Fusion proteins comprising modified glucocerebrosidase polypeptides and methods thereof

Abstract

Provided herein are proteins, which are capable of being transported across the blood-brain barrier (BBB) and comprise a glucocerebrosidase (GCase)-Fc fusion polypeptide. Certain embodiments also provide methods of using such proteins to treat a GCase deficiency.

Description

[0001] FUSION PROTEINS COMPRISING MODIFIED GLUCOCEREBROSIDASE POLYPEPTIDES AND METHODS THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application Serial No. 63 / 663,610, filed June 24, 2024, and U.S. Provisional Application Serial No. 63 / 729,160, filed December 6, 2024. The entire content of the applications referenced above are hereby incorporated by reference herein.

[0004] BACKGROUND

[0005] Glucocerebrosidase (GCase) is a lysosomal enzyme that has glucosylceramidase activity and catalyzes the breakdown of glucosylceramide to ceramide and glucose. Mutations in glucocerebrosidase are known to cause or be associated with a number of diseases including Gaucher’s disease (Brady et al., J. Clin. Invest. 5(Ty.1112- 1115, 1966), Parkinson’s disease (Balestrino et al., Neuroscientist 24(5):540-559, 2018), Parkinsonism (Lwin et al., Mol. Genet. Metab. 81 (l):70-73, 2004), and dementia with Lewy Bodies (Nalls et al., JAMA Neurol. 7Q(6' .r12rl-r1 5, 2013; Guerreiro et al., Lancet Neurol 17(l):64-74, 2018). While enzyme replacement therapies are available to patients with GCase deficiencies (e.g., imiglucerase, also known as CEREZYME™), these recombinant forms suffer from low serum stability and enzyme inactivation at physiological conditions and have little effect on the brain due to difficulties in delivering the recombinant enzyme across the blood-brain barrier (BBB). Accordingly, there is a need for more stable and effective therapies for the treatment of GCase deficiencies.

[0006] SUMMARY

[0007] Certain embodiments provide a protein comprising (a) a fusion polypeptide comprising a first Fc polypeptide linked to a glucocerebrosidase (GCase) amino acid sequence; and (b) a second Fc polypeptide; wherein the first and / or second Fc polypeptide is a modified Fc that is capable of binding (e.g., specifically binding) to a blood-brain barrier (BBB) receptor, e.g., a transferrin receptor (TfR), and wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45. In certain embodiments, the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In certain embodiments, the second Fc polypeptide is a modified Fc that is capable of binding (e.g., specifically binding) to TfR. In certain embodiments, the second Fc polypeptide is a modified Fc comprising a sequence having at least 90% identity to SEQ ID NO: 32 and is capable of specifically binding to a TfR.

[0008] For example, certain embodiments provide a protein comprising: (a) a fusion polypeptide comprising a first Fc polypeptide linked to a glucocerebrosidase (GCase) amino acid sequence; and (b) a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45.

[0009] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to the GCase amino acid sequence. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to the GCase amino acid sequence.

[0010] In certain embodiments, at least two modifications of the two or more modifications present in the GCase amino acid sequence are at positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451, wherein the positions and modifications are relative to SEQ ID NO:45. For example, in certain embodiments, the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve modifications selected from the group consisting of: (i) Arg, Glu, or Gin at position 189; (ii) Arg, Asp, Gly, His or Asn at position 190; (iii) Leu, Trp, Met, Tyr or Phe at position 191; (iv) Vai, Asp, Thr, Ala, Glu or He at position 192; (v) Gly at position 233; (vi) Lys at position 245; (vii) Ser at position 316; (viii) Ala, Asn or Thr at position 343; (ix) Phe at position 345; (x) Glu, Asn, Gin or Leu at position 346; (xi) Thr, Ser or Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451.

[0011] In certain embodiments, the second Fc polypeptide comprises at the following positions, according to EU numbering: (i) Thr at position 386; (ii) Glu at position 387; (iii) Trp at position 388; (vi) Glu at position 416; and (v) Phe at position 421.

[0012] In certain embodiments, the second Fc polypeptide further comprises one or more of the specified residues as follows, wherein the positions are according to EU numbering: (i) Glu, Trp, or Leu at position 380; (ii) Phe or Tyr at position 384; (iii) Ser, Ala, Vai or Asn at position 389; (iv) Asn or Ser at position 390; (v) Thr or Ser at position 413; (vi) Glu or Ser at position 415.

[0013] In certain embodiments, the second Fc polypeptide comprises at the following positions, according to EU numbering: (i) Glu, Trp, or Leu at position 380; (ii) Phe or Tyr at position 384; (iii) Thr at position 386; (iv) Glu at position 387; (v) Trp at position 388; (vi) Ser, Ala, Vai or Asn at position 389; (vii) Asn or Ser at position 390; (viii) Thr or Ser at position 413; (ix) Glu or Ser at position 415; (x) Glu at position 416; and (xi) Phe at position 421.

[0014] In certain embodiments, the second Fc polypeptide comprises at the following positions, according to EU numbering: (i) Glu, Trp, or Leu at position 380 (e.g., Glu); (ii) Tyr at position 384; (iii) Thr at position 386; (iv) Glu at position 387; (v) Trp at position 388; (vi) Ser or Ala at position 389 (e.g., Ala); (vii) Asn or Ser at position 390 (e.g., Asn); (viii) Thr at position 413; (ix) Glu at position 415; (x) Glu at position 416; and (xi) Phe at position 421.

[0015] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 9-16 and 21-24. In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11-16, and 21-24. In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11, 12, 21, and 22. In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:15, 16, 23, and 24.

[0016] In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108- 111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120- 121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144- 147).

[0017] In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 29-36, 38-40, and 41-44 (e.g., 29-36 and 41-44). In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31-36, 39-40, and 41-44 (e.g., 31-36 and 41-44). In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31, 32, 39, 41 and 42 (e.g., 31, 32, 41 and 42). In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:35, 36, 40, 43 and 44 (e.g., 35, 36, 43 and 44).

[0018] In certain embodiments, the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 9-16 and 21-24; and the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 29-36, 38-40 and 41-44 (e.g., 29-36 and 41-44).

[0019] In certain embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147); and wherein the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 31-36, 39-40, and 41-44 (e.g., 31-36 and 41-44). In certain embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126- 135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147); and wherein the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 39-40 and 41-44 (e.g., 41-44).

[0020] In certain embodiments, the first Fc polypeptide is not modified to bind to a blood-brain barrier (BBB) receptor and the second Fc polypeptide is modified to specifically bind to a TfR.

[0021] Certain embodiments also provide a fusion polypeptide comprising an Fc polypeptide that is linked to a glucocerebrosidase (GCase) amino acid sequence, wherein the Fc polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 12 and contains one or more modifications that promote its heterodimerization to another Fc polypeptide, and wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45. In certain embodiments, the N-terminus of the Fc polypeptide is linked to the GCase amino acid sequence. In certain embodiments, the C-terminus of the Fc polypeptide is linked to the GCase amino acid sequence. In certain embodiments, the Fc polypeptide is linked to the GCase amino acid sequence by a peptide bond or by a polypeptide linker. In certain embodiments, the polypeptide comprises from N- to C-terminus: GCase amino acid sequence; a polypeptide linker; and the Fc polypeptide. In certain embodiments, the polypeptide comprises from N- to C-terminus: the Fc polypeptide; a polypeptide linker; and the GCase amino acid sequence. In certain embodiments, the Fc polypeptide comprises T366S, L368A, and Y407V substitutions, according to EU numbering. In certain embodiments, the Fc polypeptide comprises substitutions of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329, according to EU numbering. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108- 111, 120-121, 126-135, and 144-147).

[0022] Certain embodiments provide a protein comprising 1) an Fc polypeptide that is linked to a glucocerebrosidase (GCase) amino acid sequence, wherein the Fc polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 12 and contains one or more modifications that promote its heterodimerization to another Fc polypeptide, and wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45, and 2) the other Fc polypeptide.

[0023] Certain embodiments provide a pharmaceutical composition comprising the protein or polypeptide as described herein and a pharmaceutically acceptable carrier and / or excipient.

[0024] Certain embodiments provide a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as described herein (e.g., a GCase-Fc fusion polypeptide described herein). Certain embodiments provide a vector comprising a polynucleotide as described herein. Certain embodiments provide a host cell comprising a polynucleotide or vector as described herein. In certain embodiments, the host cell further comprises a polynucleotide comprising a nucleic acid sequence encoding another polypeptide described herein (e.g., the other Fc polypeptide, such as a TfR-binding modified Fc polypeptide). Certain embodiments provide a method for producing a fusion polypeptide comprising an Fc polypeptide that is linked to a GCase amino acid sequence, comprising culturing a host cell as described herein under conditions in which the fusion polypeptide encoded by a polynucleotide as described herein is expressed. In certain embodiments, the host cell is cultured in the absence of isofagomine during protein production.

[0025] Certain embodiments provide a pair of polynucleotides comprising nucleic acid sequences encoding the fusion polypeptide and the second Fc polypeptide as described herein. In particular, certain embodiments provide a pair of polynucleotides comprising a first and a second polynucleotide, wherein the first polynucleotide comprises a first nucleic acid sequence encoding a fusion polypeptide as described herein; and the second polynucleotide comprises a second nucleic acid sequence encoding a second Fc polypeptide as described herein. Certain embodiments provide one or more vectors comprising a pair of polynucleotides as described herein. For example, certain embodiments provide a single vector comprising the pair of polynucleotides. Other embodiments provide two vectors, wherein the first vector comprises the first polynucleotide from the pair and the second vector comprises the second polynucleotide from the pair. Certain embodiments provide a host cell comprising a pair of polynucleotides as described herein, or one or more vectors as described herein. Certain embodiments provide a method for producing a protein comprising a fusion polypeptide as described herein; and a second Fc polypeptide, the method comprising culturing a host cell as described herein under conditions in which a pair of polynucleotides as described herein is expressed. In certain embodiments, the host cell is cultured in the absence of isofagomine during protein production.

[0026] Certain embodiments provide a protein produced by a method as described herein.

[0027] Certain embodiments provide a protein produced by cell expression in the absence of isofagomine, wherein the protein comprises: a. a fusion polypeptide comprising a first Fc polypeptide linked to a modified glucocerebrosidase (GCase) amino acid sequence; and b. a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); and wherein the protein has 1) a higher level of enzymatic activity as compared to a corresponding protein comprising a wild-type GCase amino acid sequence; and / or 2) an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®).

[0028] Certain embodiments provide a method of treating a GCase deficiency in a patient in need thereof, the method comprising administering a protein or polypeptide as described herein to the patient.

[0029] Certain embodiments provide a protein or polypeptide as described herein for use in treating a GCase deficiency in a patient in need thereof.

[0030] Certain embodiments provide the use of a protein or polypeptide as described herein in the preparation of a medicament for treating a GCase deficiency in a patient in need thereof.

[0031] Certain embodiments provide a method of decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency, the method comprising administering a protein or polypeptide as described herein to the patient.

[0032] Certain embodiments provide a protein or polypeptide as described herein for use in decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency.

[0033] Certain embodiments provide the use of a protein or polypeptide as described herein in the preparation of a medicament for decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency. In certain embodiments, the toxic metabolic product comprises glucosylceramide or glucosylsphingosine.

[0034] BRIEF DESCRIPTION OF THE FIGURES

[0035] Figures 1A-1B. Evaluation of cellular potency of ETV:GCase fusion proteins in GBA knockout HEK cells by measuring the reduction of glucosylsphingosine (GlcSph) (FIG. 1 A) and glucosylceramide (GlcCer) (FIG. IB).

[0036] Figures 2A-2B. Evaluation of cellular potency of ETV:GCase fusion proteins in GBA knockout HEK cells by measuring the reduction of glucosyl sphingosine (GlcSph) (FIG. 2A) and glucosylceramide (GlcCer) (FIG. 2B).

[0037] Figures 3A-3B. Evaluation of single-dose pharmacokinetics of ETV:GCase fusion proteins in GBA D409V;TfR knock in mice (“D409V; TfRmu / hu”) jnserum. Measurements out to 24 hours in 3-month old mice are shown in FIG. 3A and measurements out to 7 days in 8- month old mice are shown in FIG. 3B. All mice were dosed with 40 mg / kg of fusion protein.

[0038] Figures 4A-4B. Evaluation of single-dose pharmacokinetics of ETV:GCase fusion proteins in GBA D409V;TfR knock in mice (“D409V; TfRmu / hu”) jnliver. Measurements 7 days post-single dose in 3-month old mice are shown in FIG. 4A and measurements 7 days postsingle dose in 8-month old mice are shown in FIG. 4B. All mice were dosed with 40 mg / kg of fusion protein.

[0039] Figures 5A-5B. Evaluation of single-dose pharmacokinetics of ETV:GCase fusion proteins in GBA D409V;TfR knock in mice (“D409V; TfRmu / hu”) jnbrain. Measurements 7 days post-single dose in 3-month old mice are shown in FIG. 5 A and measurements 7 days postsingle dose in 8-month old mice are shown in FIG. 5B. All mice were dosed with 40 mg / kg of fusion protein.

[0040] Figure 6. Evaluation of pharmacodynamic response at seven (7) days post-single dose in serum in a comparative study carried out in a healthy mouse model and a GCase deficiency mouse model. Mice were dosed with 40 mg / kg of fusion protein or vehicle. The healthy mouse model is represented by TfR knock in mice (“TfRmu / hu”)anc[ the GCase deficiency mouse model is represented by TfR knock in mice harboring the GBA D409V mutation (“D409V; TfRmu / hu”). The figure displays mean ± SEM: Kruskal -Wallis with Dunnett’s multiple comparison test.

[0041] Figure 7. Evaluation of pharmacodynamic response at seven (7) days post-single dose in liver in a comparative study carried out in a healthy mouse model and a GCase deficiency mouse model. Mice were dosed with 40 mg / kg of fusion protein or vehicle. The healthy mouse model is represented by TfR knock in mice (“TfRmu / hu”)anc[ the GCase deficiency mouse model is represented by TfR knock in mice harboring the GBA D409V mutation (“D409V; TfRmu / hu”). The figure displays mean ± SEM: One way ANOVA with Dunnett’s multiple comparison test.

[0042] Figure 8. Evaluation of pharmacodynamic response at seven (7) days post-single dose in brain in a comparative study carried out in a healthy mouse model and a GCase deficiency mouse model. Mice were dosed with 40 mg / kg of fusion protein or vehicle. The healthy mouse model is represented by TfR knock in mice (“TfRmu / hu”)anc[ the GCase deficiency mouse model is represented by TfR knock in mice harboring the GBA D409V mutation (“D409V; TfRmu / hu”). The figure displays mean ± SEM: One way ANOVA with Dunnett’s multiple comparison test.

[0043] Figure 9. Illustration of an exemplary ETV:GCase fusion protein, wherein the modified GCase enzyme (e.g., having a modified GCase amino acid sequence as described herein) is linked to an Fc polypeptide.

[0044] DETAILED DESCRIPTION

[0045] There is currently a need for new therapeutics for the treatment of patients having a GCase deficiency, specifically therapeutics that treat the neurological symptoms of such diseases. Described herein is a specific enzyme replacement therapy termed ETV:GCase, which has the capability of crossing the BBB and treating both the peripheral and CNS manifestations of a GCase deficiency (e.g., Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies). As used herein, the term “ETV:GCase” refers to a protein (e.g., a dimeric protein) that is capable of being transported across the BBB and comprises a first Fc polypeptide linked (e.g., fused) to a GCase amino acid sequence; and a second Fc polypeptide.

[0046] PROTEIN MOLECULES COMPRISING A GCASE -FC FUSION POLYPEPTIDE

[0047] As described herein, certain embodiments provide a protein molecule comprising a GCase-Fc fusion polypeptide (also referred to herein as “a GCase variant-Fc fusion polypeptide”). A GCase amino acid sequence incorporated into the protein is a modified (i.e., a variant) GCase sequence, which comprises two or more modifications and is catalytically active, i.e., it retains enzymatic activity. In some aspects, a protein described herein comprises: (a) a first Fc polypeptide, which may contain modifications (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and a modified GCase amino acid sequence as described herein; and (b) a second Fc polypeptide, which may contain modifications (e.g., one or more modifications that promote heterodimerization) or may be a wild-type Fc polypeptide; and optionally a GCase amino acid sequence, wherein the first and / or second Fc polypeptide comprises modifications that result in binding to a blood-brain barrier (BBB) receptor, e.g., a transferrin receptor (TfR).

