase polypeptide comprises no more than 15 amino acid substitutions as compared to a corresponding region of a wild-type GCase. In some aspects, the GCase polypeptide comprises no more than 10 amino acid substitutions as compared to a corresponding region of a wild-type GCase.

[0265] In some aspects, the variant GCase polypeptide comprises 5-20 amino acid substitutions as compared to a corresponding region of a wild-type GCase. In some aspects, the GCase polypeptide f comprises 5-15 amino acid substitutions as compared to a corresponding region of a wild-type GCase. In some aspects, the GCase polypeptide comprises 5-10 amino acid substitutions as compared to a corresponding region of a wild-type GCase.

[0266] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 245.

[0267] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 246.

[0268] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 288.

[0269] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 374.

[0270] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 376.

[0271] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 377.

[0272] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 379.

[0273] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 385.

[0274] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 387.

[0275] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 388.

[0276] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 389.

[0277] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 390.

[0278] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 392.

[0279] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 395.

[0280] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 396.

[0281] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 399.

[0282] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 453.

[0283] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 454.

[0284] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 455.

[0285] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 393.

[0286] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 384.

[0287] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 394.

[0288] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 382.

[0289] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 479.

[0290] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 480.

[0291] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 402.

[0292] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 378.

[0293] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 380.

[0294] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 456.

[0295] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 386.

[0296] In some aspects, the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 391.

[0297] In some aspects, the variant GCase polypeptide is capable of hydrolyzing glucosylceramide. In some aspects, the variant GCase polypeptide exhibits an improved ability to hydrolyze glucosylceramide as compared to wild-type human GCase, such as compared to a GCase comprising the amino acid sequence of SEQ ID NO: 241. In some aspects, the GCase polypeptide exhibits an improved ability to hydrolyze glucosyl ceramide as compared to the variant GCase designated herein as GC119 (i.e., GCase with W312C and A341C substitutions).

[0298] In some aspects, the variant GCase polypeptide has increased protein expression in CHO cells as compared to wild-type human GCase, such as compared to a GCase comprising the amino acid sequence of SEQ ID NO: 241. In some aspects, expression of the variant GCase polypeptpide in CHO cells results in a higher titer than expression of wild-type human GCase, such as expression of a GCase comprising the amino acid sequence of SEQ ID NO: 241.

[0299] In some aspects, the variant GCase polypeptide has a melting temp (Tm) greater than 60 °C in a monovalent Fc format.

[0300] In some aspects, the variant GCase polypeptide has a melting temp (Tm) of about 60 °C to about 75 °C in a monovalent Fc format. In some aspects, the variant GCase polypeptide has a melting temp (Tm) of about 60 °C to about 70 °C in a monovalent Fc format. In some aspects, the variant GCase polypeptide has a melting temp (Tm) of about 60 °C to about 65 °C in a monovalent Fc format.

[0301] In some aspects, the variant GCase polypeptide has a half-life of at least 3 days in phosphate-buffered saline (PBS) pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of at least 4 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of at least 5 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of at least 6 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of at least 7 days in PBS pH 7.4 at 37 °C.

[0302] In some aspects, the variant GCase polypeptide has a half-life of about 3 to about 20 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 3 to about 15 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 3 to about 10 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 3 to about 8 days in PBS pH 7.4 at 37 °C.

[0303] In some aspects, the variant GCase polypeptide has a half-life of about 4 to about 20 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 4 to about 15 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 4 to about 10 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 4 to about 8 days in PBS pH 7.4 at 37 °C.

[0304] In some aspects, the variant GCase polypeptide has a half-life of about 5 to about 20 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 5 to about 15 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 5 to about 10 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 5 to about 8 days in PBS pH 7.4 at 37 °C.

[0305] In some aspects, the variant GCase polypeptide has a half-life of about 6 to about 20 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 6 to about 15 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 6 to about 10 days in PBS pH 7.4 at 37 °C. In some aspects, the variant GCase polypeptide has a half-life of about 6 to about 8 days in PBS pH 7.4 at 37 °C.II. Antigen-Binding Domains and Compositions Comprising Antigen-Binding Domains

[0306] In some aspects, compositions provided herein may comprise an antigen-binding domain.

[0307] 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.

[0308] In some aspects, compositions provided herein may comprise an antibody or an antibody fragment thereof.

[0309] 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. Fordiscussion 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.

[0310] 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).

[0311] 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. colt) or phage, as described herein.

[0312] 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.

[0313] 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 anon-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0314] 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)).

[0315] 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).

[0316] 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.

[0317] 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 by isolating 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.

[0318] 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 roc. 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 segmentsfrom 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

[0319] A composition provided herein can comprise an Fc domain or Fc region or fragment thereof. In some aspects, an Fc domain or Fc region is of the IgG class, the IgM class, or the IgA class. In some aspects, an Fc domain or Fc 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 domain or fragment thereof. In some aspects, the Fc domain or Fc region or fragment thereof is a modified Fc domain or Fc region or fragment thereof comprising one or more modifications. In some aspects, the one or more modifications reduce effector function. In some aspects, an Fc domain or region or fragment thereof is a human IgGl Fc domain or Fc region or fragment thereof. In some aspects, an Fc domain or Fc region or fragment thereof is a human IgG2 Fc domain or fragment thereof. In some aspects, the Fc domain or fragment thereof is a wild-type IgG2, such as a wild-type human IgG2. In some aspects, the Fc domain or fragment thereof is a modified IgG2 Fc comprising one or more modifications, such as one or more modifications that reduce effector function. For example, in some aspects, the IgG2 modified Fc comprises one or more amino acid substitutions relative to a wild-type IgG2. In some aspects, an Fc domain or Fc region or fragment thereof is a human IgG4 Fc domain or fragment thereof. In some aspects, the Fc domain or fragment thereof is a wild-type IgG4, such as a wild-type human IgG4. In some aspects, the Fc domain or fragment thereof is a modified IgG4 Fc comprising one or more modifications, such as one or more modifications that reduce effector function. For example, in some aspects, the IgG4 modified Fc comprises one or more amino acid substitutions (relative to a wild-type IgG4). In some aspects, the one or more amino acid substitutions in the modified IgG4 Fc are selected from IgG4-S228P or IgG4-S228P / L235E, wherein the amino acid position is according to the EU numbering convention.

[0320] 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). In some aspects, the one or more amino acid substitutions are selected from N297A (Bolt S et al. (1993) Ewr 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 comprises the amino acid substitutions L234A, L235A, and P331S (LALAPS) according to EU numbering. In some aspects, the Fc comprises N325S and L328F mutations according to EU numbering. In some aspects, the Fc comprises P329G or P329S according to EU numbering. In some aspects, the Fc comprises K322A according to EU numbering.

[0321] Any suitable Fc domain or Fc region or fragment thereof is contemplated in the variant GCase complexes described herein, and exemplary Fc domains are provided in Table 1, below.Table 1: Exemplary Fc Domains

[0322] 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.

[0323] 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.

[0324] 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.

[0325] “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.

[0326] 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.

[0327] 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

[0328] 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, such as a variant GCase polypeptide described herein) 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).

[0329] 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

[0330] 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.

[0331] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3 or 4 below (i.e. the three VH CDRs of the antibody listed in Table 3 and the three VL CDRs of the same antibody listed in Table 4). Insome aspects, an antigen-binding domain that specifically binds to human TfR comprises the six CDRs of an antibody listed in Tables 3, 4, or 5 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, or 5 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, or 5.

[0332] 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, or 5 as determined by the method in MacCallum RM et al.

[0333] 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, or 5 as determined by the AbM numbering scheme.

[0334] 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, or 5 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.

[0335] 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 antibodyvariants 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.

[0336] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VH of an antibody listed in Table 5.

[0337] In some aspects, an antigen-binding domain that specifically binds to human TfR comprises the VL of antibody listed in Table 5.

[0338] 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 (i.e., the VH of the antibody listed in Table 5 and the VL of the same antibody listed in Table 5.

[0339] 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 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 5. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (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 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.

[0340] 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, 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. Insome aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of an antibody in listed in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0341] 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 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. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0342] 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 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. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0343] 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 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 5. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0344] 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 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. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0345] 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 VHamino acid sequence of an antibody in Table 5 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 or 4. In some aspects, the antigen-binding domain that specifically binds to human TfR also comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0346] 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 and a VL amino acid sequence of the same antibody in Table 5.

[0347] 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).

[0348] 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. 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).

[0349] 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 and a VL of the same antibody in Table 5. In some aspects, the antigen-binding domain that comprises a Fab comprises the CDRs of the antibody in Tables 3 or 4 (e.g., the non-identical amino acids in the VH and / or VL are outside of the CDRs).

[0350] 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. In other aspects, the antigen-binding domain comprises a VHH comprises the heavy chain CDRs of an antibody listed in Table 3.

[0351] 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.

[0352] In some aspects, an antigen-binding domain provided herein that specifically binds to human TfR also binds to cynomolgus monkey TfR.

[0353] 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.

[0354] 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.

[0355] 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.

[0356] In some aspects, the affinity between the antigen-binding domain and TfR is measured using surface plasmon resonance, such as a BIACORE™ SPR 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.

[0357] 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 (e.g., about1 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 (e.g., 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 (e.g., about 100 nM to about 750 nM or about 100 nM to about 700 nM) and cynomolgus TfR with an affinity of about 51 nM to about 1250 nM (e.g., 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 (e.g., about 1000 nM to about 5000 nM) and cynomolgus TfR with an affinity of about 1251 nM to about 10,000 nM (e.g., 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™.

[0358] 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.

[0359] 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.

[0360] 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.

[0361] 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.

[0362] 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.

[0363] 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 providedherein 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.

[0364] 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.

[0365] 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.

[0366] 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.

[0367] 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.

[0368] 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.

[0369] In some aspects, compositions provided herein comprise a variant GCase polypeptide and an antigen-binding domain which specifically binds human TfR with a KD of about 75 nM to about 175 nM, such as about 100 nM to about 150 nM. In some aspects, compositions provided herein comprise a variant GCase polypeptide and an antigen-binding domain which specifically binds human TfR with a KD of about 350 nM to about 450 nM, such as about 375 nM to about 425 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

[0370] 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

[0371] 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 variant GCase polypeptide. In some aspects, a fusion protein or complex provided hereincomprises: (i) an antigen-binding domain that specifically binds to human TfR and (ii) a variant GCase polypeptide. In some aspects, the variant GCase polypeptide is useful in protein replacement therapy (PRT). In some aspects, the variant 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 variant GCase polypeptide or protein is an ERT enzyme or an ERT enzyme variant, or a catalytically active fragment thereof.

[0372] Exemplary formats for a fusion protein or complex as disclosed herein are depicted in Figure 1.

[0373] In some aspects, the variant GCase polypeptide in the fusion protein or complex is N- terminal to the antigen-binding domain that specifically binds to human TfR.

[0374] In some aspects, the variant GCase polypeptide in the fusion protein or complex is C- terminal to the antigen-binding domain that specifically binds to human TfR.

[0375] In some aspects, the variant GCase fusion polypeptide or fragment thereof and the antigen-binding domain that specifically binds to human TfR are directly connected via a peptide bond. In some aspects, the variant GCase fusion polypeptide or fragment thereof and the antigenbinding domain that specifically binds to human TfR are connected via a linker, e.g., a peptide linker.

[0376] In some aspects, the peptide linker comprises the amino acid sequence corresponding to SEQ ID NO: 285 (GAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKAGS).