[0048] Modified GCase Polypeptides

[0049] A GCase amino acid sequence, which may be incorporated into a fusion protein described herein, is a modified GCase sequence having two or more modifications. As described herein, such modified sequences may result in improved properties to a such a fusion protein as compared to a corresponding protein comprising a wild-type GCase sequence (e.g., SEQ ID NO: 45) or to imiglucerase (CEREZYME®). For example, in certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein may have a higher level of enzymatic activity (e.g., after serum exposure) as compared to a corresponding protein comprising a wild-type GCase sequence or to imiglucerase (CEREZYME®). Additionally, in certain embodiments, such a protein may not require the presence of, exposure to, or contact with isofagomine to achieve desired enzymatic levels. Examples of various modified GCase amino acid sequences that may be included in a protein described herein are discussed below.

[0050] For example, a GCase amino acid sequence may comprise two or more modifications at positions described herein, with respect to SEQ ID NO:45. Examples of various modifications are described below and in the Examples, wherein certain combinations may result in improved qualities of a fusion protein comprising such a GCase sequence.

[0051] In certain embodiments, the GCase amino acid sequence comprises two modifications, with respect to SEQ ID NO: 1. In certain embodiments, the GCase amino acid sequence comprises more than two modifications, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more modifications, with respect to SEQ ID NO: 1.

[0052] For example, in certain embodiments a GCase amino acid sequence comprises two or more modifications, wherein at least two modifications with respect to SEQ ID NO:45, are at positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451. In certain embodiments, the GCase amino acid sequence comprises a modification at three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451. In certain embodiments, the GCase amino acid sequence comprises a modification at three, four, five, six, seven, eight, nine, ten, eleven, or twelve positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451.

[0053] For example, in certain embodiments, the GCase amino acid sequence comprises a modification at two or three positions selected from the group consisting of: 233, 343, and 347. In certain embodiments, the GCase amino acid sequence comprises a modification at positions 233 and 343. In certain embodiments, the GCase amino acid sequence comprises a modification at positions 233 and 347. In certain embodiments, the GCase amino acid sequence comprises a modification at positions 343 and 347. In certain embodiments, the GCase amino acid sequence comprises a modification at positions 233, 343, and 347. In certain embodiments, the GCase amino acid sequence further comprises a modification at position 191 and / or 192. In certain embodiments, the GCase amino acid sequence further comprises a modification at position 191. In certain embodiments, the GCase amino acid sequence further comprises a modification at position 192. In certain embodiments, the GCase amino acid sequence comprises a modification at each of the following: 191, 192, 233, 343, and 347.

[0054] In certain embodiments, the GCase amino acid sequence may further comprise a modification at one, two, three, four, five, six, seven, or eight positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at one, two, three, four, five, six, or seven positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at one position selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at two positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at three positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at four positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at five positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at six positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at seven positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451. In certain embodiments, the GCase amino acid sequence further comprises a modification at each of the following positions: 189, 190, 245, 316, 345, 346, 425, and 451.

[0055] In certain embodiments, the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or thirteen modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 modifications) selected from the group consisting of: (i) Arg, Glu, or Gin at position 189; (ii) Arg, Asp, Gly, His or Asn at position 190; (iii) Leu, Trp, Met, Tyr or Phe at position 191; (iv) Vai, Asp, Thr, Ala, Glu or He at position 192; (v) Gly at position 233; (vi) Lys at position 245; (vii) Ser at position 316; (viii) Ala, Asn or Thr at position 343; (ix) Phe at position 345; (x) Glu, Asn, Gin or Leu at position 346; (xi) Thr, Ser or Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451.

[0056] For example, in certain embodiments, the GCase amino acid sequence comprises two or three modifications selected from the group consisting of: (i) Gly at position 233; (ii) Ala, Asn or Thr at position 343; and (iii) Thr, Ser or Pro at position 347. In particular, in certain embodiments, the GCase amino acid sequence comprises (i) Gly at position 233; and (ii) Ala, Asn or Thr at position 343. In certain embodiments, the GCase amino acid sequence comprises (i) Gly at position 233; and (ii) Thr, Ser or Pro at position 347. In certain embodiments, the GCase amino acid sequence comprises (i) Ala, Asn or Thr at position 343; and (ii) Thr, Ser or Pro at position 347. In certain embodiments, the GCase amino acid sequence comprises: (i) Gly at position 233; (ii) Ala, Asn or Thr at position 343; and (iii) Thr, Ser or Pro at position 347.

[0057] In certain embodiments, the GCase amino acid sequence further comprises: (i) Leu, Trp, Met, Tyr or Phe at position 191; and / or (ii) Vai, Asp, Thr, Ala, Glu or He at position 192.

[0058] In certain embodiments, the GCase amino acid sequence further comprises one, two, three, four, five, six, seven, or eight modifications (e.g., 2, 3, 4, 5, 6, or 7 modifications) selected from the group consisting of: (i) Arg, Glu, or Gin at position 189; (ii) Arg, Asp, Gly, His or Asn at position 190; (iii) Lys at position 245; (iv) Ser at position 316; (v) Phe at position 345; (vi) Glu, Asn, Gin or Leu at position 346; (vii) Arg at position 425; and (viii) Arg at position 451.

[0059] In certain embodiments, the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten or eleven modifications selected from the group consisting of: (i) His or Asn at position 190; (ii) Tyr or Phe at position 191; (iii) Glu or He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn or Thr at position 343; (vii) Phe at position 345; (viii) Gin or Leu at position 346; (ix) Ser or Pro at position 347; (x) Arg at position 425; and (xi) Arg at position 451. In certain embodiments, the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten or eleven modifications selected from the group consisting of: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Ser at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0060] In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Asn at position 343; (vi) Phe at position 345; (vii) Gin at position 346; and (viii) Ser at position 347.

[0061] In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Asn at position 343; (vi) Phe at position 345; (vii) Gin at position 346; (viii) Ser at position 347; (ix) Arg at position 425; and (x) Arg at position 451.

[0062] In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Ser at position 347; and (x) Arg at position 451.

[0063] In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Ser at position 347; and (x) Arg at position 425.

[0064] In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Ser at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0065] In certain embodiments, the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine or ten modifications selected from the group consisting of: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Leu at position 346; (viii) Pro at position 347; (ix) Arg at position 425; and (x) Arg at position 451.

[0066] In certain embodiments, the GCase amino acid sequence comprises: (i) Phe at position 191; (ii) He at position 192; (iii) Gly at position 233; (iv) Ser at position 316; (v) Thr at position 343; (vi) Leu at position 346; (vii) Pro at position 347; (viii) Arg at position 425; and (ix) Arg at position 451. In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Thr at position 343; (vi) Leu at position 346; (vii) Pro at position 347; (viii) Arg at position 425; and (ix) Arg at position 451.

[0067] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Pro at position 347; (viii) Arg at position 425; and (ix) Arg at position 451.

[0068] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Leu at position 346; (viii) Pro at position 347; and (ix) Arg at position 451.

[0069] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Leu at position 346; (viii) Pro at position 347; and (ix) Arg at position 425.

[0070] In certain embodiments, the GCase amino acid sequence comprises: (i) Phe at position 191; (ii) He at position 192; (iii) Gly at position 233; (iv) Thr at position 343; (v) Leu at position 346; and (vi) Pro at position 347.

[0071] In certain embodiments, the GCase amino acid sequence comprises: (i) Phe at position 191; (ii) He at position 192; (iii) Gly at position 233; (iv) Thr at position 343; and (v) Pro at position 347.

[0072] In certain embodiments, the GCase amino acid sequence comprises: (i) Gly at position 233; (ii) Thr at position 343; and (iii) Pro at position 347.

[0073] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Leu at position 346; (viii) Pro at position 347; (ix) Arg at position 425; and (x) Arg at position 451.

[0074] In certain embodiments, the GCase amino acid sequence comprises: (i) Arg at position 189; (ii) Asp at position 190; (iii) Phe at position 191; (iv) Vai at position 192; (v) Gly at position 233; (vi) Ser at position 316; (vii) Thr at position 343; (viii) Phe at position 345; (ix) Asn at position 346; (x) Ser at position 347; (xi) Arg at position 425; and (xii) Arg at position 451. In certain embodiments, the GCase amino acid sequence comprises: (i) His at position 190; (ii) Tyr at position 191; (iii) Ala at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Phe at position 345; (viii) Glu at position 346; (ix) Pro at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0075] In certain embodiments, the GCase amino acid sequence comprises: (i) Glu at position 189; (ii) Asn at position 190; (iii) Met at position 191; (iv) Thr at position 192; (v) Gly at position 233; (vi) Ser at position 316; (vii) Ala at position 343; (viii) Phe at position 345; (ix) Glu at position 346; (x) Pro at position 347; (xi) Arg at position 425; and (xii) Arg at position 451.

[0076] In certain embodiments, the GCase amino acid sequence comprises: (i) Gin at position 189; (ii) Phe at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Ser at position 347; (x) Arg at position 425; and (xi) Arg at position 451. In certain embodiments, the GCase amino acid sequence comprises: (i) Gin at position 189; (ii) Phe at position 191; (iii) Glu at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Glu at position 346; (ix) Ser at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0077] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Pro at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0078] In certain embodiments, the GCase amino acid sequence comprises: (i) Glu at position 189; (ii) Arg at position 190; (iii) Leu at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Ser at position 316; (vii) Thr at position 343; (viii) Phe at position 345; (ix) Glu at position 346; (x) Thr at position 347; (xi) Arg at position 425; and (xii) Arg at position 451.

[0079] In certain embodiments, the GCase amino acid sequence comprises: (i) Asn at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Lys at position 245; (vi) Ser at position 316; (vii) Thr at position 343; (viii) Phe at position 345; (ix) Gin at position 346; (x) Pro at position 347; (xi) Arg at position 425; and (xii) Arg at position 451.

[0080] In certain embodiments, the GCase amino acid sequence comprises: (i) Glu at position 189; (ii) Asp at position 190; (iii) Trp at position 191; (iv) Asp at position 192; (v) Gly at position 233; (vi) Ser at position 316; (vii) Thr at position 343; (viii) Phe at position 345; (ix) Glu at position 346; (x) Pro at position 347; (xi) Arg at position 425; and (xii) Arg at position 451.

[0081] In certain embodiments, the GCase amino acid sequence comprises: (i) Asp at position 190; (ii) Phe at position 191; (iii) He at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Ala at position 343; (vii) Phe at position 345; (viii) Pro at position 347; (ix) Arg at position 425; and (x) Arg at position 451.

[0082] In certain embodiments, the GCase amino acid sequence comprises: (i) Glu at position 189; (ii) Asp at position 190; (iii) Thr at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Thr at position 343; (vii) Phe at position 345; (viii) Pro at position 347; (ix) Arg at position 425; and (x) Arg at position 451.

[0083] In certain embodiments, the GCase amino acid sequence comprises: (i) Gly at position 190; (ii) Phe at position 191; (iii) Thr at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Ala at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Pro at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0084] In certain embodiments, the GCase amino acid sequence comprises: (i) Asp at position 190; (ii) Phe at position 191; (iii) Thr at position 192; (iv) Gly at position 233; (v) Ser at position 316; (vi) Asn at position 343; (vii) Phe at position 345; (viii) Gin at position 346; (ix) Thr at position 347; (x) Arg at position 425; and (xi) Arg at position 451.

[0085] In certain embodiments, the GCase amino acid sequence comprises: (i) Gly at position 233; (ii) Asn at position 343; (iii) Phe at position 345; (iv) Gin at position 346; and (v) Thr at position 347.

[0086] In certain embodiments, the GCase amino acid sequence comprises: (i) Gly at position 233; (ii) Ser at position 316; (iii) Asn at position 343; (iv) Phe at position 345; (v) Gin at position 346; (vi) Thr at position 347; (vii) Arg at position 425; and (viii) Arg at position 451.

[0087] In certain embodiments, a GCase amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 47, wherein:

[0088] (i) Xi is selected from the group consisting of: G, E, R, and Q;

[0089] (ii) X2 is selected from the group consisting of: A, N, H, D, R, and G;

[0090] (iii) X3 is selected from the group consisting of: Y, F, L, M, V, and W;

[0091] (iv) X4 is selected from the group consisting of: V, I, T, A, E, N, and D;

[0092] (v) X5 is G;

[0093] (vi) Xe is P or K; (vii) X7 is S or F;

[0094] (viii) Xs is selected from the group consisting of: T, A, and N;

[0095] (ix) X9 is selected from the group consisting of: F and S;

[0096] (x) X10 is selected from the group consisting of: K, L, Q, N, and E;

[0097] (xi) Xu is selected from the group consisting of: P, S, and T;

[0098] (xii) X12 is R or K; and

[0099] (xiii) X13 is R or H. Accordingly, such a GCase amino acid sequence would comprise a sequence having 1) the specified sequence identity to SEQ ID NO:47; and 2) a specified residue at each of positions X1-X13.

[0100] In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 85% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47. In certain embodiments, the GCase amino acid sequence consists of SEQ ID NO: 47.

[0101] In certain embodiments, Xi is G. In certain embodiments, Xi is E. In certain embodiments, Xi is R. In certain embodiments, Xi is Q.

[0102] In certain embodiments, X2 is A. In certain embodiments, X2 is N. In certain embodiments, X2 is H. In certain embodiments, X2 is D. In certain embodiments, X2 is R. In certain embodiments, X2 is G.

[0103] In certain embodiments, X3 is Y. In certain embodiments, X3 is F. In certain embodiments, X3 is L. In certain embodiments, X3 is M. In certain embodiments, X3 is V. In certain embodiments, X3 is W. In certain embodiments, X4 is V. In certain embodiments, X4 is I. In certain embodiments, X4 is T. In certain embodiments, X4 is A. In certain embodiments, X4 is E. In certain embodiments, X4 is N. In certain embodiments, X4 is D.

[0104] In certain embodiments, Xe is P. In certain embodiments, Xe is K.

[0105] In certain embodiments, X7 is S. In certain embodiments, X7 is F.

[0106] In certain embodiments, Xs is T. In certain embodiments, Xs is A. In certain embodiments, Xs is N.

[0107] In certain embodiments, X9 is F. In certain embodiments, X9 is S.

[0108] In certain embodiments, X10 is K. In certain embodiments, X10 is L. In certain embodiments, X10 is Q. In certain embodiments, X10 is N. In certain embodiments, X10 is E.

[0109] In certain embodiments, Xu is P. In certain embodiments, Xu is S. In certain embodiments, Xu is T.

[0110] In certain embodiments, X12 is R. In certain embodiments, X12 is K.

[0111] In certain embodiments, X13 is R. In certain embodiments, X13 is H.

[0112] In certain embodiments, Xi is G; X2 is: A, N, or H; X3 is V, Y or F; X4 is N, E or I; X5 is

[0113] G; Xe is P; X7 is S or F; Xs is N or T; X9 is F or S; X10 is K, L, or Q; Xu is P or S; X12 is R or K; and X13 is R or H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0114] In certain embodiments, Xi is G; X2 is H; X3 is Y; X4 is E; X5 is G; Xe is P; X7 is S or F; Xs is N; X9 is F; X10 is Q; Xu is S; X12 is R or K; and X13 is R or H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0115] In certain embodiments, Xi is G; X2 is A or N; X3 is V or F; X4 is I or N; X5 is G; Xe is P; X7 is S or F; Xs is T; X9 is S; X10 is K or L; Xu is P; X12 is R or K; and X13 is R or H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0116] In certain embodiments, Xi is G; X2 is H; X3 is Y; X4 is E; X5 is G; Xe is P; X7 is S; Xs is N; X9 is F; X10 is Q; Xu is S; X12 is R; and X13 is R. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0117] In certain embodiments, Xi is G; X2 is H; X3 is Y; X4 is E; X5 is G; Xe is P; X7 is F; Xs is N; X9 is F; X10 is Q; Xu is S; X12 is K; and X13 is H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0118] In certain embodiments, Xi is G; X2 is N; X3 is F; X4 is I; X5 is G; Xe is P; X7 is S; Xs is T; X9 is S; X10 is L; Xu is P; X12 is R; and X13 is R. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0119] In certain embodiments, Xi is G; X2 is A; X3 is F; X4 is I; X5 is G; Xe is P; X7 is F; Xs is T; X9 is S; X10 is K; Xu is P; X12 is K; and X13 is H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0120] In certain embodiments, Xi is G; X2 is A; X3 is F; X4 is I; X5 is G; Xe is P; X7 is F; Xs is T; X9 is S; X10 is L; Xu is P; X12 is K; and X13 is H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0121] In certain embodiments, Xi is G; X2 is A; X3 is V; X4 is N; X5 is G; Xe is P; X7 is F; Xs is N; X9 is F; X10 is Q; Xu is T; X12 is K; and X13 is H. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0122] In certain embodiments, Xi is G; X2 is A; X3 is V; X4 is N; X5 is G; Xe is P; X7 is S; Xs is N; X9 is F; X10 is Q; Xu is T; X12 is R; and X13 is R. In some embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the GCase amino acid sequence comprises SEQ ID NO: 47, wherein the variables are defined as indicated above. In some embodiments, the amino acid sequence consists of SEQ ID NO: 47, wherein the variables are defined as indicated above.

[0123] In certain embodiments, a GCase amino acid sequence comprises a sequence having a combination of residues at positions 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425 and 451 as recited in any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135 and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135 and 144-147). In some embodiments, the GCase amino acid sequence comprises a sequence having: 1) at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to amino acids 1-497 of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147); and 2) the recited residues of the specified sequence at each of positions 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425 and 451.

[0124] In certain embodiments, the GCase amino acid sequence comprises a sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to amino acids 1-497 of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126- 135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147). In certain embodiments, the GCase amino acid sequence comprises amino acids 1-497 of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147). In certain embodiments, the GCase amino acid sequence consists of amino acids 1-497 of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108- 111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120- 121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144- 147).

[0125] In certain embodiments, a GCase amino acid sequence as described in any of the foregoing embodiments further comprises modification at position 94, wherein the modification and position are relative to SEQ ID NO: 45. In certain embodiments, the modification at position 94 modulates binding to LIMP-2. In certain embodiments, the GCase amino acid sequence comprises Glu at position 94 (see, e.g., SEQ ID NO: 148 and 149). For example, in certain embodiments, the GCase amino acid sequence comprises: (i) Glu at position 94; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Ser at position 316; (vii) Asn at position 343; (viii) Phe at position 345; (ix) Gin at position 346; (x) Ser at position 347; (xi) Arg at position 425; and (xii) Arg at position 451. In some embodiments, the GCase amino acid sequence comprises a sequence having from about 80% identity to about 99% identity (e.g., from about 90% identity to about 99% identity, or such as about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 47, wherein the Xi is G; X2is H; X3is Y; X4is E; X5is G; X6is P; X7is S; X8is N; X9is F; Xio is Q; Xu is S; X12 is R; and X13 is R, and wherein position 94 comprises a modification relative to SEQ ID NO: 47 (e.g., comprises Glu at position 94).

[0126] Fc Polypeptide Modifications

[0127] An Fc polypeptide incorporated in a fusion protein described herein may comprise certain modifications. For example, an Fc polypeptide may comprise modifications that result in binding to a blood-brain barrier (BBB) receptor, e.g., a transferrin receptor (TfR). Additionally, an Fc polypeptide may comprise other modifications, such as modifications that promote heterodimerization, increase serum stability or serum half-life, modulate effector function, influence glycosylation, and / or reduce immunogenicity in humans. Thus, in certain embodiments, a fusion protein described herein comprises two Fc polypeptides, wherein one Fc is a wild-type Fc polypeptide, e.g., a human IgGl Fc polypeptide; and the other Fc is modified to bind to a blood-brain barrier (BBB) receptor, e.g., transferrin receptor (TfR), and optionally further comprises one or more additional modifications. In certain other embodiments, both Fc polypeptides each comprise independently selected modifications (e.g., a modification described herein). For example, in certain embodiments, a fusion protein described herein comprises two Fc polypeptides, wherein one Fc is not modified to bind to a BBB receptor but comprises one or more other modifications described herein; and the other Fc is modified to bind to a blood-brain barrier (BBB) receptor, e.g., transferrin receptor (TfR), and optionally further comprises one or more additional modifications. In certain other embodiments, a fusion protein described herein comprises two Fc polypeptides, wherein both Fc polypeptides are modified to bind to a bloodbrain barrier (BBB) receptor, e.g., transferrin receptor (TfR), and optionally each independently further comprise one or more additional modifications.

[0128] Amino acid residues designated in various Fc modifications, including those introduced in a modified Fc polypeptide that binds to a BBB receptor, e.g., TfR, are numbered herein using EU index numbering. Any Fc polypeptide, e.g., an IgG polypeptide (i.e., a IgGl, IgG2, IgG3, or IgG4 Fc polypeptide), may have modifications, e.g., amino acid substitutions, in one or more positions using EU index numbering as described herein. In some embodiments, the amino acid residues designated in various Fc modifications using EU index numbering are with respect to an IgGl Fc polypeptide.

[0129] A modified (e.g., enhancing heterodimerization and / or BBB receptor-binding) Fc polypeptide present in a fusion protein described herein can have at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to a native Fc region sequence or a fragment thereof, e.g., a fragment of at least 100 amino acids, or greater in length. In some embodiments, the native Fc amino acid sequence is the Fc region sequence of SEQ ID NO: 1. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-217 of SEQ ID NO: 1, or a fragment thereof, e.g., a fragment of at least 100 amino acids, or greater in length.

[0130] Modifications for Blood-Brain Barrier (BBB) Receptor Binding

[0131] In some aspects, provided herein are fusion proteins that are capable of being transported across the blood-brain barrier (BBB) of a subject. Such a protein comprises a modified Fc polypeptide that binds to a BBB receptor. BBB receptors are expressed on BBB endothelia, as well as other cell and tissue types. In some embodiments, the BBB receptor is a transferrin receptor (TfR).

[0132] In some embodiments a fusion protein described herein specifically binds to TfR. In some embodiments a fusion protein described herein specifically binds to TfR with an affinity of from about 50 nM to about 500 nM. In some embodiments, the protein binds (e.g., specifically binds) to a TfR with an affinity of about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nM. In some embodiments, the protein binds to a TfR with an affinity of from about 100 to about 500 nM. In some embodiments, the protein binds to a TfR with an affinity of from about 100 nM to about 300 nM, or from about 200 nM to about 300 nM, or from about 200 nM to about 450 nM. In some embodiments, the protein binds to a TfR with an affinity of about 250 nM. In some embodiments, the protein binds to a TfR with an affinity of about 300 nM. In some embodiments, the protein binds to a TfR with an affinity of from about 150 to about 400 nM, or from about 200 to about 400 nM, or from about 250 nM to about 350 nM, or from about 250 nM to about 300 nM, or from about 300 to about 350 nM. In some embodiments, the protein binds to a TfR with an affinity of from about 100 to about 400 nM. In some embodiments, the protein binds to a TfR with an affinity of from about 200 to about 400 nM. In some embodiments, affinity to a TfR is measured by surface plasmon resonance (SPR). In some embodiments, the affinity is for the apical domain of a transferrin receptor (e.g., the apical domain of a human transferrin receptor).

[0133] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises substitutions in a CH3 domain. In some embodiments, a modified Fc polypeptide comprises a human Ig CH3 domain, such as an IgG CH3 domain, that is modified for TfR- binding activity. The CH3 domain can be of any IgG subtype, i.e., from IgGl, IgG2, IgG3, or IgG4. In the context of IgG antibodies, a CH3 domain refers to the segment of amino acids from about position 341 to about position 447 as numbered according to the EU numbering scheme.

[0134] In some embodiments, a modified Fc polypeptide that specifically binds to TfR binds to the apical domain of TfR and may, e.g., bind to TfR without blocking or otherwise inhibiting binding of transferrin to TfR. In some embodiments, binding of transferrin to TfR is not substantially inhibited. In some embodiments, binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0135] In some embodiments, a modified (e.g., BBB receptor-binding) Fc polypeptide present in a fusion protein described herein comprises substitutions at amino acid positions 386, 387, 388, 416, and 421, according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide further comprises a substitution at one or more (i.e., 1, 2, 3 or 4) of the positions selected from the group consisting of: 384, 389, 413, and 415, according to the EU numbering scheme. Accordingly, in some embodiments, a modified Fc polypeptide present in a fusion protein described herein comprises substitutions at amino acid positions 384, 386, 387, 388, 389, 413, 415, 416, and 421, according to the EU numbering scheme. In certain embodiments, the modified Fc polypeptide further comprises a substitution at one or more of the positions 380 and 390, according to the EU numbering scheme.

[0136] In some embodiments, a modified (e.g., BBB receptor-binding) Fc polypeptide present in a fusion protein described herein comprises substitutions at amino acid positions 386, 387, 388, 416, and 421, according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide further comprises a substitution at one or more (i.e., 1, 2, 3, 4, 5 or 6) of the positions selected from the group consisting of: 380, 384, 389, 390, 413, and 415, according to the EU numbering scheme. In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at the following positions, according to EU numbering: (i) Thr at position 386; (ii) Glu at position 387; (iii) Trp at position 388; (vi) Glu at position 416; and (v) Phe at position 421. In some embodiments, the modified Fc polypeptide further comprises one or more (i.e., 1, 2, 3, 4, 5 or 6) of the specified residues as follows, wherein the positions are according to EU numbering: (i) Glu, Trp, or Leu at position 380; (ii) Phe or Tyr at position 384; (iii) Ser, Ala, Vai or Asn at position 389; (iv) Asn or Ser at position 390; (v) Thr or Ser at position 413; and (vi) Glu or Ser at position 415.

[0137] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at the following positions, according to EU numbering: (i) Glu, Trp, or Leu at position 380; (ii) Phe or Tyr at position 384; (iii) Thr at position 386; (iv) Glu at position 387; (v) Trp at position 388; (vi) Ser, Ala, Vai or Asn at position 389; (vii) Asn or Ser at position 390; (viii) Thr or Ser at position 413; (ix) Glu or Ser at position 415; (x) Glu at position 416; and (xi) Phe at position 421.

[0138] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at the following positions, according to EU numbering: (i) Glu, Trp, or Leu at position 380; (ii) Tyr at position 384; (iii) Thr at position 386; (iv) Glu at position 387; (v) Trp at position 388; (vi) Ser or Ala at position 389; (vii) Asn or Ser at position 390; (viii) Thr at position 413; (ix) Glu at position 415; (x) Glu at position 416; and (xi) Phe at position 421.

[0139] In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises Ala at position 389, according to EU numbering. In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at the following positions, according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, a modified Fc polypeptide that specifically binds to TfR comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0140] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-217 of SEQ ID NO:27 or SEQ ID NO: 37 (e.g., wherein Xais K). In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-217 of SEQ ID NO:27 or 37 (wherein Xais K); and comprises the amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO:27 or 37 (wherein Xais K). In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111- 216 of SEQ ID NO: 28 or 37 (wherein Xais absent). In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-216 of SEQ ID NO: 28; and comprises the amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO:27, 28, or 37. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to amino acids 111-216 of SEQ ID NO: 37 (wherein Xais absent); and comprises the amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO:27, 28, or 37.

[0141] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27, 28 or 37, and has Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421. In certain embodiments, the modified Fc polypeptide further comprises Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27, 28, or 37 and has Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27, 28, or 37, and has Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0142] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27 or 28, and has Ala at position 389, according to EU numbering. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27 or 28 and comprises at the following positions, according to EU numbering: Glu at position 380; Ala at position 389; and Asn at position 390. In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27 or 28 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421.

[0143] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:27 or 28; and comprises the amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO:27 or 28. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:27 or 28.

[0144] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:37; and comprises the amino acids at EU index positions 380, 384-390 and / or 413-421 of SEQ ID NO:37. In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of SEQ ID NO:37.

[0145] Additional Fc Polypeptide Mutations

[0146] In some aspects, a fusion protein described herein comprises two Fc polypeptides, wherein one or both Fc polypeptides each comprise independently selected modifications (e.g., a modification described herein). Non-limiting examples of other mutations that can be introduced into one or both Fc polypeptides include, e.g., mutations to increase serum stability or serum half-life, to modulate effector function, to influence glycosylation, to reduce immunogenicity in humans, and / or to provide for knob and hole heterodimerization of the Fc polypeptides. Examples of various modifications that may be included in an Fc polypeptide are described in W02019 / 070577, which is incorporated by reference herein in its entirety for all purposes.

[0147] In some embodiments, the Fc polypeptides present in the fusion protein each independently have an amino acid sequence identity of at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% to a corresponding wild-type Fc polypeptide (e.g., a human IgG polypeptide (i.e., a IgGl, IgG2, IgG3, or IgG4 Fc polypeptide)).

[0148] In some embodiments, the Fc polypeptides present in the fusion protein include knob and hole mutations to promote heterodimer formation and hinder homodimer formation. Generally, the modifications introduce a protuberance (“knob”) at the interface of one polypeptide and a corresponding cavity (“hole”) in the interface of another polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and thus 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). In some embodiments, such additional mutations are at a position in the Fc polypeptide that does not have a negative effect on binding of the polypeptide to a BBB receptor, e.g., TfR.

[0149] In one illustrative embodiment of a knob and hole approach for dimerization, position 366 (numbered according to the EU numbering scheme) of one of the Fc polypeptides present in the fusion protein comprises a tryptophan in place of a native threonine. The other Fc polypeptide in the dimer has a valine at position 407 (numbered according to the EU numbering scheme) in place of the native tyrosine. The other Fc polypeptide may further comprise a substitution in which the native threonine at position 366 (numbered according to the EU numbering scheme) is substituted with a serine and a native leucine at position 368 (numbered according to the EU numbering scheme) is substituted with an alanine. Thus, one of the Fc polypeptides of a fusion protein described herein has the T366W knob mutation and the other Fc polypeptide has the Y407V mutation, which is typically accompanied by the T366S and L368A hole mutations. In certain embodiments, the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains the T366W substitution. In certain other embodiments, the first Fc polypeptide contains the T366W substitution and the second Fc polypeptide contains the T366S, L368A, and Y407V substitutions.

[0150] In some embodiments, modifications to enhance serum half-life may be introduced. For example, in some embodiments, one or both Fc polypeptides present in a fusion protein described herein may comprise a tyrosine at position 252, a threonine at position 254, and a glutamic acid at position 256, as numbered according to the EU numbering scheme. Thus, one or both Fc polypeptides may have M252Y, S254T, and T256E substitutions. Alternatively, one or both Fc polypeptides may have M428L and N434S substitutions, as numbered according to the EU numbering scheme. Alternatively, one or both Fc polypeptides may have an N434S or N434A substitution.

[0151] In some embodiments, one or both Fc polypeptides present in a fusion protein described herein may comprise modifications that reduce effector function, i.e., having a reduced ability to induce certain biological functions upon binding to an Fc receptor expressed on an effector cell that mediates the effector function. Examples of antibody effector functions include, but are not limited to, Clq binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptor), and B-cell activation. Effector functions may vary with the antibody class. For example, native human IgGl and IgG3 antibodies can elicit ADCC and CDC activities upon binding to an appropriate Fc receptor present on an immune system cell; and native human IgGl, IgG2, IgG3, and IgG4 can elicit ADCP functions upon binding to the appropriate Fc receptor present on an immune cell.

[0152] In some embodiments, one or both Fc polypeptides present in a fusion protein described herein may also be engineered to contain other modifications for heterodimerization, e.g., electrostatic engineering of contact residues within a CH3-CH3 interface that are naturally charged or hydrophobic patch modifications.

[0153] In some embodiments, one or both Fc polypeptides present in a fusion protein described herein may include additional modifications that modulate effector function.

[0154] In some embodiments, one or both Fc polypeptides present in a fusion protein described herein may comprise modifications that reduce or eliminate effector function. In certain embodiments, one or both Fc polypeptides (e.g., both) do not have effector function. In some embodiments, one or both Fc polypeptides comprise modifications that reduce effector function. Illustrative Fc polypeptide mutations that reduce effector function include, but are not limited to, substitutions in a CH2 domain, e.g., at positions 234 and 235, according to the EU numbering scheme. For example, in some embodiments, one or both Fc polypeptides can comprise alanine residues at positions 234 and 235. Thus, one or both Fc polypeptides may have L234A and L235A (LALA) substitutions.

[0155] Additional Fc polypeptide mutations that modulate an effector function include, but are not limited to, the following: position 329 may have a mutation in which proline is substituted with a glycine, serine or arginine or an amino acid residue large enough to destroy the Fc / Fcy receptor interface that is formed between proline 329 of the Fc and tryptophan residues Trp 87 and Trp 110 of FcyRIII. Additional illustrative substitutions include S228P, E233P, L235E, N297A, N297D, and P331S, according to the EU numbering scheme. Multiple substitutions may also be present, e.g., L234A and L235A of a human IgGl Fc region; L234A, L235A, and P329G of a human IgGl Fc region; L234A, L235A, and P329S of a human IgGl Fc region; S228P and L235E of a human IgG4 Fc region; L234A and G237A of a human IgGl Fc region; L234A, L235A, and G237A of a human IgGl Fc region; V234A and G237A of a human IgG2 Fc region; L235A, G237A, and E318A of a human IgG4 Fc region; and S228P and L236E of a human IgG4 Fc region, according to the EU numbering scheme. In some embodiments, one or both Fc polypeptides may have one or more amino acid substitutions that modulate ADCC, e.g., substitutions at positions 298, 333, and / or 334, according to the EU numbering scheme.

[0156] In certain embodiments, one or both Fc polypeptides comprise the substitutions of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329, according to EU numbering.

[0157] In some embodiments, the C-terminal Lys residue is removed in an Fc polypeptide described herein (i.e., the Lys residue at position 447, according to the EU numbering scheme).