[0377] In some aspects, the fusion protein or complex comprises an antigen-binding domain that specifically binds to human TfR, a variant GCase polypeptide, and an Fc portion. In some aspects, the antigen-binding domain that specifically binds to human TfR and the variant GCase polypeptide are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0378] In some aspects, the antigen-binding domain that specifically binds to human TfR and the variant GCase polypeptide 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 variant GCase polypeptide is linked to the C-terminus of the Fc portion of the fusion protein or complex. In other aspects, the variant GCase polypeptide 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.

[0379] 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) avariant GCase polypeptide, wherein the fusion protein or complex comprises two copies of the variant GCase 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” or “knob-into-hole” or “knob-hole Fc”, as described herein). In some aspects, the single scFv, Fab or VHH antigenbinding 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 variant GCase polypeptide are linked to the N-terminus of the Fc. An example of this 2+1 format is shown in (vi) of Figure. 1.

[0380] 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 variant GCase polypeptide are linked to the C-terminus of the Fc domain. An example of this 2+1 format is shown as (v) in Figure 1.

[0381] 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 variant GCase polypeptide linked to the C-terminus of the two antibody heavy chains. An example of this 2+1 format is shown as (vii) in Figure 1.

[0382] 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 Figure 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.

[0383] 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 variant GCase polypeptide, 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 variant GCase 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 Figure 1.

[0384] 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 variant GCase polypeptide, 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 variant GCase 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 Figure 1. Such monovalent formats can have improved properties for purification and manufacturing.

[0385] 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 variant GCase polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and the variant GCase 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 Figure 1. Such N-terminal monozyme formats can result in improved serum PK (longer half-life).

[0386] 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 variant GCase polypeptide, wherein the single scFv, VHH, or Fab antigen-binding domain that specifically binds to human TfR and the variant GCase polypeptide are both linked to the C-terminus of the Fc domain.C. Antigen-Binding Domains That Bind to CD98hc

[0387] Provided herein are antigen-binding domains that specifically bind to human CD98hc.

[0388] 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.

[0389] 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 specificallybinds to human CD98hc comprises the six Chothia CDRs of an antibody listed in Tables 6A, 6B, 7 A, or 7B

[0390] 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 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 CD98hc and comprise VH and VL CDRs of an antibody listed in Table 6 A, 6B, 7 A, or 7B as determined by the method in MacCallum RM et al.

[0391] 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.

[0392] 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.

[0393] 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.

[0394] 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.

[0395] In some aspects, an antigen-binding domain that specifically binds to human CD98hc comprises the VL of antibody listed in Table 8A or 8B.

[0396] 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.

[0397] 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 non-identical 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 in Table 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.

[0398] 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).

[0399] 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).

[0400] 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).

[0401] 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).

[0402] 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 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 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).

[0403] 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).

[0404] 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

[0405] 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).

[0406] 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).

[0407] 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).

[0408] 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.

[0409] In some aspects, an antigen-binding domain provided herein that specifically binds to human CD98hc also binds to cynomolgus monkey CD98hc.

[0410] In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 500 nM and about 10 pM, such as about 500 nM to about 2.5 pM, about 500 nM to about 5 pM, about 1 pM to about 2.5 pM, about 1 pM to about 5 pM, about 1 pM to about 10 pM, about 2.5 pM to about 5 pM, about 2.5 pM to about 10 pM, about 5 pM to about 10 pM, and values and ranges there between.

[0411] In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 100 nM and about 500 nM, such as about 100 nM to about 250 nM, about 250 nM to about 500 nM, and values and ranges there between. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 200 nM and about 300 nM. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 100 nM and about 150 nM.

[0412] 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 0.1 nM to 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 there between. In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 25 nM and about 75 nM. In some aspects, the antigenbinding domain binds human CD98hc with an affinity between about 25 nM and about 100 nM.In some aspects, the antigen-binding domain binds human CD98hc with an affinity between about 50 nM and about 100 nM.

[0413] 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 CD98hc binds to human CD98hc as measured by, for example, radioimmunoassay (RIA), Western blot, or ELISA OD450.

[0414] 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 Antibodies- Ill -Table 8B: VH and VL Sequences of Anti-CD98hc AntibodiesD. Agents Comprising Anti-CD98hc Antigen-Binding Domains

[0415] Provided herein are agents comprising an antigen-binding domain that specifically binds to human CD98hc.Fusion Proteins or Complexes Comprising CD98hc binding domains

[0416] 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 variant GCase polypeptide. 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 heterologousprotein is a protein or polypeptide or fragment thereof useful in protein replacement therapy (PRT), such as a variant GCase polypeptide. 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.

[0417] Exemplary formats for a fusion protein or complex as disclosed herein are depicted in Figure 1.

[0418] In some aspects, the heterologous protein or polypeptide (e.g., a variant GCase polypeptide) in the fusion protein or complex is N-terminal to the antigen-binding domain that specifically binds to human CD98hc.

[0419] In some aspects, the heterologous protein or polypeptide (e.g., a variant GCase polypeptide) in the fusion protein or complex is C-terminal to the antigen-binding domain that specifically binds to human CD98hc.

[0420] In some aspects, in a fusion protein, complex, or polypeptide provided herein, the variant 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 variant GCase and the antigen-binding domain that specifically binds to human CD98hc are connected via a linker, e.g., a peptide linker.

[0421] 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 variant GCase polypeptide), 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 variant GCase polypeptide) are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0422] In some aspects, the antigen-binding domain that specifically binds to human CD98hc and the heterologous protein (e.g., a variant GCase polypeptide) 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 CD98hc is linked to the N-terminus of the Fc portion, and the heterologous protein or polypeptide (e.g., a variant GCase polypeptide) 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 variant GCase polypeptide) 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 CD98hc is linked to the C-terminus of the Fc portion.

[0423] 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 Figure 1.

[0424] 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 Figure 1.

[0425] 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 Figure 1.

[0426] 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 Figure 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.

[0427] 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 ofthe 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 Figure 1.

[0428] 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 Figure 1.

[0429] 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 Figure 1.

[0430] 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 and Antigen-Binding Domain that Binds to Blood-Brain Barrier Receptors or Proteins

[0431] Provided herein are compositions that specifically bind to human receptors or proteins of the blood-brain barrier.

[0432] In some aspects, the composition comprises any of the variant GCase polypeptides described herein.

[0433] In some aspects, the composition comprises a variant GCase polypeptide comprising at least one substitution. In some aspects, the at least one substitution is selected from the group consisting of V343S, V343Q, V343E, V343M, V343R, V343L, V343T, and V343K as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343S as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects,the at least one substitution comprises V343Q as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343E as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343M as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343R as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343L as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343T as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises V343K as compared to the amino acid sequence of SEQ ID NO: 241. In some aspects, the at least one substitution comprises L286C and / or A318C as compared SEQ ID NO: 241.

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

[0435] In some aspects, the composition comprises any of the fusion proteins or complexes disclosed herein.

[0436] In some aspects, the composition comprises a fusion protein or complex comprising (i) an antigen-binding domain that specifically binds to a BBB target such as human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) the variant GCase polypeptide.

[0437] In some aspects, the antigen-binding domain is an anti-TfR antigen-binding domain. In some aspects, the antigen-binding domain is an anti-CD98hc antigen-binding domain.

[0438] In some aspects, the variant GCase polypeptide in the fusion protein or complex is N- terminal to the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain.

[0439] In some aspects, the variant GCase polypeptide in the fusion protein or complex is C- terminal to the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain.

[0440] In some aspects, the variant GCase fusion polypeptide and the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain are directly connected via a peptide bond.

[0441] In some aspects, the variant GCase fusion polypeptide and the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain are connected via a linker. In some aspects, the linker is a peptide linker.

[0442] In some aspects, the fusion protein or complex further comprises an Fc portion.

[0443] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain and the variant GCase polypeptide are linked to the N-terminus of the Fc portion of the fusion protein or complex.

[0444] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain and the variant GCase polypeptide are both linked to the C-terminus of the Fc portion of the fusion protein or complex.

[0445] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain is linked to the N-terminus of the Fc portion and the variant GCase polypeptide is linked to the C-terminus of the Fc portion of the fusion protein or complex.

[0446] In some aspects, the variant GCase polypeptide is linked to the N-terminus of the Fc portion of the fusion protein or complex and the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain is linked to the C-terminus of the Fc portion.

[0447] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen-binding domain is a single scFv, or a VHH, or a Fab.

[0448] In some aspects, the fusion protein or complex comprises two copies of the variant GCase polypeptide.

[0449] In some aspects, the fusion protein or complex comprises an Fc domain.

[0450] 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 variant GCase polypeptide are linked to the N-terminus of the Fc domain.

[0451] 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 variant GCase polypeptide are linked to the C- terminus of the Fc domain.

[0452] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen binding domain comprises an anti-BBB (e.g., anti-TfR or anti-CD98hc) antibody. In some aspects, the antibody comprises two heavy chains and two light chains.

[0453] In some aspects, two copies of a variant GCase polypeptide are linked to the C-terminus of the two antibody heavy chains.

[0454] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen binding domain comprises two scFv, Fab, or VHH domains linked to the C-terminus of the heavy chain. In some aspects, two copies of the variant GCase polypeptide are linked to the N-terminus of the Fc domain.

[0455] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen binding domain comprises two scFv, Fab, or VHH antigen-binding domains that specifically bind to the BBB target (e.g., TfR or CD98hc) linked to the N-terminus of the heavy chain.

[0456] In some aspects, two copies of the variant GCase polypeptide are linked to the C- terminus of the Fc domain.

[0457] In some aspects, the anti-BBB (e.g., anti-TfR or anti-CD98hc) antigen binding domain comprises a single scFv, VHH, or Fab antigen-binding domain.

[0458] In some aspects, the fusion protein or complex further comprises an Fc domain, and a single copy of the variant GCase polypeptide.

[0459] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the C-terminus of the Fc domain and the variant GCase polypeptide is linked to N-terminus of the Fc domain.

[0460] In some aspects, the single scFv, VHH, or Fab antigen-binding domain is linked to the N-terminus of the Fc domain and the variant GCase polypeptide linked to C-terminus of the Fc domain.

[0461] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the variant GCase polypeptide are both linked to the N-terminus of the Fc domain.

[0462] In some aspects, the single scFv, VHH, or Fab antigen-binding domain and the variant GCase polypeptide are both linked to the C-terminus of the Fc domain.

[0463] In some aspects, the Fc is a single chain, engineered monovalent Fc domain.

[0464] In some aspects, the fusion protein or complex comprises any of the anti-TfR antigenbinding domains disclosed herein. In some aspects, the fusion protein or complex comprises any of the anti-CD98hc antigen-binding domains disclosed herein.

[0465] In some aspects, provided herein are complexes comprising (i) an antigen-binding domain that specifically binds to a BBB target such as human transferrin receptor (TfR) or human CD98 heavy chain (CD98hc) and (ii) a variant GCase polypeptide of fragment thereof.

[0466] In some aspects, the complex comprises a first polypeptide comprising the variant GCase polypeptide and a first Fc domain; a second polypeptide comprising a VH of the antigenbinding domain and a second Fc domain; and a third polypeptide comprising a VL of the antigenbinding domain.

[0467] In some aspects, the variant GCase polypeptide is N-terminal to the first Fc domain.

[0468] In some aspects, the first polypeptide further comprises an antibody hinge between the variant GCase polypeptide and the first Fc domain.

[0469] In some aspects, the variant GCase polypeptide is C-terminal to the second Fc domain.

[0470] In some aspects, the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation.