[0158] Illustrative Fc polypeptides comprising additional mutations

[0159] As described herein, and by way of non-limiting example, one or both Fc polypeptides present in a fusion protein described herein may comprise additional mutations, including: a knob mutation (e.g., T366W as numbered according to the EU numbering scheme, hole mutations (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)) as numbered according to the EU numbering scheme), and / or mutations that increase serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E as numbered with reference to EU numbering, or (ii) N434S with or without M428L as numbered according to the EU numbering scheme). By way of illustration, SEQ ID NOs:9-26 and 29-36, and 38-44 provide non-limiting examples of modified Fc polypeptides comprising one or more of these additional mutations.

[0160] In some embodiments, an Fc polypeptide or a modified Fc polypeptide may have a knob mutation (e.g., T366W as numbered according to the EU numbering scheme) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37. In some embodiments, an Fc polypeptide or modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 27, 28 and 37 may be modified to have a knob mutation.

[0161] In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 29, 30, and 38. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 29, 30, and 38, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the modified Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 29, 30, and 38, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO:29 or 30, and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 29 and 30.

[0162] In some embodiments, an Fc polypeptide or a modified Fc polypeptide may have a knob mutation (e.g., T366W as numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)) as numbered according to the EU numbering scheme), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37. In some embodiments, an Fc polypeptide or a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37 may be modified to have a knob mutation and mutations that modulate effector function.

[0163] In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S) as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 31-36, 39-40, and 41-44. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S) as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 31-36, 39-40, and 41-44, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the modified Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S) as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 31-36, 39-40, and 41-44, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, a modified Fc polypeptide comprises a knob mutation (e.g., T366W as numbered with reference to EU numbering) and mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S) as numbered with reference to EU numbering), has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 31-36 and 41-44 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 31-36 and 41-44.

[0164] In some embodiments, an Fc polypeptide or a modified Fc polypeptide may have hole mutations (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering scheme) and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 27, 28 and 37. In some embodiments, an Fc polypeptide or a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37 may be modified to have hole mutations.

[0165] In some embodiments, a modified Fc polypeptide comprises hole mutations (e.g., T366S, L368A, and Y407V as numbered with reference to EU numbering) and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 9 and 10. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 9 and 10.

[0166] In some embodiments, an Fc polypeptide or a modified Fc polypeptide may have hole mutations (e.g., T366S, L368A, and Y407V as numbered according to the EU numbering scheme), mutations that modulate effector function (e.g., L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)) as numbered according to the EU numbering scheme), and at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37. In some embodiments, an Fc polypeptide or a modified Fc polypeptide having the sequence of any one of SEQ ID NOs: 1, 2, 27, 28, and 37 may be modified to have hole mutations and mutations that modulate effector function.

[0167] In some embodiments, a modified Fc polypeptide comprises hole mutations (e.g, T366S, L368A, and Y407V as numbered with reference to EU numbering) and mutations that modulate effector function (e.g, L234A, L235A, and / or P329G or P329S (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S)) as numbered with reference to EU numbering), and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 11-16 and 21-24. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 11-16 and 21- 24.

[0168] FcRn Binding Sites

[0169] In certain aspects, modified (e.g., BBB receptor-binding) Fc polypeptides, or Fc polypeptides present in a fusion protein described herein that do not specifically bind to a BBB receptor, can comprise an FcRn binding site. In some embodiments, the FcRn binding site is within the Fc polypeptide or a fragment thereof.

[0170] In some embodiments, the FcRn binding site comprises a native FcRn binding site. In some embodiments, the FcRn binding site does not comprise amino acid changes relative to the amino acid sequence of a native FcRn binding site. In some embodiments, the native FcRn binding site is an IgG binding site, e.g., a human IgG binding site. In some embodiments, the FcRn binding site comprises a modification that alters FcRn binding. In some embodiments, an FcRn binding site has one or more amino acid residues that are mutated, e.g., substituted, wherein the mutation(s) increase serum half-life or do not substantially reduce serum half-life (i.e., reduce serum half-life by no more than 25% compared to a counterpart modified Fc polypeptide having the wild-type residues at the mutated positions when assayed under the same conditions). In some embodiments, an FcRn binding site has one or more amino acid residues that are substituted at positions 250-256, 307, 380, 428, and 433-436, according to the EU numbering scheme.

[0171] In some embodiments, one or more residues at or near an FcRn binding site are mutated, relative to a native human IgG sequence, to extend serum half-life of the modified polypeptide. In some embodiments, mutations are introduced into one, two, or three of positions 252, 254, and 256. In some embodiments, the mutations are M252Y, S254T, and T256E. In some embodiments, a modified Fc polypeptide further comprises the mutations M252Y, S254T, and T256E. In some embodiments, a modified Fc polypeptide comprises a substitution at one, two, or all three of positions T307, E380, and N434, according to the EU numbering scheme. In some embodiments, the mutations are T307Q and N434A. In some embodiments, a modified Fc polypeptide comprises mutations T307A, E380A, and N434A. In some embodiments, a modified Fc polypeptide comprises substitutions at positions T250 and M428, according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises mutations T250Q and / or M428L. In some embodiments, a modified Fc polypeptide comprises substitutions at positions M428 and N434, according to the EU numbering scheme. In some embodiments, the modified Fc polypeptide comprises mutations M428L and N434S. In some embodiments, a modified Fc polypeptide comprises an N434S or N434A mutation.

[0172] GCase Enzymes Linked to Fc Polypeptides

[0173] In some embodiments, a fusion protein described herein comprises two Fc polypeptides as described herein and one or both of the Fc polypeptides may further comprise a partial or full hinge region. The hinge region can be from any immunoglobulin subclass or isotype. An illustrative immunoglobulin hinge is an IgG hinge region, such as an IgGl hinge region, e.g., human IgGl hinge amino acid sequence EPKSCDKTHTCPPCP (SEQ ID NO:5) or a portion thereof (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the hinge region is at the N-terminal region of the Fc polypeptide.

[0174] In some embodiments, the first Fc polypeptide is linked to a first GCase amino acid sequence. For example, in certain embodiments, the N-terminus of the first Fc polypeptide is linked to the GCase amino acid sequence. In certain embodiments, the C-terminus of the first Fc polypeptide is linked to the GCase amino acid sequence.

[0175] In certain embodiments, a fusion protein described herein comprises a single GCase amino acid sequence.

[0176] In some embodiments, a fusion protein as described herein comprises a second GCase amino acid sequence, which may be the same or different from the first GCase amino acid sequence. For example, in certain embodiments, the second Fc polypeptide is linked to a GCase amino acid sequence. In certain embodiments, the N-terminus of the second Fc polypeptide is linked to the second GCase amino acid sequence. In certain embodiments, the C-terminus of the second Fc polypeptide is linked to the second GCase amino acid sequence.

[0177] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first GCase amino acid sequence; and the N-terminus of the second Fc polypeptide is linked to a second GCase amino acid sequence.

[0178] In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first GCase amino acid sequence; and the C-terminus of the second Fc polypeptide is linked to a second GCase amino acid sequence.

[0179] In certain embodiments, the N-terminus of the first Fc polypeptide is linked to a first GCase amino acid sequence; and the C-terminus of the second Fc polypeptide is linked to a second GCase amino acid sequence.

[0180] In certain embodiments, the C-terminus of the first Fc polypeptide is linked to a first GCase amino acid sequence; and the N-terminus of the second Fc polypeptide is linked to a second GCase amino acid sequence.

[0181] In some embodiments, an Fc polypeptide (e.g., the first Fc polypeptide) is joined to a GCase amino acid sequence by a linker, e.g., a peptide linker or polypeptide linker. In some embodiments, the Fc polypeptide (e.g., the first Fc polypeptide) is joined to the GCase amino acid sequence by a peptide bond or by a peptide linker / polypeptide linker, e.g., is a fusion polypeptide. The linker may be configured such that it allows for the rotation of the GCase amino acid sequence relative to the Fc polypeptide to which it is joined; and / or is resistant to digestion by proteases. Peptide / polypeptide linkers may contain natural amino acids, unnatural amino acids, or a combination thereof. In some embodiments, the peptide / polypeptide linker may be a flexible linker, e.g., containing amino acids such as Gly, Asn, Ser, Thr, Ala, and the like (e.g., a glycine-rich linker). Such linkers are designed using known parameters and may be of any length and contain any number of repeat units of any length (e.g., repeat units of Gly and Ser residues). For example, the linker may have repeats, such as two, three, four, five, or more Gly4-Ser (SEQ ID NO: 152) repeats or a single Gly4-Ser (SEQ ID NO: 152). In other aspects, the linker may be Gly-Ser. In some embodiments, the linker may include a protease cleavage site, e.g., that is cleavable by an enzyme present in the central nervous system.

[0182] In some embodiments, the GCase amino acid sequence is joined to the Fc polypeptide by a Gly-Ser linker, a Gly4-Ser linker (SEQ ID NO: 152) or a (Gly4-Ser)2 linker (SEQ ID NO: 151). In some embodiments, the Fc polypeptide may comprise a hinge sequence or partial hinge sequence at the N-terminus that is joined to the linker or that is directly joined to the GCase amino acid sequence.

[0183] In some embodiments, the GCase amino acid sequence is joined to the N-terminus of the Fc polypeptide, e.g., by a Gly-Ser linker, a Gly4-Ser linker (SEQ ID NO: 152) or a (Gly4- Ser)2 linker (SEQ ID NO: 151). In some embodiments, the Fc polypeptide may comprise a hinge sequence or partial hinge sequence at the N-terminus that is joined to the linker or that is directly joined to the GCase amino acid sequence.

[0184] In some embodiments, the GCase amino acid sequence is joined to the C-terminus of the Fc polypeptide, e.g., by a Gly-Ser linker, a Gly4-Ser linker (SEQ ID NO: 152) or a (Gly4- Ser)2 linker (SEQ ID NO: 151). In some embodiments, the C-terminus of the Fc polypeptide is directly joined to the GCase amino acid sequence.

[0185] In some embodiments, the GCase amino acid sequence is joined to the Fc polypeptide by a chemical cross-linking agent. Such conjugates can be generated using well-known chemical cross-linking reagents and protocols. For example, there are a large number of chemical cross-linking agents that are known to those skilled in the art and useful for crosslinking the polypeptide with an agent of interest. For example, the cross-linking agents are heterobifunctional cross-linkers, which can be used to link molecules in a stepwise manner. Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating proteins, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers. A wide variety of heterobifunctional cross-linkers are known in the art, including N-hydroxysuccinimide (NHS) or its water soluble analog N-hydroxysulfosuccinimide (sulfo-NHS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), m- maleimidobenzoyl-N-hydroxy succinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl- a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Those cross-linking agents having N-hydroxysuccinimide moieties can be obtained as the N- hydroxysulfosuccinimide analogs, which generally have greater water solubility. In addition, those cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo. In addition to the heterobifunctional cross-linkers, there exist a number of other cross-linking agents including homobifunctional and photoreactive cross-linkers. Disuccinimidyl subcrate (DSS), bismaleimidohexane (BMH) and dimethylpimelimidate. 2HC1 (DMP) are examples of useful homobifunctional cross-linking agents, and bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4’-azido-2’-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive cross-linkers.

[0186] Illustrative Protein Molecules Comprising GCase-Fc Fusion Polypeptides

[0187] In some aspects, a fusion protein described herein comprises a first Fc polypeptide that is linked to a GCase amino acid sequence; and a second Fc polypeptide; wherein the first and / or second Fc polypeptide is a modified Fc that is capable of binding (e.g., specifically binding) to a blood-brain barrier (BBB) receptor, e.g., a transferrin receptor (TfR). In certain embodiments, the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide does not include an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof. In some embodiments, the fusion protein does not include an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof. In certain embodiments, a fusion protein described herein comprises a single GCase amino acid sequence. In some other aspects, the fusion protein further comprises a second, independently selected, GCase amino acid sequence (e.g., which may be linked to the second Fc polypeptide).

[0188] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide (e.g., comprises one or more modifications described herein). For example, in some embodiments, a modified Fc polypeptide contains one or more modifications that promote its heterodimerization to the other Fc polypeptide. In some embodiments, a modified Fc polypeptide contains one or more modifications that reduce effector function. In some embodiments, a modified Fc polypeptide contains one or more modifications that extend serum half-life. In some embodiments, a modified Fc polypeptide comprises one or more modifications that confer binding BBB) receptor, e.g., transferrin receptor (TfR). For example, in certain embodiments, an Fc polypeptide that is capable of binding to a TfR comprises: Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the modified Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In certain embodiments, an Fc polypeptide that is capable of binding to a TfR comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, an Fc polypeptide that is capable of binding to a TfR receptor comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, such an Fc polypeptide specifically binds to TfR.

[0189] In some embodiments, the first Fc polypeptide is a modified Fc polypeptide. In some embodiments, the second Fc polypeptide is a modified Fc polypeptide. In some embodiments, the first and the second Fc polypeptide are each a modified Fc polypeptide. In some embodiments, the first Fc polypeptide is a modified polypeptide but does not specifically bind to TfR; and the second Fc polypeptide is a modified polypeptide that is capable of specifically binding to TfR, and optionally, further comprises one or more further modifications described herein. In other embodiments, the first Fc polypeptide is a modified polypeptide that is capable of specifically binding to TfR, and optionally, further comprises one or more further modifications described herein; and the second Fc polypeptide is a modified polypeptide but does not specifically binding to TfR. In some embodiments, the first Fc polypeptide is a modified polypeptide that is capable of specifically binding to TfR, and optionally, further comprises one or more further modifications described herein; and the second Fc polypeptide is a modified polypeptide that is capable of specifically binding to TfR, and optionally, further comprises one or more further modifications described herein.

[0190] In some embodiments, a fusion protein described herein comprises a first polypeptide chain that comprises a first Fc polypeptide comprising T366S, L368A, and Y407V (hole) substitutions linked to a GCase amino acid sequence; and a second polypeptide chain that comprises a second Fc polypeptide that comprises a T366W (knob) substitution, wherein the first and / or second Fc polypeptide is a modified polypeptide that is capable of binding to TfR. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A and L235A (LALA) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises L234A, L235A, and P329G (LALAPG) substitutions or further comprises L234A, L235A, and P329S (LALAPS) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide further comprises: 1) L234A and L235A (LALA) substitutions; L234A, L235A, and P329G (LALAPG) substitutions; or L234A, L235A, and P329S (LALAPS) substitutions; and 2) M252Y, S254T, and T256E (YTE) substitutions. In some embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises human IgGl wild-type residues at positions 234, 235, 252, 254, 256, and 366.

[0191] In some embodiments, the second Fc polypeptide is a modified polypeptide that is capable of binding to TfR. In some embodiments, the first Fc polypeptide linked to a GCase amino acid sequence is not modified to bind to TfR. In some embodiments, the second Fc polypeptide comprises the knob, LALA / LALAPG / LALAPS, and / or YTE mutations, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:29-36 and 38. In some embodiments, the second Fc polypeptide comprises the knob, LALA / LALAPG / LALAPS, and / or YTE mutations, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:29-36 and 38, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In some embodiments, the second Fc polypeptide comprises the knob, LALA / LALAPG / LALAPS, and / or YTE mutations, has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:29-36 and 38, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 29-36 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421; or comprises the sequence of any one of SEQ ID NOs: 29-36. In some embodiments, the first Fc polypeptide comprises the hole, LALA / LALAPG / LALAPS, and / or YTE mutations, and has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:9-16; or comprises the sequence of any one of SEQ ID NOs:9-16. In some embodiments, the second Fc polypeptide comprises any one of SEQ ID NOs:29-36 and 38, and the first Fc polypeptide comprises any one of SEQ ID NOs:9- 16. In some embodiments, the N-terminus of the first Fc polypeptide and / or the second Fc polypeptide includes a portion of an IgGl hinge region (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-40 and 41-44. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-40 and 41-44, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-40 and 41-44, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 41-44 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs:41-44. In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 21-24, or comprises the sequence of any one of SEQ ID NOs:21-24.

[0192] In some embodiments, a GCase amino acid sequence, present in a fusion protein described herein is linked to a first polypeptide chain that comprises a first Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 9-16, or comprises the sequence of any one of SEQ ID NOs: 9-16 (e.g., as a fusion polypeptide). In some embodiments, the GCase amino acid sequence is linked to the first Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the N-terminus of the first Fc polypeptide includes a portion of an IgGl hinge region (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:21-24, or comprises the sequence of any one of SEQ ID NOs:21-24. In some embodiments, the GCase amino acid sequence comprises an amino acid sequence having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to SEQ ID NO: 47, or comprises SEQ ID NO: 47. In some embodiments, the GCase amino acid linked to the first Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:64-67, 72-75, 78-105, 108-111, 120-121, 126- 135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or of SEQ ID NOs:64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147), or comprises the sequence of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs:64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147). In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 29-36 and 38. In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 29-36 and 38, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415, according to EU numbering. In some embodiments, the fusion protein comprises a second Fc polypeptide having at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 29-36 and 38, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, according to EU numbering. In some embodiments, the second polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 29-36 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs: 29-36. In some embodiments, the N-terminus of the second Fc polypeptide includes a portion of an IgGl hinge region (e.g., DKTHTCPPCP; SEQ ID NO:6). In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs:39-40 and 41-44. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-40 and 41-44, and comprises Thr at position 386; Glu at position 387; Trp at position 388; Glu at position 416; and Phe at position 421, according to EU numbering. In certain embodiments, the second Fc polypeptide further comprises: Glu, Trp, or Leu at position 380; Phe or Tyr at position 384; Ser, Ala, Vai or Asn at position 389; Asn or Ser at position 390; Thr or Ser at position 413; and / or Glu or Ser at position 415, according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85%, at least 90%, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to any one of SEQ ID NOs: 39-40 and 41-44, and comprises: Glu, Trp, or Leu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser or Ala at position 389; Asn or Ser at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, according to EU numbering. In some embodiments, the second Fc polypeptide has at least 85% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence of any one of SEQ ID NOs: 41-44 and comprises at the following positions, according to EU numbering: Glu at position 380; Tyr at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ala at position 389; Asn at position 390; Thr at position 413; Glu at position 415; Glu at position 416; and Phe at position 421, or comprises the sequence of any one of SEQ ID NOs:41-44. In some embodiments, a second GCase amino acid sequence is linked to the second Fc polypeptide by a linker, such as a flexible linker, and / or a hinge region or portion thereof (e.g., DKTHTCPPCP; SEQ ID N0:6).

[0193] In certain embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135 (e.g., to any one of SEQ ID NOs: 64-67, 72-75, 78-79, 82-85, 90-105, 108-111, 120-121, and 126-135; or to any one of SEQ ID NOs: 64-65, 94-95, 98-105, 108-111, 120-121, and 126-135, such as to any one of SEQ ID NOs: 94-95 and 98-105). In certain embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147 (e.g., to any one of SEQ ID NOs: 64-67, 72-75, 78-79, 82-85, 90-105, 108-111, 120-121, 126-135, and 144- 147; or to any one of SEQ ID NOs: 64-65, 94-95, 98-105, 108-111, 120-121, 126-135, and 146- 147, such as to any one of SEQ ID NOs: 94-95, 98-105, and 146-147). In certain embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108- 111, 120-121, 126-135, and 144-149 (e.g., to any one of SEQ ID NOs: 64-67, 72-75, 78-79, 82- 85, 90-105, 108-111, 120-121, 126-135, and 144-149; or to any one of SEQ ID NOs: 64-65, 94- 95, 98-105, 108-111, 120-121, 126-135, and 146-149, such as to any one of SEQ ID NOs: 94- 95, 98-105, and 146-149). For example, in certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 64 or 65. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 66 or 67. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 72 or 73. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 74 or 75. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 78 or 79. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 80 or 81. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 82 or 83. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 84 or 85. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 86 or 87. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 88 or 89. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 90 or 91. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 92 or 93. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 94 or 95. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 96 or 97. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 98 or 99. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 100 or 101. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 102 or 103. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 104 or 105. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 108 or 109. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 110 or 111. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 120 or 121. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 126 or 127. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 128 or 129. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 130 or 131. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 132 or 133. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 134 or 135. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 144 or 145. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 146 or 147. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 148 or 149. In certain embodiments, the fusion protein comprises a second Fc polypeptide comprising an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31-36, 39-40, and 41-44 (e.g., SEQ ID NOs:41-44). For example, in certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31, 32, 39, 41 and 42. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:35, 36, 40, 43 and 44. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 39, wherein Xais Lys. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 39, wherein Xais absent. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 41. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 42. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 40, wherein Xais Lys. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 40, wherein Xais absent. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 43. In certain embodiments, the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149 (e.g., SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, and 126-135; or SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-147); and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 31-36, 39-40, and 41-44 (e.g., any one of SEQ ID NOs: 41-44).

[0194] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:94 or 95, and wherein the sequence comprises with respect to SEQ ID NO:94: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:94 or 95, and wherein the sequence comprises with respect to SEQ ID NO:94: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 94-95; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 94-95; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 94; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 95; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0195] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:98 or 99, and wherein the sequence comprises with respect to SEQ ID NO:98: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:98 or 99, and wherein the sequence comprises with respect to SEQ ID NO:98: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 98-99; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 98-99; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 98; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 99; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0196] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 100 or 101, and wherein the sequence comprises with respect to SEQ ID NO: 100: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 100 or 101, and wherein the sequence comprises with respect to SEQ ID NO: 100: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 100-101; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 100-101; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 100; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 101; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 102 or 103, and wherein the sequence comprises with respect to SEQ ID NO: 102: (i) Gly at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 102 or 103, and wherein the sequence comprises with respect to SEQ ID NO: 102: (i) Gly at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 102-103; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 102-103; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 102; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 103; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0197] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 104 or 105, and wherein the sequence comprises with respect to SEQ ID NO: 104: (i) Gly at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 104 or 105, and wherein the sequence comprises with respect to SEQ ID NO: 104: (i) Gly at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 104-105; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 104-105; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 104; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 105; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0198] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 146 or 147, and wherein the sequence comprises with respect to SEQ ID NO: 146: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Thr at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 146 or 147, and wherein the sequence comprises with respect to SEQ ID NO: 146: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Thr at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 146-147; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 146-147; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 146; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 147; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0199] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 148 or 149, and wherein the sequence comprises with respect to SEQ ID NO: 148: (i) Glu at position 94; (ii) Gly at position 189; (iii) His at position 190; (iv) Tyr at position 191; (v) Glu at position 192; (vi) Gly at position 233; (vii) Pro at position 245; (viii) Ser at position 316; (ix) Asn at position 343; (x) Phe at position 345; (xi) Gin at position 346; (xii) Ser at position 347; (xiii) Arg at position 425; and (xiv) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 148 or 149, and wherein the sequence comprises with respect to SEQ ID NO: 148: (i) Glu at position 94; (ii) Gly at position 189; (iii) His at position 190; (iv) Tyr at position 191; (v) Glu at position 192; (vi) Gly at position 233; (vii) Pro at position 245; (viii) Ser at position 316; (ix) Asn at position 343; (x) Phe at position 345; (xi) Gin at position 346; (xii) Ser at position 347; (xiii) Arg at position 425; and (xiv) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 148-149; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 148-149; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 148; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 149; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0200] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to amino acids 1-497 of SEQ ID NO:64 and wherein the sequence comprises with respect to SEQ ID NO:64 or 65: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In certain embodiments, the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:64 or 65 and wherein the sequence comprises with respect to SEQ ID NO:64: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 64-65; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 64; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 65; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0201] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:66 or 67, and wherein the sequence comprises with respect to SEQ ID NO:66: (i) Arg at position 189; (ii) Asp at position 190; (iii) Phe at position 191; (iv) Vai at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Asn at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 66-67; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 66; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 67; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0202] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:72 or 73, and wherein the sequence comprises with respect to SEQ ID NO:72: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Ala at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Glu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0203] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 72-73; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 72; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 73; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0204] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:74 or 75, and wherein the sequence comprises with respect to SEQ ID NO:74: (i) Glu at position 189; (ii) Asn at position 190; (iii) Met at position 191; (iv) Thr at position 192; (v) Gly at position 233; (vi) Pro at position 245;

[0205] (vii) Ser at position 316; (viii) Ala at position 343; (ix) Phe at position 345; (x) Glu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0206] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 74-75; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 74; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 75; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0207] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:78 or 79, and wherein the sequence comprises with respect to SEQ ID NO:78: (i) Gin at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245;

[0208] (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Glu at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 78-79; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 78; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 79; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0209] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:80 or 81, and wherein the sequence comprises with respect to SEQ ID NO:80: (i) Glu at position 189; (ii) Asp at position 190; (iii) Vai at position 191; (iv) Thr at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 80-81; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 80; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 81; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0210] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:82 or 83, and wherein the sequence comprises with respect to SEQ ID NO:82: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0211] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 82-83; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 82; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 83; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0212] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:84 or 85, and wherein the sequence comprises with respect to SEQ ID NO:84: (i) Glu at position 189; (ii) Arg at position 190; (iii) Leu at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245;

[0213] (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Glu at position 346; (xi) Thr at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0214] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 84-85; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 84; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 85; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0215] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:86 or 87, and wherein the sequence comprises with respect to SEQ ID NO:86: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Lys at position 245;

[0216] (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 86-87; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 86; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 87; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0217] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:88 or 89, and wherein the sequence comprises with respect to SEQ ID NO:88: (i) Glu at position 189; (ii) Asp at position 190; (iii) Trp at position 191; (iv) Asp at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Phe at position 345; (x) Glu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 88-89; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 88; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 89; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:90 or 91, and wherein the sequence comprises with respect to SEQ ID NO:90: (i) Gly at position 189; (ii) Asp at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Ala at position 343; (ix) Phe at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0218] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 90-91; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 90; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 91; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0219] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:92 or 93, and wherein the sequence comprises with respect to SEQ ID NO:92: (i) Gly at position 189; (ii) Gly at position 190; (iii) Phe at position 191; (iv) Thr at position 192; (v) Gly at position 233; (vi) Pro at position 245;

[0220] (vii) Ser at position 316; (viii) Ala at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168;

[0221] (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 92-93; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 92; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 93; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0222] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:96 or 97, and wherein the sequence comprises with respect to SEQ ID NO:96: (i) Gly at position 189; (ii) Asp at position 190; (iii) Phe at position 191; (iv) Thr at position 192; (v) Gly at position 233; (vi) Pro at position 245;

[0223] (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Thr at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 96-97; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 96; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 97; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0224] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 108 or 109, and wherein the sequence comprises with respect to SEQ ID NO: 108: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Lys at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 108-109; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 108; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 109; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0225] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 110 or 111, and wherein the sequence comprises with respect to SEQ ID NO: 110: (i) Gly at position 189; (ii) His at position 190; (iii) Tyr at position 191; (iv) Glu at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Ser at position 347; (xii) Arg at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 110-111; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 110; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 111; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0226] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 120 or 121, and wherein the sequence comprises with respect to SEQ ID NO: 120: (i) Gly at position 189; (ii) Ala at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 120-121; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 120; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 121; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0227] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 126 or 127, and wherein the sequence comprises with respect to SEQ ID NO: 126: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 126-127; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 126; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 127; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0228] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 128 or 129, and wherein the sequence comprises with respect to SEQ ID NO: 128: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 128-129; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 128; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 129; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0229] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 130 or 131, and wherein the sequence comprises with respect to SEQ ID NO: 130: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) Arg at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 130-131; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 130; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 131; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0230] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 132 or 133, and wherein the sequence comprises with respect to SEQ ID NO: 132: (i) Gly at position 189; (ii) Asn at position 190; (iii) Phe at position 191; (iv) He at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Ser at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Leu at position 346; (xi) Pro at position 347; (xii) Arg at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 132-133; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 132; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 133; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0231] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 134 or 135, and wherein the sequence comprises with respect to SEQ ID NO: 134: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 34, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 134 or 135, and wherein the sequence comprises with respect to SEQ ID NO: 134: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Thr at position 343; (ix) Ser at position 345; (x) Lys at position 346; (xi) Pro at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 134-135; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 134; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 135; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44.

[0232] In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 144 or 145, and wherein the sequence comprises with respect to SEQ ID NO: 144: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Thr at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 34, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO: 144 or 145, and wherein the sequence comprises with respect to SEQ ID NO: 144: (i) Gly at position 189; (ii) Ala at position 190; (iii) Vai at position 191; (iv) Asn at position 192; (v) Gly at position 233; (vi) Pro at position 245; (vii) Phe at position 316; (viii) Asn at position 343; (ix) Phe at position 345; (x) Gin at position 346; (xi) Thr at position 347; (xii) Lys at position 425; and (xiii) His at position 451; and a second Fc polypeptide comprising an amino acid sequence having at least about 85% identity, (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identity) to SEQ ID NO:43 or 44, and wherein the sequence comprises with respect SEQ ID NO:43: (i) Glu, Trp, or Leu at position 160; (ii) Tyr at position 164; (iii) Thr at position 166; (iv) Glu at position 167; (v) Trp at position 168; (vi) Ser or Ala at position 169; (vii) Asn or Ser at position 170; (viii) Thr at position 193; (ix) Glu at position 195; (x) Glu at position 196; and (xi) Phe at position 201. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 144-145; and a second Fc polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 43-44. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 144; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 43. In some embodiments, the fusion protein comprises a fusion polypeptide comprising a first Fc polypeptide linked to a GCase amino acid sequence, wherein the fusion polypeptide comprises an amino acid sequence of SEQ ID NO: 145; and a second Fc polypeptide comprising an amino acid sequence of SEQ ID NO: 44. In some embodiments, a fusion protein described herein comprises 1) a first Fc polypeptide linked to a GCase amino acid sequence; and 2) a second Fc polypeptide; wherein each polypeptide consists of an amino acid sequence as recited in a foregoing embodiment.

[0233] Fusion proteins and other compositions described herein may have a range of binding affinities. For example, in some embodiments, a protein has an affinity for a transferrin receptor (TfR), ranging anywhere from about 50 nM to about 500 nM, or from about 100 nM to about 500 nM, or from about lOOnM to about 400nM. In some embodiments, the affinity for TfR ranges from about 50 nM to about 300 nM. In some embodiments, the affinity for TfR ranges from about 100 nM to about 350 nM. In some embodiments, the affinity for TfR ranges from about 100 nM to about 400 nM. In some embodiments, the affinity for TfR ranges from about 150 nM to about 400 nM. In some embodiments, the affinity for TfR ranges from about 100 nM to about 500 nM. In some embodiments, the affinity for TfR ranges from about 200 nM to about 400 nM. In some embodiments, the affinity for TfR ranges from about 200 nM to about 300 nM. In some embodiments, the affinity for TfR ranges from about 250 nM to about 300 nM. In some embodiments, the affinity for TfR ranges from about 200 nM to about 450 nM. In some embodiments, the affinity for TfR is a monovalent affinity. In some embodiments, the affinity is for the apical domain of a transferrin receptor (e.g., the apical domain of a human transferrin receptor).

[0234] EVALUATION OF PROTEIN ACTIVITY

[0235] Fusion proteins comprising modified GCase sequences as described herein may have improved properties as compared to a corresponding protein comprising a reference GCase sequence (e.g., a wild-type GCase sequence, such as SEQ ID NO: 45) or to imiglucerase (CEREZYME®). For example, in certain embodiments, a fusion protein comprising a modified GCase amino acid sequence may have a higher level of enzymatic activity (e.g., during or after serum exposure) as compared to a corresponding protein comprising a reference GCase sequence or an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®). Additionally, in certain embodiments, such a protein may not require the presence of, exposure to, or contact with isofagomine to achieve desired enzymatic levels.

[0236] Accordingly, in certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein has a higher level of enzymatic activity as compared to a corresponding protein comprising a reference GCase amino acid sequence (e.g., wildtype GCase amino acid sequences, such as SEQ ID NO: 45). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein has an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®). In certain embodiments, the level of enzymatic activity of a fusion protein comprising a modified GCase amino acid sequence as described herein is higher by at least about 1.25-fold, 1.5-fold, 1.75- fold, 2-fold, 3-fold, 4-fold or more relative to that of a corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45). In certain embodiments, the level of enzymatic activity of a fusion protein comprising a modified GCase amino acid sequence as described herein is higher by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold, 3- fold, 4-fold or more relative to that of imiglucerase (CEREZYME®). Thus, certain embodiments provide a protein comprising: (a) a fusion polypeptide comprising a first Fc polypeptide linked to a glucocerebrosidase (GCase) amino acid sequence; and (b) a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); wherein the protein has a higher level of enzymatic activity as compared to a corresponding protein comprising a reference GCase sequence (e.g., a wild-type GCase amino acid sequence, such as SEQ ID NO: 45) and / or an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®). As described below, in certain embodiments, the enzymatic activity of the fusion protein is observed 1) in the absence of exposure to or contact with isofagomine; and / or 2) in the presence of, or after the fusion protein is contacted with, serum.

[0237] Isofagomine (D-tartrate) is an iminosugar that has been shown to facilitate folding and transport of the enzyme (Steet, et al., Proc Natl Acad Sci U S A. 2006 Sep 12; 103(37): 13813-8). As described herein, a fusion protein comprising a modified GCase amino acid sequence as described herein may not require the presence of, exposure to, or contact with isofagomine to achieve certain desired enzymatic levels (see, e.g., Examples 2 and 4; and Table 3). Thus, in certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine (e.g., has not been exposed to or contacted with isofagomine in vitro during cell culture production or in vivo via co-administration). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein has a higher level of enzymatic activity as compared to a corresponding protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO: 45), wherein 1) the fusion protein and the corresponding fusion protein have not been exposed to or contacted with isofagomine; or 2) the fusion protein has not been exposed to or contacted with isofagomine and the corresponding fusion protein has been exposed to or contacted with isofagomine. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein has an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®), wherein the fusion protein has not been exposed to or contacted with isofagomine. In certain embodiments, the level of enzymatic activity is higher by at least about 1.25-fold, 1.5-fold, 1.75- fold, 2-fold or more.