[0471] In some aspects, the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

[0472] In some aspects, the second polypeptide is an antibody heavy chain.

[0473] In some aspects, the third polypeptide further comprises a CL domain. In some aspects, the third polypeptide is an antibody light chain.

[0474] 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.

[0475] In some aspects, the complex comprises a first polypeptide comprising an antibody heavy chain and a variant GCase polypeptide and a second polypeptide comprising an antibody light chain, wherein the antibody heavy chain and the antibody light chain comprise the antigenbinding domain.

[0476] In some aspects, the variant GCase polypeptide is C-terminal to the antibody heavy chain.

[0477] In some aspects, the complex further comprises a third polypeptide comprising a Fc domain.

[0478] In some aspects, the antibody heavy chain comprises a knob mutation and the Fc domain comprises a hole mutation.

[0479] In some aspects, the antibody heavy chain comprises a hole mutation and the Fc domain comprises a knob mutation.

[0480] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0481] In some aspects, the antigen-binding domain and the variant GCase polypeptide are contained in a single polypeptide.

[0482] In some aspects, the variant GCase polypeptide and the antigen-binding domain are directly connected via a peptide bond.

[0483] In some aspects, the variant GCase polypeptide and the antigen-binding domain are connected via a linker.

[0484] In some aspects, the linker is a peptide linker.

[0485] In some aspects, the variant GCase polypeptide is N-terminal to the antigen-binding domain.

[0486] In some aspects, the variant GCase polypeptide is C-terminal to the antigen-binding domain.

[0487] In some aspects, the complex further comprises an Fc domain.

[0488] In some aspects, the Fc domain comprises a CH2 and a CH3.

[0489] In some aspects, the antigen-binding domain and the variant GCase polypeptide are both N-terminal to the Fc domain.

[0490] In some aspects, the antigen-binding domain and the variant GCase polypeptide are both C-terminal to the Fc domain.

[0491] In some aspects, the antigen-binding domain is N-terminal to the Fc domain and the variant GCase polypeptide is C-terminal to the Fc domain or fragment thereof.

[0492] In some aspects, the variant GCase polypeptide 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.

[0493] In some aspects, the variant GCase polypeptide has greater monomeric purity than a variant GCase polypeptide of SEQ ID NO: 241 when expressed under the same conditions.

[0494] In some aspects, the variant GCase polypeptide has increased protein expression in CHO cells as compared to wild-type human GCase.

[0495] In some aspects, the complex comprises any of the variant GCase polypeptides disclosed herein.

[0496] In some aspects, the antigen binding domain of the complex is a Fab.

[0497] In some aspects, the antigen-binding domain of the complex is an scFv.

[0498] In some aspects, the antigen-binding domain of the complex is a VHH.VI. Polynucleotides and Methods of Making the Same

[0499] 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 variant GCase polypeptide. 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 variant GCase polypeptide. In some aspects, the human BBB protein or receptor is TfR. In some aspects, the human BBB protein is CD98hc.

[0500] 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 recombinantexpression 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.

[0501] 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.

[0502] 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.

[0503] 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 or complex 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 -methyl -pseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 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- 1-deaza-pseudoisocyti dine, 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-methylinosine, 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.

[0504] In certain aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding the compositions described herein, or a 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).

[0505] 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 variant GCase polypeptide. 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.

[0506] Accordingly, also provided herein are cells, e.g. host cells, comprising polynucleotides and / or vectors for recombinantly expressing the compositions described herein and a variant GCase polypeptide. In some aspects, for the expression of double-chained antigen-binding proteins, vectors encoding both the heavy and light chains, individually, can be co-expressed 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.

[0507] In some aspects, provided herein are methods for producing the compositions described herein, or a domain thereof and a variant GCase polypeptide described herein in a host cell.

[0508] In some aspects, the variant GCase polypeptide described herein comprises a signal peptide. In some aspects, the signal peptide may be derived from a wild-type GBA1 sequence, a wild-type IgG signal peptide, or may be from any other suitable source.

[0509] 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.

[0510] 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 Pi chia) 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, NSO, 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 is pOptiVEC™ 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.

[0511] 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 cancontribute 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, BT20 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.

[0512] 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.

[0513] 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 Variant GCase Polypeptide and Antigen Binding Domains That Bind TfR and Methods of Making the Same

[0514] 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 variant GCase polypeptide.

[0515] 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 variant GCase polypeptide. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0516] 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.

[0517] 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.

[0518] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the variant GCase polypeptide as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding a domain of the variant GCase polypeptide as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding one or more domains of the variant GCase polypeptide as provided herein.

[0519] 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 Figure 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.

[0520] 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 Figure 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.

[0521] 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 thecomplex comprises two copies of the light chain, and wherein a variant 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 Figure 1 (vii), wherein the complex comprises an antigen-binding domain that binds to TfR.

[0522] In some aspects, disclosed herein are polynucleotides encoding a fusion protein or complex disclosed herein comprising a variant GCase polypeptide. In some aspects, provided herein is a polynucleotide that encodes a variant GCase polypeptide, 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 variant GCase polypeptide, 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 antigenbinding domain that bind to human TfR provided herein. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0523] 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.

[0524] 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 variant GCase polypeptide.

[0525] 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 variant GCase polypeptide.

[0526] 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 variant GCase polypeptide described herein in a host cell.

[0527] 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 variant GCase polypeptide.

[0528] 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 Variant GCase Polypeptide and Antigen Binding Domains That Bind CD98hc and Methods of Making the Same

[0529] 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 variant GCase polypeptide.

[0530] 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 variant GCase polypeptide. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0531] 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.

[0532] 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. 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.

[0533] In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding the variant GCase polypeptide as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding a domain of a variant GCase polypeptide as provided herein. In some aspects, a polynucleotide provided herein comprises a nucleic acid molecule encoding one or more domains of a variant GCase polypeptide as provided herein.

[0534] 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 Figure 1 (ii), (vi), or (vii), wherein the complexcomprises 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.

[0535] 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 Figure 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.

[0536] 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 variant 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 Figure 1 (vii), wherein the complex comprises an antigen-binding domain that binds to CD98hc.

[0537] In some aspects, disclosed herein are polynucleotides encoding a fusion protein or complex disclosed herein comprising a variant GCase polypeptide. In some aspects, provided herein is a polynucleotide that encodes a variant GCase polypeptide, 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 variant GCase polypeptide, 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 antigenbinding domain that bind to human CD98hc provided herein. In some aspects, the polynucleotide is DNA. In some aspects, the polynucleotide is RNA or mRNA.

[0538] In certain aspects, provided herein are vectors e.g., expression vectors) comprising polynucleotides comprising the compositions that bind CD98hc described herein, or a domainthereof described herein, for recombinant expression in a host cell, e.g., in a mammalian host cell.

[0539] 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 variant GCase polypeptide.

[0540] 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 variant GCase polypeptide.

[0541] 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 variant GCase polypeptide described herein in a host cell.

[0542] 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 variant GCase polypeptide.

[0543] A variety of host-expression vector systems and host cells are described herein.

[0544] 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).C. Vectors and Viral Particles

[0545] In some aspects, provided herein are recombinant viral vectors comprising a transgene encoding any one of the variant GCase polypeptides disclosed herein. Suitable viral vectors include those derived from any suitable AAV, lentivirus, adenovirus, retrovirus, herpes simplex virus, pox virus, or vaccinia virus. In some aspects, the viral vector is a recombinant AAV (rAAV) vector.

[0546] In some aspects, provided herein are rAAV viral particles encapsulating an rAAV vector comprising a transgene encoding one of the variant GCase polypeptides disclosed herein. The transgene encoding one of the variant GCase polypeptides disclosed herein may be codon- optimized for expression in a target cell.

[0547] In some aspects, the rAAV viral particles comprise (i) an expression cassette comprising the transgene encoding any one of the variant GCase polypeptides disclosed herein and (ii) a capsid protein. In some aspects, the expression cassette is flanked by a 5' inverted terminal repeat sequence and a 3' inverted terminal repeat sequence.

[0548] rAAV vectors can further comprise regulatory elements suitable for the expression of the variant GCase transgene in a host cell. These regulatory elements may be selected on the basis of the host cell to be used for expression and should be operably linked to the transgene. The term “operably linked” as used herein refers to the functional relationship and position of the regulatory element sequence relative to a polynucleotide of interest (e.g., a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence). Typically, an operably-linked promoter is contiguous with the sequence of interest. However, enhancers need not be contiguous with the sequence of interest to control its expression. The term “promoter,” as used herein, refers to a nucleic acid fragment that functions to control the transcription of one or more polynucleotides, located upstream of the polynucleotide sequence(s), and which is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites, and any other DNA sequences including, but not limited to, transcription factor binding sites, repressor binding sites, and activator protein binding sites, and any other sequences of nucleotides known in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “tissue-specific” promoter is only substantially active in specific types of differentiated cells or tissues.

[0549] In some aspects, the rAAV vector further comprises expression control sequences including, but not limited to, appropriate transcription sequences (i.e., initiation, termination, promoter, and / or enhancer sequences), efficient RNA processing signals (e.g., splicing and polyadenylation (poly A) signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (e.g., Kozak consensus sequence), and sequences that enhance protein stability. Many expression control sequences, including promoters that are native, constitutive, inducible, or tissue-specific, are known in the art and contemplated for use for expressing the variant GCase polypeptides disclosed herein. Regulatory elements described herein include promoters, enhancers, internal ribosomal entry sites (IRES), and any other expression control elements (e.g., transcription termination signals, such as polyA signals and poly-U sequences). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest. Regulatory elements can also direct expression in a temporal-dependent manner, such as in a cell-cycle-dependent or developmental stage-dependent manner, which may or may not be tissue or cell-type specific.

[0550] In some aspects, the transgene is operably linked to a promoter. In some aspects, the promoter can be constitutive. Suitable tissue-specific and constitutive promoters are well-known in the art. For example, suitable neuronal tissue- or cell-specific promoters include GFAP promoters (astrocytes), SYN1 promoters (neurons), and NSE / RU5’ promoters (mature neurons). In some aspects, provided herein is an expression cassette comprising a promoter operably linked to the trannsgene. In some aspects, the expression cassette further comprises an enhancer. In some aspects, the expression cassette further comprises an intron. The inclusion of an intron in the expression cassette can improve gene expression in animals. See, for example, Miao et al., Inclusion of the hepatic locus control region, an intron, and untralsted region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro, Mol. Ther. 1, 522-532 (2000). In some aspects, the expression cassette further comprises a polyadenylation signal (poly A signal). Selection of an appropriate poly A signal, such as one of beta-globin, SV40, or bovine growth hormone (BGH), can optimize transgene expression and stability. In some aspects, the expression cassette further comprises a stuffer nucleic acid. rAAV payloads have an ideal cargo size, and the inclusion of a stuffer sequence can ensure the payload is of the appropriate size, which can improve packaging efficiency. Those of skill in the art may select from a wide variety of compatible nucleic acid sequences when selecting a stuffer sequence, including those with functional properties such as a stuffer nucleic acid encoding a reporter polypeptide or a mixture of functional sequence (such as an exon) and non-functional sequence (such as an intron). In some aspects, the expression cassette may be incorporated into a vector, such as a viral vector, such as an rAAV vector.

[0551] In some aspects, the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences (e.g., a 5' ITR and a 3' ITR). Functional AAV ITRs promote the replication and packaging of the vector into rAAV viral particles. Any suitable wild-type or mutant AAV ITR is contemplated for use in the rAAV vectors described herein and may be derived from any suitable AAV serotype. In some aspects, the AAV ITRs are selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAV.rhlO, AAV11, AAV12, or any other suitable AAV ITR. In some aspects, the ITRs are AAV2 ITRs.