[0238] In certain embodiments, enzymatic activity is measured at neutral pH and room temperature. In certain embodiments, enzymatic activity is measured in the presence of, or after the fusion protein is contacted with, serum. In certain embodiments, enzymatic activity is measured under physiological conditions. Physiological conditions are typically at neutral pH (e.g., about pH 6.8 to 7.4, or about pH 7 to 7.2) and temperatures of about 37°C. In some embodiments, physiological conditions include the presence of serum (e.g., mammalian serum, such as mouse, rabbit, monkey, or human serum). Thus, in certain embodiments, enzymatic activity is measured in the presence of, or after the fusion protein is contacted with, serum under physiological conditions. Exposures to physiological conditions to assess the enzymatic activity of a fusion protein described herein can range from, e.g., about 24 hours to 168 hours (e.g. for about 24 hours, 48 hours, 72 hours, or 168 hours) in duration. In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity, whereas the corresponding comparator fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) may or may not have been exposed to or contacted with isofagomine.

[0239] For example, in certain embodiments, enzymatic activity is measured in the presence of, or after the fusion protein is contacted with, serum. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has a higher level of enzymatic activity in the presence of, or after being contacted with, serum as compared to a corresponding protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO: 45). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has an equivalent or a higher level of enzymatic activity in the presence of, or after being contacted with, serum as compared to imiglucerase (CEREZYME®). In certain embodiments, the level of enzymatic activity is higher by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold or more. In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity. In certain embodiments, a corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) has been exposed to or contacted with isofagomine prior to measuring the enzymatic activity.

[0240] In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains enzymatic activity better than a corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) in the presence of, or after contact with, serum. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains enzymatic activity better than imiglucerase (CEREZYME®) in the presence of, or after contact with, serum. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 65%, 75%, 80%, 85%, 90%, 95%, 96%, 87%, 98%, or 99% of its baseline enzymatic activity in the presence of, or after the fusion protein is contacted with, serum. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its baseline enzymatic activity in the presence of, or after the fusion protein is contacted with, serum. In certain embodiments, in the presence of, or after being contacted with, serum, a fusion protein comprising a modified GCase amino acid sequence as described herein has an equivalent level of enzymatic activity to its baseline enzymatic activity. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has a higher level of enzymatic activity in the presence of, or after being contacted with, serum as compared to its baseline enzymatic activity (e.g., higher by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold or more). In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity, and wherein any corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) has optionally been exposed to or contacted with isofagomine. In the foregoing embodiments, baseline enzymatic activity is measured at t=0 (at initial exposure) or immediately prior to exposure to serum.

[0241] In certain embodiments, enzymatic activity is measured under physiological conditions. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has a higher level of enzymatic activity after exposure to physiological conditions as compared to a corresponding protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO: 45). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has an equivalent or a higher level of enzymatic activity after exposure to physiological conditions as compared to imiglucerase (CEREZYME®). In certain embodiments, the enzymatic activity is improved by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold or more relative to the corresponding protein comprising a reference GCase amino acid sequence or to imiglucerase (CEREZYME®). In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity. In certain embodiments, the corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) has been exposed to or contacted with isofagomine prior to measuring the enzymatic activity.

[0242] In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains enzymatic activity better than a corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) under physiological conditions. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains enzymatic activity better than imiglucerase (CEREZYME®) under physiological conditions, which may include, e.g., contact with serum at physiological conditions. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 65%, 75%, 80%, 85%, 90%, 95%, 96%, 87%, 98%, or 99% of its baseline enzymatic activity under physiological conditions (e.g., neutral pH and about 37°C). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein retains at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its baseline enzymatic activity under physiological conditions (e.g., neutral pH and about 37°C). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence as described herein has an equivalent level of enzymatic activity as compared to its baseline enzymatic activity under physiological conditions. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has a higher level of enzymatic activity as compared to its baseline enzymatic activity (e.g., higher by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold or more) under physiological conditions. In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity, and wherein any corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) has optionally been exposed to or contacted with isofagomine. In the foregoing embodiments, baseline enzymatic activity is measured at t=0 (at initial exposure) or immediately prior to exposure to physiological conditions. In certain embodiments, enzymatic activity is measured in the presence of, or after the fusion protein is contacted with, serum at physiological conditions. In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has a higher enzymatic activity in the presence of, or after being contacted with, serum at physiological conditions as compared to a corresponding protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO: 45). In certain embodiments, a fusion protein comprising a modified GCase amino acid sequence has an equivalent or a higher level of enzymatic activity in the presence of, or after being contacted with, serum at physiological conditions as compared to imiglucerase (CEREZYME®). In certain embodiments, the level of enzymatic activity is higher by at least about 1.25-fold, 1.5-fold, 1.75-fold, 2-fold or more. In certain embodiments, a fusion protein as described herein has not been exposed to or contacted with isofagomine prior to measuring the enzymatic activity. In certain embodiments, the corresponding fusion protein comprising a reference GCase amino acid sequence (e.g., SEQ ID NO:45) has been exposed to or contacted with isofagomine prior to measuring the enzymatic activity.

[0243] In certain embodiments, the serum is mouse serum. In certain embodiments, the serum is human serum.

[0244] In certain embodiments, the serum concentration ranges from about 10% to about 100% (v / v) in a suitable buffer at neutral pH e.g., PBST). In certain embodiments, the serum concentration (v / v) is about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, the serum concentration is about 20% (v / v). In certain embodiments, the serum concentration is about 50% (v / v). In certain embodiments, the serum concentration is about 80% (v / v). In certain embodiments, the serum concentration is about 100% (v / v).

[0245] In certain embodiments, the fusion protein is contacted with serum for at least about 30 minutes, such as for between about 30 minutes to about 168 hours (e.g., 30, 60, 90, or 120 minutes; or 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, 24, 36, 48, 72, 96 or 168 hours). In certain embodiments, the fusion protein is contacted with serum for between about 4 to about 168 hours; about 4 to about 72 hours; about 4 to about 48 hours; about 4 to about 24 hours; or about 4 to about 12 hours. In certain embodiments, a fusion protein as described herein is contacted with serum for about 30 minutes or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 60 minutes or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 90 minutes or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 4 hours or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 6 hours or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 12 hours or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 24 hours or longer before the enzymatic activity of the protein is evaluated. In certain embodiments, a fusion protein as described herein is contacted with serum for about 72 hours or longer before the enzymatic activity of the protein is evaluated.

[0246] In certain embodiments, a fusion protein as described herein is contacted with serum at a temperature of about 4°C. In certain embodiments, a fusion protein as described herein is contacted with serum at a temperature of about room temperature to about 37°C. For example, in certain embodiments, a fusion protein as described herein is contacted with serum at a temperature ranging from about 20°C to about 37°C (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37°C). In certain embodiments, a fusion protein as described herein is contacted with serum at room temperature (i.e., about 20°C to about 22°C). In certain embodiments, a fusion protein as described herein is contacted with serum at about 30°C. In certain embodiments, a fusion protein as described herein is contacted with serum at about 37°C.

[0247] In certain embodiments, a fusion protein as described herein is contacted with serum at a temperature of about 37°C for about 4 to about 168 hours; about 4 to about 72 hours; about 4 to about 48 hours; about 4 to about 24 hours; or about 4 to about 12 hours. In certain embodiments, a fusion protein as described herein is contacted with serum at a temperature of about 37°C for about 24 hours. In certain embodiments, a fusion protein as described herein is contacted with serum at a temperature of about 37°C for about 72 hours.

[0248] To assess enzymatic activity, an assay described herein or known in the art may be used. For example, assays for evaluating enzymatic activity include, e.g., detection with a probe cap surrogate for GCase activity, ELISA methods involving a fluorogenic substrate, and GCase substrate correction assays (e.g., in vitro glucosylceramide or glycosyl sphingosine assays). Alternatively, assays that measure activity in vitro using a fluorogenic artificial substrate, such as those described in the Examples section (e.g., 4-Methylumbelliferyl P-D-glucopyranoside as described in Example 2), may also be used. As such, in certain embodiments, enzymatic activity is measured by a fluorescence-based assay, such as a fluorescence-based assay comprising an in vitro reaction with 4-MUG (4-Methylumbelliferyl P-D-glucopyranoside) substrate. In some embodiments, GCase substrate correction assays can be used, e.g., by detecting a correction in the levels of one or both of glucosyl sphingosine and glucosylceramide in a GBA knockout cell or cell line (e.g. GBA knock-out HEK293 cells). In other embodiments, a tissue sample is evaluated (e.g., brain, liver, heart, quadricep muscle, kidney, lung, spleen, plasma, serum, cerebrospinal fluid (CSF), and urine). In some embodiments, CSF samples from a patient receiving an GCase-Fc fusion protein described herein may be evaluated. A tissue sample can be evaluated using an assay, wherein multiple freeze-thaw cycles, e.g., 2, 3, 4, 5, or more, are typically included before a sonication step to ensure that microvesicles are broken open. Mass spectrometry -based methods for measuring glucosyl sphingosine and glucosylceramide are described in, e.g., Merrill, Jr. et al. 2005. Methods 36(2):207-224; and Logan et al. 2021. Cell 184: 1-18. Additional methods for measuring glucosyl sphingosine and glucosylceramide are known in the art. When comparing enzymatic activity levels, a modified GCase-Fc fusion protein being tested and a reference GCase-Fc fusion protein (e.g., comprising SEQ ID NO:45) are assayed under identical conditions, unless specifically indicated otherwise. The Examples provided herein are not limiting; other methods for measuring enzymatic activity are known in the art and can be used for the purposes described herein.

[0249] NUCLEIC ACIDS, VECTORS, AND HOST CELLS

[0250] Polypeptide chains contained in the fusion proteins as described herein are typically prepared using recombinant methods. Accordingly, in some aspects, the present disclosure provides isolated nucleic acids comprising a nucleic acid sequence encoding any of the polypeptide chains comprising Fc polypeptides as described herein, and host cells into which the nucleic acids are introduced that are used to replicate the polypeptide-encoding nucleic acids and / or to express the polypeptides. In some embodiments, the host cell is eukaryotic, e.g., a human cell.

[0251] In another aspect, polynucleotides are provided that comprise a nucleotide sequence that encodes one or more of the polypeptide chains described herein. In some embodiments, the polynucleotide encodes one of the polypeptide sequences described here (e.g., a first Fc polypeptide linked to a GCase amino acid sequence; or a second Fc polypeptide). In some embodiments, the polynucleotide encodes two of the polypeptide sequences described herein (e.g., a first Fc polypeptide linked to a GCase amino acid sequence; and a second Fc polypeptide). The polynucleotides may be single-stranded or double-stranded. In some embodiments, the polynucleotide is DNA. In particular embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0252] Some embodiments also provide a pair of nucleic acid sequences, wherein each nucleic acid sequence encodes a polypeptide described herein. For example, certain embodiments provide a pair of nucleic acid sequences, wherein a first nucleic acid sequence in the pair encodes a first Fc polypeptide linked to a GCase amino acid sequence; and a second nucleic acid sequence in the pair encodes a second Fc polypeptide, wherein the first and / or second Fc polypeptide is a modified Fc that is capable of binding (e.g., specifically binding) to a bloodbrain barrier (BBB) receptor, e.g., a transferrin receptor (TfR).

[0253] In some embodiments, the polynucleotide is included within a nucleic acid construct or the pair of polynucleotides is included within one or more nucleic acid constructs. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non-episomal mammalian vector.

[0254] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one series of embodiments, the nucleic acid expression constructs are adapted for use as a surface expression library. In some embodiments, the library is adapted for surface expression in yeast. In some embodiments, the library is adapted for surface expression in phage. In another series of embodiments, the nucleic acid expression constructs are adapted for expression of the polypeptide in a system that permits isolation of the polypeptide in milligram or gram quantities. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0255] Expression vehicles for production of a recombinant polypeptide include plasmids and other vectors. For instance, suitable vectors include plasmids of the following types: pBR322- derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express the recombinant polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors. Additional expression systems include adenoviral, adeno-associated virus, and other viral expression systems.

[0256] Vectors may be transformed into any suitable host cell. In some embodiments, the host cells, e.g., bacteria or yeast cells, may be adapted for use as a surface expression library. In some cells, the vectors are expressed in host cells to express relatively large quantities of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cells are mammalian cells, such as Chinese Hamster Ovary (CHO) cell, baby hamster kidney (BHK) cell, NSO cell, YO cell, HEK293 cell, COS cell, Vero cell, or HeLa cell.

[0257] Thus, certain embodiments provide a method for producing a protein or polypeptide as described herein, the method comprising culturing a host cell as described herein under conditions in which one or more polypeptides encoded by one or more polynucleotides as described herein are expressed. For example, a host cell transfected with an expression vector(s) encoding one or more Fc polypeptide chains as described herein can be cultured under appropriate conditions to allow expression of the one or more polypeptides to occur. In certain embodiments, the host cell is cultured in the absence of isofagomine during protein production. The polypeptides may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptides may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.

[0258] Further, certain embodiments provide a protein produced by a method as described herein. For example, certain embodiments provide a protein produced by cell expression (e.g., via culturing a host cell as described herein under conditions suitable for expression of the polypeptides) in the absence of isofagomine (i.e., the protein is not expose to or contacted with isofagomine), wherein the protein comprises: a. a fusion polypeptide comprising a first Fc polypeptide linked to a modified glucocerebrosidase (GCase) amino acid sequence as described herein; and b. a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); and wherein the protein has 1) a higher level of enzymatic activity as compared to a corresponding protein comprising a wild-type GCase amino acid sequence (e.g., after exposure to serum); and / or 2) an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®), including, e.g., after exposure to serum.

[0259] THERAPEUTIC METHODS

[0260] A fusion protein or polypeptide as described herein may be used therapeutically to treat a GCase deficiency in a patient in need thereof. In certain embodiments, the GCase deficiency is associated with a disease or disorder, such as Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies. As such, a fusion protein or polypeptide as described herein may be used to treat these diseases / disorders.

[0261] Accordingly, certain embodiments provide a method of treating a GCase deficiency in a patient in need thereof, the method comprising administering a protein or polypeptide as described herein to the patient.

[0262] Certain embodiments provide a protein or polypeptide as described herein for use in treating a GCase deficiency in a patient in need thereof.

[0263] Certain embodiments provide the use of a protein or polypeptide as described herein in the preparation of a medicament for treating a GCase deficiency in a patient in need thereof.

[0264] Certain embodiments also provide a method of decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency, the method comprising administering a protein or polypeptide as described herein to the patient.

[0265] Certain embodiments provide a protein or polypeptide as described herein for use in decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency.

[0266] Certain embodiments provide the use of a protein or polypeptide as described herein in the preparation of a medicament for decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency.

[0267] In certain embodiments, the toxic metabolic product comprises glucosylceramide. In certain embodiments, the toxic metabolic product comprises glucosylsphingosine.

[0268] In some embodiments, administration of the protein (e.g., linked to a GCase amino acid sequence) improves (e.g., increases) Cmax of GCase in the brain as compared to the uptake of GCase in the absence of being linked to a fusion protein described herein or as compared to the uptake of GCase linked to a reference protein (e.g., a fusion protein as described herein, which does not have the modifications to the second Fc polypeptide that result in TfR binding).

[0269] In some embodiments, Cmax of GCase in the brain is improved (e.g., increased) by at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2- fold, 2.2-fold, 2.4-fold, 2.6-fold, 2.8-fold, 3-fold, 4-fold, 5-fold, 6-fold, or more, as compared to the uptake of GCase in the absence of being linked to a fusion protein described herein or as compared to the uptake of GCase linked to a reference protein (e.g., a fusion protein as described herein, which does not have the modifications to the second Fc polypeptide that result in TfR binding).

[0270] A protein or polypeptide described herein is administered to a subject at a therapeutically effective amount or dose.

[0271] In various embodiments, a protein or polypeptide described herein is administered parenterally. In some embodiments, the protein or polypeptide is administered intravenously.

[0272] In some parenteral embodiments, a protein or polypeptide as described herein is administered intraperitoneally, intradermally, or intramuscularly. In some embodiments, the protein or polypeptide as described herein is administered intrathecally, such as by epidural administration, or intracerebroventricularly.

[0273] PHARMACEUTICAL COMPOSITIONS AND KITS

[0274] In other aspects, pharmaceutical compositions and kits comprising a fusion protein or polypeptide described herein are provided.

[0275] Pharmaceutical Compositions

[0276] Guidance for preparing formulations for use in the present disclosure can be found in any number of handbooks for pharmaceutical preparation and formulation that are known to those of skill in the art.