[0552] In some aspects, the transgene encodes a variant GCase enzyme comprising one or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the one or more amino acid substitutions comprise V343S, V343Q, V343E, V343M, V343R, V343L, or V343K, with reference to numbering of SEQ ID NO:241. In some aspects, the transgene encodesa variant GCase enzyme comprising a sequence exhibiting at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:242-247, 286- 289, 372-374, 376-396, 399, 402, 453-456, 479-480, or 483-385. In some aspects, the transgene encodes a variant GCase enzyme comprising the sequence of any one of SEQ ID NOs:242-247, 286-289, 372-374, 376-396, 399, 402, 453-456, 479-480, or 483-385. In some aspects, the transgene encodes a variant GCase enzyme comprising a signal peptide that is cleaved after protein expression to generate mature variant GCase comprising a sequence exhibiting at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:242-247, 286-289, 372-374, 376-396, 399, 402, 453-456, 479-480, or 483-385. In some aspects, the transgene encodes a variant GCase enzyme comprising a signal peptide that is cleaved after protein expression to generate mature variant GCase comprising a sequence of any one of SEQ ID NOs:242-247, 286-289, 372-374, 376-396, 399, 402, 453-456, 479-480, or 483- 385. In some aspects, the signal peptide may be derived from a wild-type GBA1 sequence, a wild-type IgG signal peptide, or may be from any other suitable source.

[0553] The expression cassette may be encapsidated in a viral particle, such as a recombinant AAV viral particle. The AAV capsid may be selected, in part, based on the target cell to be transduced, such as liver cells, macrophages, or CNS cells. An rAAV viral particle or vector may comprise viral capsid proteins of one AAV serotype and viral nucleic acids of the same serotype or a different serotype (a chimeric or pseudotyped rAAV). For example, the rAAV viral particle may comprise AAV9 capsid proteins and AAV2 ITRs (i.e., an AAV2 / 9 viral vector that is a virus containing a genome derived from serotype 2 and the capsid derived from serotype 9). In some aspects, the capsid protein may be selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, or AAV12, any derivatives thereof, or any other suitable capsid protein.

[0554] In some aspects, the rAAV viral particle may further comprise a targeting moiety, such as a peptide which targets the rAAV viral particle to the transferrin receptor, CD98hc, or any other binding partner that can be present on the surface of a target cell. See, e.g., Huang, Q., et al., Scient 384(6701): 1220-1227 (2024). Binding partners include, but are not limited to, nucleic acids, proteins, peptides, sugars, fats, or any combination thereof or any other molecule or molecules present on the surface of a target cell. In some aspects, the binding partner is unique to a cell type, cell state, or group of related cell types or states. In some aspects, the binding partner is a receptor, such as the transferrin receptor, a channel, or other complex present on the surface of a target cell. In some aspects, the rAAV viral particle capsid is engineered to comprise thetargeting moiety. In some aspects, the engineered capsid can comprise variants of wild-type AAV capsids. For example, an engineered capsid can include one or more of variants of wildtype VP1, wild-type VP2, and / or wild-type VP3 capsid proteins. Such targeting moieties may be useful to target the rAAV viral particle to desired cell types or to cross the blood-brain barrier by providing an altered or tuned tropism to the rAAV viral particle as compared to the parent wildtype capsid.VII. Pharmaceutical Compositions

[0555] Provided herein are pharmaceutical compositions comprising the variant GCase polypeptide and compositions that bind to a BBB target (e.g., human TfR or 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 variant GCase polypeptide 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.

[0556] In some aspects, a pharmaceutical composition comprises a variant GCase polypeptide and compositions that bind to a BBB target (e.g., human TfR or human CD98hc) 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)). 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.

[0557] In some aspects, provided herein are pharmaceutical compositions comprising a polynucleotide encoding a variant GCase polypeptide and a composition that binds to a BBB target (e.g., human TfR or human CD98hc) as described herein. In some aspects, the polynucleotide is RNA. In some aspects, the polynucleotide is a synthetic mRNA. In someaspects, the polynucleotide is a modified mRNA. In some aspects, the pharmaceutical composition comprising a polynucleotide further comprises a lipid-based transfection reagent.

[0558] 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.

[0559] In some aspects, provided herein are pharmaceutical compositions comprising any of the viral particles disclosed herein and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition comprises rAAV viral particles comprising rAAV vector genomes encoding any one of the variant GCase polypeptides disclosed herein.

[0560] The dose of viral particles, such as rAAV viral vectors, may be expressed in terms of vector genomes, such as vector genomes per kilogram of body weight (vg / kg). The number of vector genomes in the pharmaceutical compositions disclosed herein may be optimized based on route of administration, level of expression required to achieve a therapeutic effect, the specific disease or disorder being treated, the frequency of dosing, and the stability of the variant GCase polypeptide in the target cell type, among other factors. One of skill in the art can determine the viral particle dose range to treat a subject having a particular disease or disorder based on these and other factors known in the art. For rAAV vectors, an effective amount is generally in the range of about 10 pL to about 100 mL of solution containing from about 109to about 1016genome copies or about 1011to about 1014vg / kg. Any method known in the art may be used to determine the genome copy number of the viral compositions.

[0561] The pharmaceutical compositions comprising any of the viral particles disclosed herein may be formulated using one or more physiologically acceptable carriers and / or excipients. In some aspects, the pharmaceutical composition is formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Injection formulation may be presented in unit dosage form (e.g., in ampoules or multi-dose containers). The viral compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing, or dispersing agents.

[0562] 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.

[0563] 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 Variant GCase Polypeptide and Antigen- Binding Domains that Bind to Blood-Brain Barrier Receptors or Proteins

[0564] Compositions provided herein comprise (i) a BBB antigen-binding domain (e.g., an anti-TfR or anti-CD98hc antigen-binding domain) and (ii) a variant GCase polypeptide that can advantageously be transported across a blood-brain barrier.

[0565] Accordingly, provided herein are methods of administering or transporting compositions comprising (i) an antigen-binding protein that specifically binds to a BBB target (e.g., human TfR or human CD98hc) and (ii) a variant GCase polypeptide across the blood-brain barrier of a subject comprising administering to the subject such compositions.

[0566] In view of the ability of compositions provided herein to specifically bind to a BBB target (e.g., 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 a BBB target (e.g., human TfR or human CD98hc) and (ii) a variant GCase polypeptide.

[0567] 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, or CNS 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, a subject with Parkinson’s Disease has a GBA gene mutation. 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.

[0568] 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.

[0569] In some aspects, provided herein is a method of transporting a variant GCase polypeptide 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.

[0570] 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 variant GCase polypeptides 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.

[0571] In some aspects, the lysosomal storage disease or disorder is Gaucher’s disease.

[0572] 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 variant GCase polypeptides 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.

[0573] In some aspects, the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia.

[0574] In some aspects, the method reduces brain lipid accumulation in the subject.

[0575] In some aspects, the method reduces liver glucosyl sphingosine (GlcSph) accumulation in the subject.

[0576] In some aspects, the method enhances peripheral GCase activity in the subject.

[0577] In some aspects, provided herein is a method of reducing glucosyl sphingosine (GlcSph) accumulation in a subject comprising administering to the subject any of the variant GCase polypeptides 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.

[0578] In some aspects, the method reduces GlcSph accumulation in the liver. In some aspects, the method reduces GlcSph accumulation in the brain.

[0579] In some aspects, provided herein is a method of enhancing peripheral P- glucocerebrosidase (GCase) in a subject comprising administering to the subject any of the variant GCase polypeptides 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.

[0580] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety.

[0581] 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.IX. Methods of Using Expression Cassettes

[0582] Provided herein are methods of treating a neurological disease or disorder by administering an effective amount of an expression cassette, such as an expression cassette delivered by an rAAV particle, to express one of the variant GCase polypeptides disclosed herein at a target site, such as a CNS cell. The neurological disease or disorder can be, for example, a neuropathy disorder, a neurodegenerative disease, cancer, an ocular disease or disorder, a seizure disorder, a lysosomal storage disease, amyloidosis, a viral or microbial disease, ischemia, a behavioral disorder, or CNS 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, and spinal muscular atrophy), a tauopathy (such as Alzheimer’s 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, a 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, a subject with Parkinson’s disease has a GBA gene mutation. 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. In some aspects, the neurological disease or disorder is Parksinon’s disease or Lewy Body Dementia.

[0583] Provided herein are methods of treating a lysosomal storage disease by administering an effective amount of an expression cassette, such as an expression cassette delivered by an rAAV particle, to express one of the variant GCase polypeptides disclosed herein at a target site, such as a liver cell. 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.). In some aspects, the lysosomal storage disease is Gaucher disease.

[0584] Provided herein are methods of treating a disease or disorder such as Gaucher disease by administering an effective amount of an expression cassette, such as an expression cassette delivered by an rAAV particle, to express one of the variant GCase polypeptides disclosed herein at a target site, such as a macrophage cell.EXAMPLESExample 1: Generation of GCase Variants

[0585] To generate GCase variants that have improved enzymatic activity, better stability, pharmacokinetics, and / or manufacturability, a GCase variant, designated "dGCase3" (also known as "GC3") (see Pokoma 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), was selected as a starting point. This variant, which was generated from a computer-based algorithm, has 55 mutations compared to wild-type GCase, and shows improved expression, yield, and thermal stability. This high percentage of mutations (-15% of the total wild-type sequence) represents a potential liability for use in human patients, as extensively mutated proteins may be recognized as foreign by the immune system, often leading to the production of anti-drug antibodies. Structural and functional studies have shown that the residues Glu235, Glu340, and Cys342 lining the catalytic site are critical for GCase activity (Romero et al., Mechanism of glucocerebrosidase activation and dysfunction in Gaucher disease unraveled by molecular dynamics and deep learning. Proc Natl Acad Sci U S A. 2019 Mar 12; 116(11):5086-5095). However, as shown below, the valine at position 343 near, but facing away from, the active site can be engineered to enhance activity of GCase enzyme.

[0586] To dissect the minimal and critical beneficial mutations, several approaches and multiple protein engineering iterations were used. Gene fragments that contain either a subset of dGCase3 mutations or chimera GCase sequences (that is, domains swapped between wild-type GCase and dGCase3) were synthesized by IDT (Integrated DNA Technologies, Inc., Iowa, USA) and cloned into mammalian expression vectors. The initial expression and protein purification results showed that GCase variants that have only dGCase3 domain III sequence (a variant designated GC7) or a variant that has a subset of dGCase3 mutations (designated GC15) had increased GCase protein expression yield and relieved a GCase protein aggregation problem observed with wild-type protein. However, GC15 completely lost GCase activity in a cell-free enzyme activity assay.

[0587] To regain the GCase activity, a new template with only the GC15 domain III mutations (termed GC15v2a) was used in a round of mutagenesis. However, GC144, GC145, GC146, and GC147 have wild-type LI 03 compared to GC15v2a. All GC 15v2a-derived variants were screened in a FACS enzymatic assay in glucosylceramidase beta (GBA) -I- HEK293T cells. Table 9 discloses wild-type (WT) human GCase and GCase variant sequences.Table 9. WT Human GCase and GCase variant sequences

[0588] Briefly, the recombinant proteins were evaluated for their rate of conversion of an artificial substrate, 4-Methylumbelliferyl-P-D-glucopyranosiduronic acid (4-MUG, Sigma), into the fluorescent compound 4-Methylumbelliferone (4-MU). One hundred (100) ng of recombinant GCase enzyme were mixed with 1.67 mM 4-MUG in Mcllvaine buffer (77 mM Na2HPO4, 61.5 mM citric acid, pH 4.0). The samples were placed in a plate reader (BioTek) pre-warmed to37°C. Every 4 minutes for 1 hour, fluorescence elicited by 355 nm light was detected at 460 nm. The rate of conversion of 4-MUG to 4-MU was reported as the arbitrary fluorescence unit of 4- MU fluorescence per minute. Variants with improved FACS signal were expressed and purified. As shown in Table 10, below, among all variants tested, GC62 (GC15v2a with V343T mutation) regained the most significant enzyme activity as measured by 4-MUG assay and had improved purity as assessed by monomer percentage.Table 10. Activity of GCase variants*N.T. = not tested

[0589] To confirm if V343T is a significant activity-enhancing mutation, a GCase variant was generated and tested with only the V343T mutation (variant designated GC113) and, indeed, V343T alone improved enzyme activity by 17-fold over wild-type (data not shown). When V343T was combined with disulfide mutations W312C / A341C (designated GC119), the new variant (GC279, which is V343T / W312C / A341C) showed improved enzyme activity with similar stability as GC119.

[0590] However, from screening all V343X mutations in combination with W312C / A341C (GC119) background, results showed that only a subset of mutations, V343M (as in GC109), V343L (as in GC111), V343R (as in GC110), and V343K (as in GC112) in this background showed comparable or better activity compared to GC119 alone, while improving protein stability (Table 11). Table 12 discloses additional GCase variant sequences.Table 11. Activity of Variant GCasesTable 12. Variant GCase sequences

[0591] Contrary to the results from combining with GC119 (W312C / A341C), V343M and V343L mutations in the wild-type GCase background completely lost GCase activity. Instead, mutants with V343S and V343A showed the best activity, superior to the GC113 variant, as shown in Table 13, below.Table 13. Activity of GCase variants.

[0592] As shown in Table 14, below, when combined with different stabilizing mutations like GC130 and GC126, V343S had significantly improved activity in the tested backgrounds. Table 15 discloses additional variant GCase sequences.Table 14. Activity of GCase variants.Table 15. GCase variant sequences

[0593] These results demonstrate that position V343 is a critical activity-modulating position of GCase enzyme.

[0594] Introduction of disulfide crosslinks, by structure-based design (see Sowdhamini et al., Stereochemical modeling of disulfide bridges. Criteria for introduction into proteins by site- directed mutagenesis, Protein Eng. 1989 Nov:3(2):95-103; Craig and Dombkowski, Disulfide by Design 2.0: a web-based tool for disulfide engineering in proteins, BMC Bioinformatics, 2013 Dec: 14(346)) is a promising method to improve the stability of proteins and enzymes. Therefore, disulfide bond engineering was performed to screen for variants that could significantly improve the production yield, enzymatic activity, thermal, and stress stability compared to the wild-type enzyme. Atomic structures of GCase with PDB Codes: 6TJQ (see: Rowland, R.J., et al., (2020) Acta Crystallogr D Struct Biol 76: 565-580), 3GXI, 3GXM, 3GXD (see Lieberman, R.L. et al., (2009) Biochemistry 48: 4816-4827), and 2NT1 (see Lieberman, R.L. et al., (2007) Nat Chem Biol 3: 101-107) were analyzed using MOE suite (Molecular Operating Environment; Chemical Computing Group, Canada). Predicted and designed disulfide variants were further extensively analyzed by manual visualization, and a total of 51 disulfide mutants of GCase were generated and experimentally investigated for production feasibility and stability. Many of these GCase- disulfide variants, like GC108 (T43C / S488C / V343S), while showing a higher activity compared to the wild-type enzyme, gave lower production yield and were less stable. However, GC100 containing Cys mutations at positions Leu286 and Ala318 showed significant improvement in production yield and purity while maintaining enzyme activity comparable to the wild-type GCase enzyme.

[0595] To further enhance enzymatic activity for GC100, a series of activity mutants GC100_V343X containing a new amino acid at position V343 were generated by overlapping PCR, and the enzyme activity was checked in a cell-free GCase assay. Several of the GC100_V343X GCase triple mutants such as GC102 (L286C / A318C / V343A), GC103(L286C / A318C / V343S), GC104 (L286C / A318C / V343Q), GC105 (L286C / A318C / V343E), and GC107 (L286C / A318C / V343M) showed improved production yield, enhanced enzymatic activity, and stability.

[0596] In addition to the structure-based approach described above, the machine-learning (ML) algorithm ThermoMPNN was employed (see Dieckhaus, Henry, et al. "Transfer learning to leverage larger datasets for improved prediction of protein stability changes." Proceedings of the National Academy of Sciences 121.6 (2024): e2314853121) to design mutations to improve the activity and stability of wild-type GCase. Specifically, the fine-tuned ThermoMPNN ProteinMPNN (see Dauparas, Justas, et al. "Robust deep learning-based protein sequence design using ProteinMPNN." Science 378.6615 (2022): 49-56) was used on the megascale (Tsuboyama, Kotaro, et al. "Mega-scale experimental analysis of protein folding stability in biology and design." Nature 620.7973 (2023): 434-444) stability dataset. This adapted ProteinMPNN from its original purpose of mapping protein structure to sequence to a new purpose of mapping structure and sequence to predictions of (AAG), a metric for predicting how a single point mutation will affect protein stability. To identity sites to improve GCase stability, fine-tuned ThermoMPNN analyses of GCase crystal structure (PDB code: 6TJQ) were performed and several potentially stabilizing mutations were experimentally tested. Among those tested, the T379I mutation when combined with GC103 (which is L286C / A318C / V343S) triple-mutant (designated GC125, which has mutations L286C / A318C / V343S / T379I), significantly increased GCase in activity and stability compared to the wild-type enzyme (see Figure 3 and stability data for GC125 in following examples).Example 2: Expression and Purification of Monovalent Fc-GCase Variants (mvFc-GCase)

[0597] Monovalent Fc Glucocerebrosidase variant (mv-Fc-GCase) proteins were produced recombinantly via cloning the engineered DNA sequence into the respective vector, followed by transient transfection and expression ExpiCHO cells. Samples were harvested five days after transfection. The two constructs included a signal peptide, followed by a monovalent Fc, linker, and GCase variant sequence of interest, respectively. The mv-Fc-GCase mutants were purified using HiTrap Protein A column followed by cation exchange chromatography and a preparative size exclusion column, as needed. As the final step, the variants were taken through a preparative size exclusion column to get the samples into the formulation buffer. Tables 16 and 17 disclose mv-Fc-GCase variant sequences.Table 16. mvFc-GCase variant sequencesTable 17. Combined sequences of mvFc-GCase variantsTable 18: Main Peak Purity and Yield after Protein A Recovery.Example 3: Enzymatic Activity of Recombinant GCase Enzymes

[0598] To increase the ability of recombinant GCase to clear toxic substrate accumulation in vivo, the stability of the enzyme was enhanced, such that GCase is active longer, and has enhanced enzymatic activity, such that it can process more substrate in a set amount of time. This property may allow for lower doses of GCase to be used to reduce the levels of toxic substrate accumulated in the brain of patients. The recombinant proteins from the present disclosure were evaluated for their rate of conversion of an artificial substrate 4-Methylumbelliferyl-P-D- glucopyranosiduronic acid (4-MUG, Sigma) into the fluorescent compound 4- Methylumbelliferone (4-MU). Ten (10) nM of recombinant GCase enzyme was mixed with 1.67 mM 4-MUG in Mcllvaine buffer (77 mM Na2HPC>4, 61.5 mM citric acid, pH 4.0). The samples were placed in a plate reader (BioTek) pre-warmed to 37°C. Every 4 minutes, for 1 hour, fluorescence elicited by 355 nm light was detected at 460 nm. The fluorescence intensity was graphed as a function of time, and the Vmax was determined by measuring the maximum slope of the curve. The Vmax was then converted from arbitrary units to 4-MU nmol using a standard curve of 4-MU concentration vs fluorescence.

[0599] As shown in Figure 3, creation of a disulfide bridge between position 286 and 318 (GC100) increased the Vmax of GCase by 7-fold compared to wild-type (WT) GCase. Mutating the valine at position 343 can enhance activity of the wild-type GCase enzyme background without enhancing its stability (GC101). Addition of a point mutation at position 343 to GC100 further enhanced activity to 24- to 83-fold over WT (GC102-107). However, not all amino acid combinations were able to achieve this increase in activity. In this assay, V343I had no effect (data not shown), and V343Q and V343M (GC104 and 107, respectively) were not as active as V343S, V343A, or V343E (GC103, GC102, and GC105, respectively). In addition to position 343, mutating position 379 from threonine to isoleucine further enhanced the activity of GC103by 50% (GC125). Another variant containing a disulfide bridge between positions 43 and 488 combined with a serine at position 343 had a Vmax 139-fold greater than that of WT enzyme (GC108). Mutating position 343 to M, R, or K can also enhance the Vmax of GC119 another 2 to 12-fold (GC109, GC110, and GC112, respectively) while the V343L mutation did not further enhance activity (GC111) in this assay.

[0600] Thus, these results demonstrate increased GCase activity of GC109 over GC119 and several other variants tested.

[0601] Surprisingly, this data demonstrates that the Vmax can be enhanced to the same order of magnitude as dGCase3 (55 mutations) with as few as 3 mutated positions. A greater number of mutations increases the risk of rendering the protein immunogenic in patients. Therefore, it is preferable to limit the number of mutations as much as possible to reach a desired activity.Example 4: Thermal Stability of Engineered GCase Variants

[0602] To facilitate stability studies, GCase variants in the monovalent mv-Fc format were expressed at 200 mL scale in ExpiCHO cells and purified using mAbSPrismA, Resources, and Superdex200 columns following standard procedures. The melting temperatures (Tms) of purified mv-Fc-GCase variants were measured in the 25 mM sodium citrate pH 5.0, 150 mM sodium chloride, and 0.1% tween-80 buffer using the UNCLE (Unchained Labs, USA) instrument that facilitates simultaneous monitoring of fluorescence and static-light scattering along with dynamic-light scattering at the beginning and end of the temperature ramping. A temperature from 25°C to 95°C was used with a ramping rate of 0.5°C per minute. Data were analyzed using Lunatic analysis software.Table 19. Tm and Tonset for GCase variants in the monovalent mvFc-format

[0603] The thermal -melt Tm values for the GCase wild-type and dGCase3 are 57.5 °C and72.2 °C, respectively. Tm values of GCase single-variants, in the wild-type background, V343T (GC113) and V343A (GC101) are slightly lower than that of wild-type itself. GC108 is also comparable to the wild-type GCase enzyme indicating that the introduction of a disulfide bridge at the T43C and S488C positions did not increase the stability of GCase. The Tm value for the disulfide-linked double mutant GC119 (W312C / A341C) is 64.7 °C. Introduction of V343M to GC119 (i.e., GC109) slightly increased the Tm to 64.9 °C, whereas V343R (GC110), V343L (GC111) and V343K (GC112) mutants have lower Tm values of 61.0 °C, 62.3 °C, and 60.5 °C, respectively. The Tm for the disulfide mutant GC100 (L286C / A318C) is 65.0°C, which is an approximately 10 °C increase compared to the wild-type GCase enzyme. Introduction of the activity mutants on the GC100 background such as V343A (GC102), V343S (GC103), and V343Q (GC104) on the GC100 (L286C / A318C) background generally maintained the Tm value near 65 °C, whereas V343E (GC105) and V343M (GC107) show slightly lower Tm values of62.3 °C and 63.1 °C, respectively. Overall, the disulfide GCase variants and their triple activityenhancing mutants show improvements of 7° C to 10° C in their thermal-melt Tm values.Example 5: Forced Degradation and Half-life Estimation of Engineered GCase Variants in PBS pH 7.4 at 37°C.