[0277] In some embodiments, a pharmaceutical composition comprises a fusion protein or polypeptide as described herein and further comprises one or more pharmaceutically acceptable carriers and / or one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers and one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that do not interfere with or otherwise inhibit the activity of the active agent.

[0278] Dosages and desired drug concentration of pharmaceutical compositions described herein may vary depending on the particular use envisioned.

[0279] Kits

[0280] In some embodiments, a kit for use in treating a GCase deficiency, comprising a fusion protein or polypeptide as described herein, is provided.

[0281] In some embodiments, the kit further comprises one or more additional therapeutic agents. For example, in some embodiments, the kit comprises a fusion protein or polypeptide as described herein and further comprises one or more additional therapeutic agents for use in the treatment of one or more symptoms of a GCase deficiency (e.g., one or more symptoms associated with Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies). In some embodiments, the kit further comprises instructional materials containing directions (i.e., protocols) for the practice of the methods described herein (e.g., instructions for using the kit for administering a fusion protein comprising a modified GCase amino acid sequence across the blood-brain barrier). While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD-ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

[0282] Certain Definitions

[0283] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” may include two or more such molecules, and the like.

[0284] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0285] The term “subject,” “individual,” and “patient,” as used interchangeably herein, refer to a mammal, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human. In some embodiments, the human is a patient in need of treatment for a disease or disorder associated with GCase deficiency (e.g., Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies). In some embodiments, the patient has one or more signs or symptoms associated with GCase deficiency (e.g., Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies).

[0286] The term “pharmaceutically acceptable excipient” refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as but not limited to a buffer, carrier, or preservative.

[0287] The term “administer” refers to a method of delivering agents (e.g., a therapeutic agent, such as an ETV:GCase protein described herein), compounds, or compositions (e.g., pharmaceutical composition) to the desired site of biological action. These methods include, but are not limited to, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, or intraperitoneal delivery. In one embodiment, the polypeptides or proteins described herein are administered intravenously.

[0288] The term “GCase deficiency” as used herein refers to a condition wherein a subject expresses levels of GCase that are less than that of a control or population of control subjects and / or wherein the enzymatic activity level of the subject’s expressed GCase is less than that of a control or population of control subjects.

[0289] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0290] The phrase “effective amount” means an amount of a compound described herein that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0291] A “therapeutically effective amount” of a substance / molecule disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

[0292] “Glucocerebrosidase” or “GCase” is also known as “P-glucocerebrosidase”, “GBA” or “glucosylceramidase” (EC 3.2.1.45). The term as used herein refers to a lysosomal enzyme that has glucosylceramidase activity and catalyzes the breakdown of glucosylceramide to ceramide and glucose. Deficiency of GCase is associated with Gaucher’s disease, Parkinson’s disease, Parkinsonism, or dementia with Lewy Bodies. The term “GCase” as used herein is catalytically active and encompasses functional variants, including allelic and splice variants, and catalytically active fragments of wild-type GCase and its functional variants. A catalytically active GCase 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 enzymatic activity of the corresponding full-length GCase or variant thereof, e.g., when assayed under identical conditions. Similarly, a GCase variant is functional and has at least 50% (e.g., or 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 a corresponding wild-type GCase, e.g., when assayed under identical conditions. Enzymatic activity can be measured according to methods known in the art, including those described herein. The sequence of human GCase, long isoform, which is designated as the canonical sequence, is available under UniProt entry P04062-1 and is encoded by the human GBA gene at lq22. The full-length sequence is provided as SEQ ID NO:46. A “mature” GCase sequence as used herein refers to a form of a polypeptide chain that lacks the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human GCase polypeptide is provided as SEQ ID NO:45, which corresponds to amino acids 40-536 of the full-length human sequence. In certain embodiments, a modified GCase amino acid sequence for use in a fusion protein described herein is a modified, mature human GCase amino acid sequence. Commercially available forms of recombinant GCase include imiglucerase (CEREZYME®), velaglucerase alfa (VPRIV®), and taliglucerase alfa (ELELYSO®).

[0293] Assays for evaluating enzymatic activity include, e.g., ELISA methods involving a fluorogenic substrate and GCase substrate correction assays. For example, assays that measure activity in vitro using a fluorogenic artificial substrate, such as those described in the Examples section (e.g., 4-Methylumbelliferyl a-D-glucopyranoside as described in Example 2), may be used. In some embodiments, GCase substrate correction assays can be used, e.g., by detecting a correction in the levels of glucosylceramide or glucosyl sphingosine in a GBA knockout cell or cell line (e.g. GBA knock-out HEK293 cells) (see, e.g., Example 3). Mass spectrometry-based methods for measuring glucosylceramide or glucosylsphingosine levels are described in, e.g., Logan et al. 2021. Cell 184: 1-18. Additional methods for measuring glucosylceramide or glucosyl sphingosine levels are known in the art.

[0294] The term “serum” refers to the liquid fraction of clotted blood, which is generally depleted of cells, fibrin and clotting factors. For example, serum may be prepared by centrifuging until the clot and remaining blood cells are separated from the liquid phase. Fusion proteins as described herein may be contacted with serum to assess the impact that exposure to serum has on the enzymatic activity of the protein. Thus, to determine if a fusion protein comprising a modified GCase amino acid sequence retains or has improved enzymatic activity in the presence of serum, the fusion protein and a reference protein may be contacted with serum for a period of time (e.g., under conditions described herein) and subsequently assessed using an assay described herein or known in the art.

[0295] A “transferrin receptor” or “TfR” as used herein refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO:7. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, accession number XP_003310238.1; rhesus monkey, NP_001244232.1; dog, NP_001003111.1; cattle, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term “transferrin receptor” also encompasses allelic variants of exemplary reference sequences, e.g., human sequences, that are encoded by a gene at a transferrin receptor protein 1 chromosomal locus. 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. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID N0:8.

[0296] A “fusion protein” or “GCase enzyme-Fc fusion protein” or “GCase-Fc fusion protein” as used herein refers to a protein (e.g., a dimeric protein) comprising a first Fc polypeptide that is linked (e.g., fused) to a GCase amino acid sequence (i.e., an “GCase-Fc fusion polypeptide”); and a second Fc polypeptide (e.g., that forms an Fc dimer with the first Fc polypeptide). The second Fc polypeptide may also be linked e.g., fused) to a GCase amino acid sequence. The first Fc polypeptide and / or the second Fc polypeptide may be linked to the GCase amino acid sequence by a peptide bond or by a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that promote its heterodimerization to the other Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that confer binding to a transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that reduce effector function. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide do not have effector function. The first Fc polypeptide and / or the second Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that extend serum half-life. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide do not include an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof. In certain embodiments, the first Fc polypeptide and the second Fc polypeptide do not include an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof.

[0297] A “fusion polypeptide” or “GCase enzyme-Fc fusion polypeptide” or “GCase-Fc fusion polypeptide” as used herein refers to an Fc polypeptide that is linked (e.g., fused) to a GCase amino acid sequence. The Fc polypeptide may be linked to the GCase amino acid sequence by a peptide bond or by a polypeptide linker. The Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that promote its heterodimerization to another Fc polypeptide. The Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that confer binding to a transferrin receptor. The Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that reduce effector function. The Fc polypeptide may be a modified Fc polypeptide that contains one or more modifications that extend serum half-life.

[0298] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide that is characterized by an Ig fold as a structural domain. An Fc polypeptide contains constant region sequences including at least the CH2 domain and / or the CH3 domain and may contain at least part of the hinge region. In general, an Fc polypeptide does not contain a variable region.

[0299] A “modified Fc polypeptide” refers to an Fc polypeptide that has at least one mutation, e.g., a substitution, deletion or insertion, as compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0300] The term “FcRn” refers to the neonatal Fc receptor. Binding of Fc polypeptides to FcRn reduces clearance and increases serum half-life of the Fc polypeptide. The human FcRn protein is a heterodimer that is composed of a protein of about 50 kDa in size that is similar to a major histocompatibility (MHC) class I protein and a P2-microglobulin of about 15 kDa in size.

[0301] As used herein, an “FcRn binding site” refers to the region of an Fc polypeptide that binds to FcRn. In human IgG, the FcRn binding site, as numbered using the EU index, includes T250, L251, M252, 1253, S254, R255, T256, T307, E380, M428, H433, N434, H435, and Y436. These positions correspond to positions 20 to 26, 77, 150, 198, and 203 to 206 of SEQ ID NO: 1.

[0302] As used herein, a “native FcRn binding site” refers to a region of an Fc polypeptide that binds to FcRn and that has the same amino acid sequence as the region of a naturally occurring Fc polypeptide that binds to FcRn. In certain embodiments, the first Fc polypeptide and / or the second Fc polypeptide comprises a native FcRn binding site.

[0303] The terms “CH3 domain” and “CH2 domain” as used herein refer to immunoglobulin constant region domain polypeptides. For purposes of this application, a CH3 domain polypeptide refers to the segment of amino acids from about position 341 to about position 447 as numbered according to EU, and a CH2 domain polypeptide refers to the segment of amino acids from about position 231 to about position 340 as numbered according to the EU numbering scheme and does not include hinge region sequences. CH2 and CH3 domain polypeptides may also be numbered by the IMGT (ImMunoGeneTics) numbering scheme in which the CH2 domain numbering is 1-110 and the CH3 domain numbering is 1-107, according to the IMGT Scientific chart numbering (IMGT website). CH2 and CH3 domains are part of the Fc region of an immunoglobulin. An Fc region refers to the segment of amino acids from about position 231 to about position 447 as numbered according to the EU numbering scheme, but as used herein, can include at least a part of a hinge region of an antibody. An illustrative hinge region sequence is the human IgGl hinge sequence EPKSCDKTHTCPPCP (SEQ ID NO: 5).

[0304] “Naturally occurring,” “native” or “wild type” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified in the laboratory, is naturally occurring. Furthermore, “wild-type” refers to the normal gene, or organism found in nature without any known mutation. For example, the terms “wild-type,” “native,” and “naturally occurring” with respect to a CH3 or CH2 domain are used herein to refer to a domain that has a sequence that occurs in nature. In other examples, the terms “wild-type,” “native,” and “naturally occurring” with respect to GCase are used herein to refer to a GCase polypeptide that has a sequence that occurs in nature. In some embodiments, the wild-type GCase polypeptide is a wild-type human GCase polypeptide.

[0305] As used herein, the term “mutant” with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with “variant.” A variant with respect to a given wildtype reference sequence (e.g., CH3 or CH2 domain reference sequence; or a GCase reference sequence) can include naturally occurring allelic variants. Accordingly, a “non-naturally” occurring CH3 or CH2 domain refers to a variant or mutant domain that is not present in a cell in nature and that is produced by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native CH3 domain or CH2 domain polynucleotide or polypeptide. Similarly, a “non-naturally” occurring GCase refers to a variant or mutant domain that is not present in a cell in nature and that is produced by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native GCase polynucleotide or polypeptide. A “variant” includes any domain comprising at least one amino acid mutation with respect to wild-type. Mutations may include substitutions, insertions, and deletions.

[0306] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0307] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxy glutamate and O- phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally-occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D- Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D- Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D- asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0308] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0309] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0310] The term “protein” as used herein refers to either a polypeptide or a dimer (z.e, two) or multimer (z.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.

[0311] The term “conservative substitution,” “conservative mutation,” or “conservatively modified variant” refers to an alteration that results in the substitution of an amino acid with another amino acid that can be categorized as having a similar feature. Examples of categories of conservative amino acid groups defined in this manner can include: a “charged / polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R), and His (Histidine or H); an “aromatic group” including Phe (Phenylalanine or F), Tyr (Tyrosine or Y), Trp (Tryptophan or W), and (Histidine or H); and an “aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Vai (Valine or V), Leu (Leucine or L), He (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T), and Cys (Cysteine or C). Within each group, subgroups can also be identified. For example, the group of charged or polar amino acids can be sub-divided into sub-groups including: a “positively-charged sub-group” comprising Lys, Arg and His; a “negatively-charged sub-group” comprising Glu and Asp; and a “polar sub-group” comprising Asn and Gin. In another example, the aromatic or cyclic group can be sub-divided into subgroups including: a “nitrogen ring sub-group” comprising Pro, His and Trp; and a “phenyl subgroup” comprising Phe and Tyr. In another further example, the aliphatic group can be subdivided into sub-groups, e.g., an “aliphatic non-polar sub-group” comprising Vai, Leu, Gly, and Ala; and an “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys. Examples of categories of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained. In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve hydrophobicity.

[0312] The terms “identical” or percent “identity,” in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. In some embodiments, a sequence that has a specified percent identity relative to a reference sequence differs from the reference sequence by one or more conservative substitutions.

[0313] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used. The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. Thus, for example, an amino acid residue in a modified Fc polypeptide “corresponds to” an amino acid in SEQ ID NO: 1, when the residue aligns with the amino acid in SEQ ID NO: 1 when optimally aligned to SEQ ID NO: 1. Similarly, an amino acid residue in a modified GCase polypeptide / amino acid sequence “corresponds to” an amino acid in SEQ ID NO:45, when the residue aligns with the amino acid in SEQ ID NO: 45 when optimally aligned to SEQ ID NO:45. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0314] The term “polynucleotide” and “nucleic acid” interchangeably refer to chains 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 chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having mixtures of single- and double-stranded DNA and RNA.

[0315] A “binding affinity” as used herein refers to the strength of the non-covalent interaction between two molecules, e.g., a single binding site on a polypeptide and a target, e.g., transferrin receptor, to which it binds. Thus, for example, the term may refer to 1 : 1 interactions between a polypeptide and its target, unless otherwise indicated or clear from context. Binding affinity may be quantified by measuring an equilibrium dissociation constant (KD), which refers to the dissociation rate constant (kd, time'1) divided by the association rate constant (ka, time'1M'X). KD can be determined by measurement of the kinetics of complex formation and dissociation, e.g., using Surface Plasmon Resonance (SPR) methods, e.g., a Biacore™ system; kinetic exclusion assays such as KinExA®; and BioLayer interferometry (e.g., using the ForteBio® Octet® platform). As used herein, “binding affinity” includes not only formal binding affinities, such as those reflecting 1 : 1 interactions between a polypeptide and its target, but also apparent affinities for which KD’S are calculated that may reflect avid binding.

[0316] As used herein, the term “specifically binds” or “selectively binds” to a target, e.g., TfR, when referring to an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR- binding fusion protein as described herein, refers to a binding reaction whereby the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein binds to the target with greater affinity, greater avidity, and / or greater duration than it binds to a structurally different target. In typical embodiments, the engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or greater affinity for a specific target, e.g., TfR, compared to an unrelated target when assayed under the same affinity assay conditions. The term “specific binding,” “specifically binds to,” or “is specific for” a particular target (e.g., TfR), as used herein, can be exhibited, for example, by a molecule having an equilibrium dissociation constant KD for the target to which it binds of, e.g., 10'4M or smaller, e.g., 10'5M, 10'6M, 10'7M, 10'8M, 10'9M, IO'10M, 10'11M, or IO’12M. In some embodiments, an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein specifically binds to an epitope on TfR that is conserved among species, e.g., structurally conserved among species), e.g., conserved between non-human primate and human species e.g., structurally conserved between nonhuman primate and human species). In some embodiments, an engineered TfR-binding polypeptide, TfR-binding peptide, or TfR-binding fusion protein may bind exclusively to a human TfR.

[0317] The term “variable region” or “variable domain” refers to a domain in an antibody heavy chain or light chain that is derived from a germline Variable (V) gene, Diversity (D) gene, or Joining (J) gene (and not derived from a Constant (Cp and C5) gene segment), and that gives an antibody its specificity for binding to an antigen. Typically, an antibody variable region comprises four conserved “framework” regions interspersed with three hypervariable “complementarity determining regions.”

[0318] The terms “antigen-binding portion” and “antigen-binding fragment” are used interchangeably herein and refer to one or more fragments of an antibody that retains the ability to specifically bind to an antigen via its variable region. Examples of antigen-binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL, and CHI domains), a F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a single chain Fv (scFv), a disulfide- linked Fv (dsFv), complementarity determining regions (CDRs), a VL (light chain variable region), and a VH (heavy chain variable region).