[0604] The mv-Fc-GCase mutant proteins, at 0.4 to 1.0 mg / mL, were dialyzed against PBS pH 7.4 at 4°C overnight with at least one additional buffer exchange using slide- A-lyzer 10 kD molecular weight cut off (MWCO; 0.5 mL cups). Concentrations of dialyzed proteins were measured using Lunatic (Unchained labs). About 80 to 120 pL of mv-Fc-GCase variant proteins in PBS pH 7.4 were incubated at 37 °C for up to 7 days. Samples were frozen at various time points and analyzed on Superdex 200 Increase 5 / 150 GL column mounted on an Agilent 1260 Infinity II HPLC using 25 mM sodium acetate pH 5.0 and 150 mM sodium chloride. Area under the GCase peak corresponding to its monomeric conformation was used to estimate their half-life in PBS pH 7.4 at 37°C.Table 20. Estimated half-life of mvFc-GCase mutants in PBS pH 7.4 at 37 °C

[0605] Engineered GCase variants, in the monovalent-Fc format, were incubated at 37 °C in PBS at pH 7.4 for 7 days and analyzed at various time intervals by analytical SEC to estimate their half-life (Table 20). The wild-type GCase enzyme has a short half-life of about 6 hours. Point mutations of GCase such as V343A (GC101) and V343T (GC113) have half-life of less than a day. The disulfide mutant GC119 showed a half-life of greater than 7-days. The V343M (GC109), V343R (GC110), and V343L (GC111) GCase variants also showed a half-life of greater than 7 days, and V343K (GC112) had a half-life of about 7 days. Thus, GC109, GC110, GC111, and GC112 variants of GCase exhibited good stress stability, in addition to, increased activity. The half-life for the disulfide engineered GCase mutant GC100 (L286C, A318C) is greater than 4-days, but less than 7-days. On this GC100 background, V343S (GC103) and V343S / T379I (GC125), which demonstrated significantly enhanced activity, showed half-lives of less than 4 days and approximately 4 days, respectively. V343A (GC102), V343Q (GC104), V343E (GC105), and V343M (GC107) variants on the GC100 background each showed a halflife of about 7 days. Thus, all variants listed in Table 20 show significantly extended half-life and stress stability in units of days compared to wild-type GCase enzyme, which has a half-life of only about 6 hours.Example 6: Animal Study

[0606] The following N-terminal monozyme-TfR antibody constructs will be administered in animals.Table 21. Constructs To Be TestedTable 22. Combined Sequences of Constructs To Be TestedExample 7: Enzymatic Activity of an Engineered GCase Variant in N-terminal Monozyme Format

[0607] The activity of GC103 in an N-terminal monozyme format was assessed. GC103 was linked to the N-terminus of a LALAP329S.knob Fc domain (SEQ ID NO:437), the VH and CHI of the antibody designated hTfR.15.WH8.1.42Q.L7-2 was linked to the N-terminus of a LALAP329S Fc domain in a hole format (SEQ ID NO:429), and a third polypeptide comprising the VL and CL of hTfR.15.WH8.1.42Q.L7-2 (SEQ ID NO:431) together form the N-term monozyme GC103 hTfR.15.WH8.1.42Q.L7-2 (see Example 6, above).

[0608] GC103 activity in the N-terminal monozyme format was compared to wild-type GCase in the N-terminal monozyme format using the cell-free activity assay described in Example 3, above, except that construct concentrations were 30 nM. In this assay, GC103 (comprising the GCase mutations L286C / A318C / V343S) exhibited a Vmax approximately 9-fold higher than the wild-type enzyme.

[0609] Next, engineered GCase variants were fused to a full length Fc and an anti-TfR Fab to confirm that the activity measured in monovalent-Fc format was consistent with the N-terminal monozyme format (as shown in Figure 2A). The engineered GCase variants were N-term monozyme WT-mTfRl, N-term monozyme GC119-TfR.15.WH8.L42Q, and N-term monozyme GC109-TfR.15.WH8.1.42Q (mTfRl refers to a surrogate mouse TfR antibody).

[0610] The engineered GCase variants were tested for GCase activity using the same 4-MUG substrate as described above. Thirty (30) nanomolar of recombinant enzyme fusions were incubated in Mcllvaine buffer (77 mM Na2HPC>4, 61.5 mM citric acid, pH 4.0, supplemented with 9 mM sodium taurocholate) with variable concentrations of 4-MUG ranging from 10 to 5900 pM. The samples were placed in a plate reader (BioTek) pre-warmed to 37°C. For 50 minutes, fluorescence elicited by 355 nm light was detected at 460 nm every 4 minutes. The fluorescence intensity was graphed as a function of time, and the activity was determined by measuring the maximum slope of the curve over 16 minutes. The activity was converted from arbitrary units per minute to 4-MU pmol per minute using a standard curve of 4-MUconcentration vs fluorescence. The GCase activity was graphed against the 4-MUG substrate concentration, and a Michaelis-Menten regression analysis was performed to determine Vmax (GraphPad Prism VI 0).

[0611] As shown in Figure 6, N-term monozyme GC119-hTfR.15.WH8.1.42Q maintained higher GCase activity (~25-fold) than wild-type enzyme (N-term monozyme WT-mTfRl), while N-term monozyme GC109-TfR.15.WH8.1.42Q exhibited ~48-fold more activity than wild-type enzyme (N-term monozyme WT-mTfRl) in this format. This data confirms that engineered N- terminal monozyme maintained the increased activity over GCase wild-type enzyme observed in the monovalent Fc format. The fold-increase over WT was largely dependent on GCase wildtype enzyme activity levels, which may vary 2-3-fold between experiments, but the fold-increase between GCase variants was consistent between both formats (i.e., GC109 was ~2-fold more active than GC119).Example 8: Evaluation of Pharmacokinetic, Toxicokinetic, and Pharmacodynamic Parameters of GCase-TfR in Non-Human Primates (NHPs)

[0612] Three- to four-year old female Cynomolgus monkeys (3 per group) were injected intravenously weekly for 23 days with the treatments in Table 23.Table 23: NHP Study Groups

[0613] At multiple time points before and after each injection event, blood samples were collected to measure reticulocyte count, hemoglobin level, and red blood cell (RBC) count. Engagement of TfR with an antibody can lead to depletion of reticulocytes and loss of red cell mass. To prevent this, the GCase-TfR constructs comprise a silenced Fc domain, preventing engagement of Fc receptors on immune cells or ac...

Claims

WHAT IS CLAIMED IS:

1. A variant glucocerebrosidase (GCase) polypeptide comprising one or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the one or more amino acid substitutions comprises: V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241.

2. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343S with reference to numbering of SEQ ID NO: 241.

3. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substiutions comprises V343Q with reference to numbering of SEQ ID NO: 241.

4. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343E with reference to numbering of SEQ ID NO: 241.

5. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343M with reference to numbering of SEQ ID NO: 241.

6. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343R with reference to numbering of SEQ ID NO: 241.

7. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343L with reference to numbering of SEQ ID NO: 241.

8. The variant GCase polypeptide of claim 1, wherein the one or more amino acid substitutions comprises V343K with reference to numbering of SEQ ID NO: 241.

9. The variant GCase polypeptide of any one of claims 1-8, wherein the wild-type GCase polypeptide is a mammalian GCase.

10. The variant GCase polypeptide of any one of claims 1-9, wherein the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

11. The variant GCase polypeptide of any one of claims 1-10, wherein the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

12. The variant GCase polypeptide of any one of claims 1-11, wherein the GCase polypeptide further comprises two or more amino acid substitutions capable of forming an intramolecular disulfide bond.

13. The variant GCase polypeptide of claim 12, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L286C and A318C with reference to SEQ ID NO: 241.

14. The variant GCase polypeptide of claim 12, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L240C and G250C with reference to SEQ ID NO: 241.

15. The variant GCase polypeptide of claim 12, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise T43C and S488C with reference to SEQ ID NO: 241.

16. The variant GCase polypeptide of any one of claims 1-14, wherein the variant Gcase polypeptide does not comprise T43C and / or S488C with reference to SEQ ID NO:241.

17. The variant GCase polypeptide of claim 12, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise W312C and A341C with reference to SEQ ID NO: 241.

18. The variant GCase polypeptide of any one of claims 1-17, wherein the variant GCase polypeptide does not comprise W312C and / or A341C with reference to SEQ ID NO: 241.

19. The variant GCase polypeptide of claim 12, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise G83C and L383C with reference to SEQ ID NO: 241.

20. The variant GCase polypeptide of any one of claims 1-18, wherein the variant GCase polypeptide does not comprise G83C and / or L383C with reference to SEQ ID NO: 241.

21. The variant GCase polypeptide of any one of claims 1-20, wherein the variant GCase polypeptide further comprises a T379I substitution with reference to SEQ ID NO: 241.

22. The variant GCase polypeptide of any one of claims 1-15, wherein the variant GCase polypeptide further comprises a A338H substitution with reference to SEQ ID NO: 241.

23. The variant GCase polypeptide of any one of claims 1, 2, and 9-22, wherein variant GCase polypeptide comprises V343S, L286C, and A318C substitutions with reference to SEQ ID NO: 241.

24. The variant GCase polypeptide of any one of claims 1, 2, and 9-23, wherein the variant GCase polypeptide comprises V343S, L286C, A318C, and T379I substitutions with reference to SEQ ID NO: 241.

25. A variant GCase polypeptide comprising two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to a wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L286C and A318C with reference to the numbering of SEQ ID NO: 241.

26. The variant GCase polypeptide of claim 25, wherein the wild-type GCase polypeptide is a mammalian GCase.

27. The variant GCase polypeptide of claim 25 or 26, wherein the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

28. The variant GCase polypeptide of any one of claims 25-27, wherein the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

29. The variant GCase polypeptide of any one of claims 25-28, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L286C and A318C with reference to SEQ ID NO: 241.

30. A variant GCase polypeptide comprising two or more amino acid substitutions capable of forming an intramolecular disulfide bond as compared to an wild-type GCase polypeptide, wherein the two or more amino acid substitutions comprise at least one of L240C and G250C with reference to SEQ ID NO: 241.

31. The variant GCase polypeptide of claim 30, wherein the wild-type GCase polypeptide is a mammalian GCase.

32. The variant GCase polypeptide of claim 30 or 31, wherein the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

33. The variant GCase polypeptide of any one of claims 30-32, wherein the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

34. The variant GCase polypeptide of any one of claims 30-33, wherein the two or more amino acid substitutions capable of forming an intramolecular disulfide bond comprise L240C and G250C with reference to SEQ ID NO: 241.

35. The variant GCase polypeptide of any one of claims 25-34, wherein the variant GCase polypeptide further comprises V343S, V343Q, V343E, V343M, V343R, V343L, or V343K with reference to numbering of SEQ ID NO: 241.

36. The variant GCase polypeptide of any one of claims 25-34, wherein the variant GCase polypeptide further comprises V343A or V343T with reference to SEQ ID NO: 241.

37. A variant GCase polypeptide comprising three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343T, W312C, and A341C with reference to SEQ ID NO: 241.

38. A variant GCase polypeptide comprising three or more amino acid substitutions as compared to a wild-type GCase polypeptide, wherein the three or more amino acid substitutions comprise V343M, W312C, and A341C with reference to SEQ ID NO: 241.

39. The variant GCase polypeptide of claim 37 or 38, wherein the wild-type GCase polypeptide is a mammalian GCase.

40. The variant GCase polypeptide of any one of claims 37-39, wherein the wild-type GCase comprises (i) the amino acid sequence set forth in SEQ ID NO: 241 or (ii) a sequence of amino acids that has at least 95% amino acid sequence identity to SEQ ID NO: 241.

41. The variant GCase polypeptide of any one of claims 37-40, wherein the variant GCase polypeptide comprises at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 241.

42. The variant GCase polypeptide of any one of claims 1-41, wherein the variant GCase polypeptide does not comprise an amino acid change at any of positions Glu235, Glu340, and Cys342 with reference to SEQ ID NO: 241.

43. The variant GCase polypeptide of any one of claims 1-42, wherein the variant GCase polypeptide comprises at least 96% amino acid sequence identity to SEQ ID NO: 241.

44. The variant GCase polypeptide of claim 43, wherein the variant GCase polypeptide comprises at least 97% amino acid sequence identity to SEQ ID NO: 241.

45. The variant GCase polypeptide of claim 44, wherein the variant GCase polypeptide comprises at least 98% amino acid sequence identity to SEQ ID NO :241.

46. The variant GCase polypeptide of claim 45, wherein the variant GCase polypeptide comprises at least 99% amino acid sequence identity to SEQ ID NO: 241.

47. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 453, 454, or 455.

48. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 395, 396, 288, or 246.

49. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 399.

50. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 395.

51. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 392.

52. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 379, 245, 377, 376, or 374.

53. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 245.

54. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 385, 388, or 387.

55. The variant GCase polypeptide of claim 1, wherein the variant GCase polypeptide comprises the amino acid sequence of SEQ ID NO: 389 or 390.

56. The variant GCase polypeptide of any one of claims 1-55, wherein the variant GCase polypeptide is capable of hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell- free enzyme assay.

57. The variant GCase polypeptide of claim 56, wherein the variant GCase polypeptide is at least as efficient as a polypeptide of SEQ ID NO: 241 in hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell-free enzyme assay.

58. The variant GCase polypeptide of claim 57, wherein the variant GCase polypeptide is at least ten times as efficient as a polypeptide of SEQ ID NO: 241 in hydrolyzing a glycolipid glucosylceramide (Glc-Cer) in a cell-free enzyme assay.

59. The variant GCase polypeptide of any one of claims 1-58, wherein the variant GCase polypeptide is capable of converting 4-Methylumbelliferyl-P-D-glucopyranosiduronic acid (4-MUG) into 4-Methylumbelliferone (4-MU).

60. The variant GCase polypeptide of claim 59, wherein the variant GCase polypeptide is at least as efficient as a polypeptide of SEQ ID NO: 241 in converting 4-MUG into 4-MU.

61. The variant GCase polypeptide of claim 60, wherein the variant GCase polypeptide is at least two times as efficient as a polypeptide of SEQ ID NO:241 in converting 4-MUG into 4-MU.

62. The variant GCase polypeptide of claim 60, wherein the variant GCase polypeptide is at least five times as efficient as a polypeptide of SEQ ID NO:241 in converting 4-MUG into 4-MU.

63. The variant GCase polypeptide of claim 60, wherein the variant GCase polypeptide is at least ten times as efficient as a polypeptide of SEQ ID NO: 241 in converting 4-MUG into 4-MU.

64. The variant GCase polypeptide of any one of claims 1-63, wherein the variant GCase polypeptide is expressed at a level equivalent to or greater than the expression level of a polypeptide of SEQ ID NO: 241 in ExpiCHO cells.

65. The variant GCase polypeptide of any one of claims 1-64, wherein the GCase polypeptide has a melting temperature equivalent to or greater than the melting temperature of a polypeptide of SEQ ID NO: 241.

66. he variant GCase polypeptide of any one of claims 1-65, wherein the GCase polypeptide has a half-life equivalent to or longer than the half-life of a polypeptide of SEQ ID NO: 241 in PBS pH 7.4 at 37 °C.

67. A fusion protein comprising an Fc domain and the variant GCase polypeptide of any one of claims 1-66.

68. The fusion protein of claim 67, wherein the variant GCase polypeptide is N-terminal to the Fc domain.

69. The fusion protein of claim 67 or 68, further comprising a linker between the Fc domain and the variant GCase polypeptide.

70. The fusion protein of claim 67 or 69, wherein the Fc domain is N-terminal to the variant GCase polypeptide.

71. The fusion protein of any one of claims 67-70, wherein the Fc domain comprises a CH2 and a CH3 domain.

72. A complex comprising the variant GCase polypeptide of any one of claims 1-66 and an antigen-binding domain that specifically binds to a blood brain barrier (BBB) target.

73. The complex of claim 72, wherein the BBB target is human transferrin receptor (TfR).

74. The complex of claim 73, 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, VL CDR2, and VL 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;(xxxvi) SEQ ID NOs: 10, 22, 28; and 54, 58, and 62; respectively;(xxxvii) SEQ ID NOs: 10, 22, 30, 50, 58, and 62, respectively;(xxxviii) SEQ ID NOs: 10, 22, 461, 50, 58, and 62, respectively;(xxxix) SEQ ID NOs: 10, 22, 28, 463, 58, and 62, respectively;(xl) SEQ ID NOs: 10, 22, 28, 464, 58, and 62, respectively;(xli) SEQ ID NOs: 10, 22, 28, 465, 58, and 62, respectively;(xlii) SEQ ID NOs: 10, 22, 28, 466, 58, and 62, respectively;(xliii) SEQ ID NOs: 10, 22, 462, 50, 58, and 62, respectively;(xliv) SEQ ID NOs: 8, 14, 25, 41, 55, and 61, respectively;(xlv) SEQ ID NOs: 8, 15, 25, 467, 55, and 61, respectively;(xlvi) SEQ ID NOs: 8, 15, 25, 468, 55, and 61, respectively; or(xlviii) SEQ ID NOs: 8, 14, 25, 469, 55, and 61, respectively.

75. The complex of claim 74, wherein the antigen-binding domain that specifically binds to human TfR comprises a VH and a VL comprising 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) SEQ ID NOs: 77 and 131, respectively;(xxiv) SEQ ID NOs: 80 and 140, respectively;(xxv) SEQ ID NOs: 81 and 141, respectively;(xxvi) SEQ ID NOs: 82 and 131, respectively;(xxvii) SEQ ID NOs: 83 and 142, respectively;(xxviii) SEQ ID NOs: 77 and 143, respectively;(xxix) SEQ ID NOs: 75 and 131, respectively;(xxx) SEQ ID NOs: 75 and 144, respectively;(xxxi) SEQ ID NOs: 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) SEQ ID NOs: 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) SEQ ID NOs: 88 and 131, respectively;(xli) SEQ ID NOs: 75 and 149, respectively;(xlii) SEQ ID NOs: 89 and 150, respectively;(xliii) SEQ ID NOs: 90 and 151, respectively;(xliv) SEQ ID NOs: 77 and 152, respectively;(xlv) SEQ ID NOs: 79 and 153, respectively;(xlvi) SEQ ID NOs: 77 and 139, respectively;(xlvii) SEQ ID NOs: 91 and 131, respectively;(xlviii) SEQ ID NOs: 92 and 131, respectively;(xlix) SEQ ID NOs: 79 and 154, respectively;(1) SEQ ID NOs: 93 and 155, respectively;(li) SEQ ID NOs: 80 and 131, respectively;(lii) SEQ ID NOs: 94 and 131, respectively;(liii) SEQ ID NOs: 95 and 131, respectively;(liv) SEQ ID NOs: 66 and 156, respectively;(Iv) SEQ ID NOs: 97 and 138, respectively;(Ivi) SEQ ID NOs: 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;(cii) SEQ ID NOs: 117 and 178, respectively;(ciii) SEQ ID NOs: 118 and 174, respectively;(civ) SEQ ID NOs: 470 and 174, respectively;(cv) SEQ ID NOs: 117 and 471, respectively;(cvi) SEQ ID NOs: 117 and 472, respectively;(cvii) SEQ ID NOs: 117 and 473, respectively;(cviii) SEQ ID NOs: 117 and 474, respectively;(cix) SEQ ID NOs: 475 and 174, respectively;(ex) SEQ ID NOs: 101 and 154, respectively;(cxi) SEQ ID NOs: 102 and 476, respectively;(cxii) SEQ ID NOs: 102 and 477, respectively; or(cxiii) SEQ ID NOs: 101 and 478, respectively.

76. The complex of claim 72, wherein the BBB target is human CD98 heavy chain (CD98hc).

77. The complex of 76, wherein the antigen-binding domain that specifically binds to human CD98 comprises heavy chain variable region (VH) complementarity determining region (CDR) 1, VH CDR2, VH CDR3 and light chain variable region (VL) CDR1, VL CDR2, and VL 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;(ix) SEQ ID NOs: 184, 234, 193, 197, 198, and 204; respectively,(x) SEQ ID NOs: 184, 291, 193, 197, 198, and 204, respectively;(xi) SEQ ID NOs: 184, 292, 193, 197, 198, and 204, respectively;(xii) SEQ ID NOs: 184, 293, 193, 197, 198, and 204, respectively;(xiii) SEQ ID NOs: 184, 294, 193, 197, 198, and 204, respectively;(xiv) SEQ ID NOs: 184, 295, 193, 197, 198, and 204, respectively;(xv) SEQ ID NOs: 184, 296, 193, 197, 198, and 204, respectively;(xvi) SEQ ID NOs: 184, 297, 193, 197, 198, and 204, respectively;(xvii) SEQ ID NOs: 184, 190, 193, 318, 198, and 204, respectively;(xviii) SEQ ID NOs: 184, 190, 193, 319, 198, and 204, respectively;(xix) SEQ ID NOs: 184, 190, 193, 320, 198, and 204, respectively;(xx) SEQ ID NOs: 184, 190, 193, 321, 198, and 204, respectively;(xxi) SEQ ID NOs: 184, 190, 193, 322, 198, and 204, respectively;(xxii) SEQ ID NOs: 184, 190, 193, 323, 198, and 204, respectively;(xxiii) SEQ ID NOs: 184, 190, 193, 324, 198, and 204, respectively;(xxiv) SEQ ID NOs: 184, 293, 193, 320, 198, and 204, respectively;(xxv) SEQ ID NOs: 184, 298, 193, 197, 198, and 204, respectively;(xxvi) SEQ ID NOs: 184, 190, 299, 197, 198, and 18, respectively;(xxvii) SEQ ID NOs: 184, 190, 300, 197, 198, and 204, respectively;(xxviii) SEQ ID NOs: 184, 190, 301, 197, 198, and 204, respectively;(xxix) SEQ ID NOs: 184, 190, 302, 197, 198, and 204, respectively;(xxx) SEQ ID NOs: 184, 190, 303, 197, 198, and 204, respectively;(xxxi) SEQ ID NOs: 184, 190, 304, 197, 198, and 204, respectively;(xxxii) SEQ ID NOs: 184, 190, 305, 197, 198, and 204, respectively;(xxxiii) SEQ ID NOs: 184, 190, 306, 197, 198, and 204, respectively;(xxxiv) SEQ ID NOs: 184, 190, 307, 197, 198, and 204, respectively;(xxxv) SEQ ID NOs: 184, 190, 193, 325, 198, and 204, respectively;(xxxvi) SEQ ID NOs: 184, 190, 193, 326, 198, and 204, respectively;(xxxvii) SEQ ID NOs: 184, 190, 193, 327, 198, and 204, respectively;(xxxviii) SEQ ID NOs: 184, 190, 193, 328, 198, and 204, respectively;(xxxix) SEQ ID NOs: 184, 190, 193, 329, 198, and 204, respectively;(xl) SEQ ID NOs: 184, 190, 193, 330, 198, and 204, respectively;(xli) SEQ ID NOs: 184, 190, 193, 197, 198, and 308, respectively;(xlii) SEQ ID NOs: 184, 190, 193, 197, 198, and 309, respectively;(xliii) SEQ ID NOs: 184, 190, 193, 197, 198, and 310, respectively;(xliv) SEQ ID NOs: 184, 190, 193, 197, 198, and 311, respectively;(xlv) SEQ ID NOs. 184, 190, 193, 197, 198, and 312, respectively;(xlvi) SEQ ID NOs: 184, 190, 193, 197, 198, and 313, respectively;(xlvii) SEQ ID NOs: 184, 190, 193, 197, 198, and 314, respectively;(xlviii) SEQ ID NOs: 184, 190, 193, 197, 198, and 315, respectively;(xlix) SEQ ID NOs: 184, 190, 193, 197, 198, and 316, respectively;(1) SEQ ID NOs: 184, 190, 193, 197, 198, and 317, respectively;(li) SEQ ID NOs: 290, 190, 193, 197, 198, and 204, respectively; or(lii) SEQ ID NOs: 290, 190, 193, 197, 198, and 317, respectively.

78. The complex of claim 77, wherein the antigen-binding domain that specifically binds to human CD98 comprises a VH and a VL comprising 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;(ix) SEQ ID NOs: 237 and 216, respectively;(x) SEQ ID NOs: 331 and 216, respectively;(xi) SEQ ID NOs: 332 and 216, respectively;(xii) SEQ ID NOs: 333 and 216, respectively;(xiii) SEQ ID NOs: 334 and 216, respectively;(xiv) SEQ ID NOs: 335 and 216, respectively;(xv) SEQ ID NOs: 336 and 216, respectively;(xvi) SEQ ID NOs: 337 and 216, respectively;(xvii) SEQ ID NOs: 210 and 338, respectively;(xviii) SEQ ID NOs: 210 and 339, respectively;(xix) SEQ ID NOs: 210 and 340, respectively;(xx) SEQ ID NOs: 210 and 341, respectively;(xxi) SEQ ID NOs: 210 and 342, respectively;(xxii) SEQ ID NOs: 210 and 343, respectively;(xxiii) SEQ ID NOs: 210 and 344, respectively;(xxiv) SEQ ID NOs: 333 and 340, respectively;(xxv) SEQ ID NOs: 345 and 216, respectively;(xxvi) SEQ ID NOs: 346 and 216, respectively;(xxvii) SEQ ID NOs: 347 and 216, respectively;(xxviii) SEQ ID NOs: 348 and 216, respectively;(xxix) SEQ ID NOs: 349 and 216, respectively;(xxx) SEQ ID NOs: 350 and 216, respectively;(xxxi) SEQ ID NOs: 351 and 216, respectively;(xxxii) SEQ ID NOs: 352 and 216, respectively;(xxxiii) SEQ ID NOs: 353 and 216, respectively;(xxxiv) SEQ ID NOs: 354 and 216, respectively;(xxxv) SEQ ID NOs: 210 and 355, respectively;(xxxvi) SEQ ID NOs: 210 and 356, respectively;(xxxvii) SEQ ID NOs: 210 and 357, respectively;(xxxviii) SEQ ID NOs: 210 and 358, respectively;(xxxix) SEQ ID NOs: 210 and 359, respectively;(xl) SEQ ID NOs: 210 and 360, respectively;(xli) SEQ ID NOs: 210 and 361, respectively;(xlii) SEQ ID NOs: 210 and 362, respectively;(xliii) SEQ ID NOs: 210 and 363, respectively;(xliv) SEQ ID NOs: 210 and 364, respectively;(xlv) SEQ ID NOs: 210 and 365, respectively;(xlvi) SEQ ID NOs: 210 and 366, respectively;(xlvii) SEQ ID NOs: 210 and 367, respectively;(xlviii) SEQ ID NOs: 210 and 368, respectively;(xlix) SEQ ID NOs: 210 and 369, respectively;(1) SEQ ID NOs: 210 and 370, respectively;(li) SEQ ID NOs: 371 and 215, respectively; or(lii) SEQ ID NOs: 371 and 370, respectively.

79. The complex of any one of claims 72-78, wherein the complex comprises a first polypeptide comprising the variant GCase polypeptide 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.

80. The complex of claim 79, wherein the variant GCase polypeptide is N-terminal to the first Fc domain.

81. The complex of claim 80, wherein the first polypeptide further comprises an antibody hinge between the variant GCase polypeptide or fragment thereof and the first Fc domain.

82. The complex of any one of claims 79-81, wherein the first Fc domain comprises a knob mutation and the second Fc comprises a hole mutation.

83. The complex of any one of claims 79-81, wherein the first Fc domain comprises a hole mutation and the second Fc comprises a knob mutation.

84. The complex of any one of claims 79-83, wherein the second polypeptide chain is an antibody heavy chain.

85. The complex of any one of claims 79-84, wherein the third polypeptide further comprises a CL domain, optionally wherein the third polypeptide is an antibody light chain.

86. The complex of any one of claims 79-85, wherein the first Fc domain comprises a CH2 and a CH3 and / or wherein the second Fc domain comprises a CH2 and a CH3.

87. The complex of claim 76 comprising the amino acid sequence of SEQ ID NO: 510, 511, 514, or 517.

88. The complex of claim 72, wherein the complex comprises the amino acid sequences of SEQ ID NOs: 437, 429, and 431; SEQ ID NOs: 526, 429, and 431; SEQ ID NOs: 526, 528, and 529; SEQ ID NOs: 526, 429, and 430; SEQ ID NOs: 437, 429, and 430; SEQ ID NOs: 526, 429, and 428; or SEQ ID NOs: 437, 429, and 428.

89. The complex of claim 72, wherein the complex comprises the amino acid sequences of SEQ ID NOs: 526, 429, and 430.

90. A viral vector comprising an expression cassette comprising a nucleotide sequence encoding the variant GCase polypeptide of any one of claims 1-66.

91. A recombinant adeno-associated virus (rAAV) vector comprising an expression cassette comprising a nucleotide sequence encoding the variant GCase polypeptide of any one of claims 1-66.

92. The rAAV vector of claim 91, wherein the nucleotide encoding the variant GCase polypeptide is operably linked to a promoter.

93. The rAAV vector of claim 92, wherein the promoter induces expression of the variant GCase polypeptide in the central nervous system.

94. The rAAV vector of claim 92, wherein the promoter induces expression of the variant GCase polypeptide in macrophages.

95. The rAAV vector of any one of claims 91-94, wherein the nucleotide encoding the variant GCase polypeptide is operably linked to an enhancer.

96. The rAAV vector of any one of claims 91-95, wherein the nucleotide encoding the variant GCase polypeptide is codon optimized for expression in a target cell.

97. An rAAV viral particle comprising an AAV capsid protein encapsidating the rAAV vector of any one of claims 91-96.

98. The rAAV viral particle of claim 97, wherein the viral particle comprises an AAV9 capsid protein.

99. The rAAV viral particle of claim 97, wherein the viral particle comprises an AAVrhlO capsid protein.

100. The rAAV viral particle of claim 97, wherein the viral particle comprises an AAV2 capsid protein.

101. The rAAV viral particle of claim 97, wherein the viral particle comprises an AAV8 capsid protein.

102. The rAAV viral particle of any one of claims 97-101, wherein the capsid further comprises a binding moiety which targets human TfR.

103. A polynucleotide or combination of polynucleotides comprising a nucleotide sequence or combination of nucleotide sequences encoding the variant GCase polypeptide of any one of claims 1-66, the fusion protein of any one of claims 67-71, or the complex of any one of claims 72-89.

104. A host cell comprising the polynucleotide or combination of polynucleotides of claim 103.

105. A method of producing the variant GCase polypeptide of any one of claims 1-66, the fusion protein of any one of claims 67-71, or the complex of any one of claims 72-89 comprising culturing the host cell of claim 104.

106. The variant GCase polypeptide, fusion protein, or complex produced by the method of claim 105.

107. A host cell comprising the variant GCase polypeptide, fusion protein or complex of any one of claims 1-89 and 106.

108. A composition comprising the variant GCase polypeptide, fusion protein, or complex of any one of claims 1-89 and 106 or the polynucleotide or combination of polynucleotides of claim 103.

109. A pharmaceutical composition comprising (a) the variant GCase polypeptide, fusion protein, or complex of any one of claims 1-89 and 106 or the polynucleotide or combination of polynucleotides of claim 103 and (b) a pharmaceutically acceptable carrier, excipient or stabilizer.

110. A pharmaceutical composition comprising the rAAV viral particle of any one of claims 97-102 and a pharmaceutically acceptable carrier, excipient, or stabilizer.

111. The pharmaceutical composition of claim 110 comprising a dose of about 1011vg / kg to about 1014vg / kg.

112. A method of treating a lysosomal storage disease or disorder in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides, or composition of any one of claims 1- 89, 103, 106and 108-111.

113. The method of claim 112, wherein the lysosomal storage disease or disorder is Gaucher’s disease.

114. A method of reducing glucosyl sphingosine (GlcSph) accumulation in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition of any one of claims 1-89, 103, 106, and 108-111.

115. The method of claim 114, wherein the method reduces GlcSph accumulation in the liver.

116. The method of claim 114, wherein the method reduces GlcSph accumulation in the brain.

117. A method of enhancing peripheral P-glucocerebrosidase (GCase) in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition of any one of claims 1- 89, 103, 106, and 108-111.

118. A method of treating a CNS disease or disorder in a subject comprising administering to the subject the variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition of any one of claims 1-89, 103, 106, and 108-111.

119. The method of claim 118, wherein the CNS disease or disorder is Parkinson’s Disease or Lewy Body Dementia.

120. The method of claim 119, wherein the CNS disorder is early Parkinson’s Disease and the subject has a GBA gene mutation.

121. The method of any one of claims 112-120, wherein the method reduces brain lipid accumulation in the subject.

122. The method of any one of claims 112-116 and 118-121, wherein the method enhances peripheral Gcase activity in the subject.

123. The method of any one of claims 112, 113, and 117-122, wherein the method reduces liver glucosyl sphingosine (GlcSph) accumulation in the subject.

124. The method of any one of claims 112, 113, and 117-122, wherein the method reduces brain glucosyl sphingosine (GlcSph) accumulation in the subject.

125. The method of any one of claims 112-124, wherein the administration is intravenous administration.

126. The method of any one of claims 112-124, wherein the administration is subcutaneous administration.

127. The variant GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition of any one of claims 1-89, 103, 106, and 108-111 for use in the method of any one of claims 112-126.

128. Use of the GCase polypeptide, fusion protein, complex, polynucleotide or combination of polynucleotides or composition of any one of claims 1-89, 103, 106, and 108-111 in the method of any one of claims 112-126.

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