[0319] The following Examples are intended to be non-limiting. EXAMPLE 1 : Construction of Fusion Proteins Containing Glucocerebrosidase (GCase) Sequences

[0320] Design and cloning

[0321] Fusion proteins (“ETV:GCase Fusion”) were designed that contain (i) a first fusion polypeptide where a glucocerebrosidase (GCase) sequence is fused to a human IgGl fragment that includes the Fc region (a “GCase-Fc fusion polypeptide”), and (ii) a modified human IgGl fragment which contains mutations in the Fc region that confer transferrin receptor (TfR) binding (a “modified Fc polypeptide”). In certain exemplary fusion proteins, the first fusion polypeptide (i) included an engineered GCase amino acid sequence fused to a human IgGl Fc polypeptide (a “GCase variant-Fc fusion polypeptide”). Exemplary engineered GCase amino acid sequences with modifications relative to wild-type human GCase sequence (SEQ ID NO:45) are listed in Table 1. GCase variant-Fc fusion polypeptides were created in which the GCase variant sequences were fused to the N-terminus of the human IgGl Fc region. In all constructs, the signal peptide MGWSCIILFLVATATGAYA (SEQ ID NO: 150) was inserted upstream to facilitate secretion. The fragment of the human IgGl Fc region used corresponds to amino acids D104-K330 of the sequence in UniProtKB ID P01857 (positions 221-447, EU numbering, which includes 10 amino acids of the hinge (positions 221-230)). Expression vectors that separately encode (i) the GCase variant-Fc fusion polypeptide (or GCase-Fc fusion polypeptide) and (ii) the modified Fc polypeptide were generated and co-transfected into Chinese Hamster Ovary (CHO) cells to generate heterodimeric fusion proteins containing the GCase sequence (a “monozyme”). In all constructs, the IgGl fragments contained additional mutations to facilitate heterodimerization of the two Fc regions, as well as modifications to reduce effector function.

[0322] Table 1. Exemplary Engineered GCase Sequences

[0323]

[0324]

[0325] WT = wild-type GCase (SEQ ID NO: 45)

[0326] * GCase sequence also contains L94E modification relative to SEQ ID NO:45

[0327] A GCase-Fc fusion polypeptide comprising the wild-type human GCase amino acid sequence (SEQ ID NO: 45) was fused to the N-terminus of an IgGl Fc polypeptide sequence with hole and LALA mutations (e.g., SEQ ID NO:21) The GCase amino acid sequence was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 152) and the N-terminus of the Fc polypeptide included a portion of an IgGl hinge region (DKTHTCPPCP; SEQ ID NO:6). As described herein, other linkers may also be used, for example, the GCase amino acid sequence may alternatively be joined to the Fc polypeptide by a GS linker or a (GGGGS)? linker (SEQ ID NO:151).

[0328] GCase variant-Fc fusion polypeptides comprising an engineered GCase amino acid sequence were fused to the N-terminus of an IgGl Fc polypeptide sequence with hole and LALA or LALAPS mutations (e.g., SEQ ID NO:21 or 23). The GCase amino acid sequence was joined to the Fc polypeptide by a GGGGS linker (SEQ ID NO: 152) and the N-terminus of the Fc polypeptide included a portion of an IgGl hinge region (DKTHTCPPCP; SEQ ID NO:6). As described herein, other linkers may also be used, for example, the GCase amino acid sequence may alternatively be joined to the Fc polypeptide by a GS linker or a (GGGGS)? linker (SEQ ID NO:151).

[0329] A TfR-binding modified Fc polypeptide with knob and LALA mutations has the sequence of SEQ ID NO:41 or 42 and the TfR-binding modified Fc polypeptide that further includes the P329S mutation has the sequence of SEQ ID NO:43 or 44. The N-terminus of the modified Fc polypeptide included a portion of an IgGl hinge region (DKTHTCPPCP; SEQ ID NO:6).

[0330] A GCase-Fc fusion protein (“ETV:GCase Fusion 1”) was generated comprising a TfR- binding modified Fc polypeptide having the sequence of SEQ ID NO:41 and a GCase-Fc fusion polypeptide having the sequence of SEQ ID NO:48. The GCase-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide in the fusion protein has the sequence of SEQ ID NO:42, and / or the GCase-Fc fusion polypeptide comprises an Fc polypeptide having the sequence of SEQ ID NO:49. Thus, as used herein, the term ETV:GCase Fusion 1 may be used to refer to protein molecules having unprocessed sequences (i.e., comprising SEQ ID NOs: 41 and 48); protein molecules comprising one or more processed sequences (i.e., selected from SEQ ID NOs:42 and 49); or to a mixture comprising processed and unprocessed protein molecules.

[0331] A first comparator GCase-Fc fusion protein (“ETV:GCase Fusion 3”) was generated that includes a modified GCase sequence that contains the point mutations W312C and A341C (numbering relative to SEQ ID NO:45, reference publication WO 2022 / 023761). ETV:GCase Fusion 3 comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:41 and a GCase-Fc fusion polypeptide having the sequence of SEQ ID NO:52. The GCase-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide in the fusion protein has the sequence of SEQ ID NO:42, and / or the GCase-Fc fusion polypeptide comprises an Fc polypeptide having the sequence of SEQ ID NO:53. Thus, as used herein, the term ETV:GCase Fusion 3 may be used to refer to protein molecules having unprocessed sequences (i.e., comprising SEQ ID NOs: 41 and 52); protein molecules comprising one or more processed sequences (i.e., selected from SEQ ID NOs:42 and 53); or to a mixture comprising processed and unprocessed protein molecules.

[0332] A second comparator GCase-Fc fusion protein (“ETV:GCase Fusion 64”) was generated that includes a modified GCase sequence that contains the point mutation E233Q (numbering relative to SEQ ID NO:45, reference publication WO 2022 / 023761). ETV:GCase Fusion 64 comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:41 and a GCase-Fc fusion polypeptide having the sequence of SEQ ID NO:54. The GCase-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide in the fusion protein has the sequence of SEQ ID NO:42, and / or the GCase-Fc fusion polypeptide comprises an Fc polypeptide having the sequence of SEQ ID NO: 55. Thus, as used herein, the term ETV:GCase Fusion 64 may be used to refer to protein molecules having unprocessed sequences (i.e., comprising SEQ ID NOs: 41 and 54); protein molecules comprising one or more processed sequences (i.e., selected from SEQ ID NOs:42 and 55); or to a mixture comprising processed and unprocessed protein molecules.

[0333] A GCase variant-Fc fusion protein (“ETV:GCase Fusion 2”) was generated that includes a modified GCase sequence that contains the modifications listed in Table 1. ETV:GCase Fusion 2 comprises a TfR-binding modified Fc polypeptide having the sequence of SEQ ID NO:41 and a GCase-Fc fusion polypeptide having the sequence of SEQ ID NO:50. The GCase-Fc fusion protein may also be further processed during cell culture production such that the TfR-binding modified Fc polypeptide in the fusion protein has the sequence of SEQ ID NO:42, and / or the GCase-Fc fusion polypeptide comprises an Fc polypeptide having the sequence of SEQ ID NO: 51. Thus, as used herein, the term ETV:GCase Fusion 2 may be used to refer to protein molecules having unprocessed sequences (i.e., comprising SEQ ID NOs: 41 and 50); protein molecules comprising one or more processed sequences (i.e., selected from SEQ ID NOs:42 and 51); or to a mixture comprising processed and unprocessed protein molecules.

[0334] GCase variant-Fc fusion proteins (“ETV:GCase Fusions 4 through 63, and ETV:GCase Fusions 65 through 67”) were generated comprising a TfR-binding modified Fc polypeptide (e.g., having the sequence of SEQ ID NO: 43) and a GCase variant-Fc fusion polypeptide (e.g., a GCase enzyme variant with the modifications listed in Table 1 fused via a GGGGS linker (SEQ ID NO: 152) to an Fc polypeptide having the sequence of SEQ ID NO: 23). The GCase variant-Fc fusion protein may also be further processed during cell culture production such that the TfR- binding modified Fc polypeptide in the fusion protein has the sequence of SEQ ID NO: 44, and / or the GCase variant-Fc fusion polypeptide comprises an Fc polypeptide having the sequence of SEQ ID NO: 24. Thus, as used herein, the term ETV:GCase Fusion X, wherein X may be a number selected from 4 to 63 or a number selected from 65 to 67, may be used to refer to protein molecules having unprocessed sequences (i.e., comprising SEQ ID NOs:23 and 43); protein molecules comprising one or more processed sequences (i.e., selected from SEQ ID NOs:24 and 44); or to a mixture comprising processed and unprocessed protein molecules.

[0335] A composition comprising ETV:GCase (e.g., any of the fusion proteins described above) may be used to refer to a composition comprising protein molecules having unprocessed sequences; protein molecules comprising one or more processed sequences; or to a mixture comprising processed and unprocessed protein molecules.

[0336] EXAMPLE 2: Protein Expression (+ / - Isofagomine) and Specific Activity of ETV: GCase fusion proteins

[0337] Isofagomine (IFG) is an iminosugar that acts as a competitive inhibitor and pharmacological chaperone of GCase enzyme. IFG is able to increase the global stability of wild-type GCase enzyme (see, Komhaber, et al. 2008. Chembiochem 9(16):2643-2649). ETV:GCase fusion proteins (Example 1) were generated in the absence of IFG, and specific activity was measured.

[0338] Results

[0339] ETV:GCase Fusion 1 generated in the absence of isofagomine displayed low levels of specific activity (data not shown). The presence of isofagomine during CHO cell expression is able to restore some of the specific activity of ETV: GCase Fusion 1 (Table 3, specific activity < 200); however, the restored specific activity remains lower than that of a commercial recombinant ERT (Cerezyme® / imiglucerase; Table 3, specific activity approx. 650). ETV:GCase Fusions 3 and 64, which contain modified GCase sequences disclosed in WO 2022 / 023761, expressed in the absence of isofagomine also illustrated specific activity that was lower than that of Cerezyme® / imiglucerase. In addition, about one-third of the ETV:GCase fusion proteins, when generated in the absence of isofagomine, also exhibited specific activity levels lower than that of Cerezyme® / imiglucerase. In contrast, certain ETV:GCase fusion proteins e.g., ETV:GCase Fusions 8-9, 12, 15, 16, 18, 26-28, 30-33, 35-38, 41-44, 47-50, 52, 53, 55-58, and 65-67) generated in the absence of isofagomine exhibited approximately equivalent or higher levels of specific activity relative to Cerezyme® / imiglucerase. Recombinant protein expression and purification

[0340] To express recombinant GCase-Fc fusion proteins, CH0-K1 -derived cells were transfected with relevant DNA constructs using TransIT-PRO Transfection Reagent according to manufacturer’s instructions (Minis Bio, cat#MIR5700). Cells were grown in BalanCD Transfectory CHO expression media (Irvine Scientific, cat# 91147-1L) at 37 °C, 5% CO2 and 125 rpm in an orbital shaker (Infers HT Multitron). After transfection, cells were temperature shifted to 32°C. Twenty-four (24) hours post transfection cells were provided a commercially available nutrient feed. Where isofagomine (IFG) was used in cell culture (e.g., for generation of ETV:GCase Fusion 1), IFG was also added to a final concentration of 100 pM 24 hours after transfection. Transfected cell culture supernatants were harvested 168 hours post transfection by centrifugation at 4000 rpm for 10 minutes.

[0341] The GCase-Fc fusion proteins were purified from cell culture supernatants using Protein A affinity chromatography. Supernatants were loaded onto a HiTrap MabSelect SuRe Protein A affinity column (GE Healthcare Life Sciences using an Akta Pure System). The column was then washed with 10 column volumes (CVs) of PBS. Bound proteins were eluted using 50 mM citrate / NaOH buffer pH 3.0 containing 100 mM NaCl. Immediately after elution, fractions were neutralized using 1 M Tris pH 8. In some cases, the eluates from Protein A affinity chromatography were neutralized by passing through a HiTrap Desalting column (GE Healthcare Life Sciences using an Akta Pure System) into 50 mM citrate, 50 mM phosphate, 100 mM NaCl, pH 6.5 buffer. Homogeneity of GCase-Fc fusions in eluted fractions was assessed by a number of techniques including reducing and non-reducing SDS-PAGE and HPLC-SEC.

[0342] Where additional purification was carried out, the Protein A pool was diluted into 20 mM sodium acetate at pH 5.0 and further purified by cation-exchange chromatography (CEX) over a ResourceQ column (Cytivas). Briefly, after binding, the column was washed with 10 CV of 20 mM sodium acetate and 5 mM NaCl at pH 5.0. Bound proteins were eluted using a linear gradient of 5 mM NaCl to 500 mM NaCl in 20 mM sodium acetate at pH 5.0. Homogeneity of post-CEX purified ETV:GCase was assessed by SDS-PAGE and analytical SEC-HPLC. Fractions with >95% purity were pooled and dialyzed over 20 mM sodium acetate, 150 mM NaCl, at pH 5 overnight.

[0343] Where delipidation of GCase-Fc fusions was carried out, the Protein A pool was mixed with 20% n-butanol (v / v) and the mixture was incubated at 25°C for 1 hour. The aqueous layer of the mixture was then separated and purified by size exclusion chromatography over a Superdex 200 increase column (Cytivas). Homogeneity of post-SEC purified ETV:GCase was assessed by SDS-PAGE and analytical SEC-HPLC. Fractions with >95% purity were pooled for further analysis.

[0344] Specific Activity Measurement.

[0345] Enzymatic activity of ETV:GCase fusion proteins was measured in 384-well plates as follows. Varying concentrations of ETVGCase fusion proteins were incubated with a fixed concentration of 4-Methylumbelliferyl P-D-glucopyranoside substrate (4-MUG, Thermo Fisher 10815-812) in assay buffer (100 mM phosphate citrate buffer, pH 5.2, 0.5% sodium taurocholate, 0.25% Triton X-100). An 11-point titration of GCase protein was made by serially diluting GCase two-fold in the assay buffer, with a 12th point set containing only assay buffer as blank. After incubation for one hour at room temperature in the dark, the reactions were quenched by addition of “stop buffer” (0.5 A / sodium carbonate, 0.5 A / sodium bicarbonate, pH 10.3). Fluorescence readout was measured using a Biotek Synergy Neo2 multimode plate reader using excitation wavelength 365 nm and emission wavelength 445 nm Signal from blank controls were subtracted as background from each plate. Background subtracted values for each fusion protein were normalized to expression titer measured for the corresponding fusion protein.

[0346] EXAMPLE 3: Cellular potency of ETV:GCase fusion proteins

[0347] Certain ETV:GCase fusion proteins (Example 1) were evaluated in GBA1 knock-out (“GBA KO”) HEK cells to ascertain if the fusion proteins were able to reduce endogenous substrate (e.g. glucosyl sphingosine, glucosylceramide).

[0348] Results

[0349] ETVGCase Fusion 1 illustrated approximate cellular potencies (ECso) values of from 0.64 to 1.56 nM for glucosyl sphingosine (GlcSph) in GBA KO cells and from 0.123 to 0.47 nM for glucosylceramide (GlcCer). A comparator construct (ETVGCase Fusion 3, which contains a modified GCase amino acid sequence disclosed in WO 2022 / 023761) displayed weaker cellular potencies for GlcSph and GlcCer (3.26 nM and 0.599 nM, respectively) (FIGS. 1A-1B). In contrast, ETV:GCase Fusions 27 and 30 exhibited stronger cellular potencies for GlcSph and GlcCer (FIGS. 1A-1B). ETWGCase Fusions 31, 32, and 33 were also assessed for cellular potency of endogenous substrate in GBA KO cells, and all displayed strong cellular potency values (ECso < 50 pM) for GlcSph and GlcCer (Table 2, FIGS. 2A-2B). The results are consistent with the specific activity results for the fusion proteins (Example 2), and indicate that the ETV:GCase fusion proteins are able to effectively reduce endogenous substrate in GBA KO cells.

[0350] Table 2. Cellular potency of ETV:GCase fusion proteins for reducing endogenous substrate

[0351] Cellular potency assay

[0352] All cells were cultured throughout in standard culture media comprised of DMEM (Thermo 11965) supplemented with 10% FBS (VWR), unless otherwise noted. GBA1 KO (and WT control) HEK293T cells were seeded in poly-D-lysine coated 96-well plates (Corning 354640) and allowed to adhere overnight at 37°C. Cells were treated the following day via complete media change into standard media supplemented with indicated concentrations of ETV:GCase fusion protein. After 24 hours, media containing ETV:GCase fusion protein was removed and replaced with fresh standard media, and cells were cultured for an additional 48 hours. Cell extracts were then prepared for analysis via LC-MS / MS by removal of media from the wells and rinsing once with IX PBS. 50pL of an extraction buffer (MeOH supplemented with internal standards) was added to each well, and wells were scraped vigorously. The plates were sealed and agitated on a plate shaker for 20 minutes at room temperature, and cellular debris was removed by centrifugation at room temperature. The resulting cleared extract was transferred to a 96-well plate with glass inserts for LC-MS / MS (Analytical Sales 27350). Extracts were dried completely under a stream of nitrogen gas at room temperature before being resuspended in a 200uL volume of assay buffer (92% ACN, 5% IP A, 2.5% H2O, 0.5% FA, 5mM NH4HCO2). Unless otherwise noted, all sample preparation steps were performed on ice or at 4°C. GlcSph and GlcCer were measured using an LC-MS / MS protocol as previously described in Logan et al. 2021. Cell 184: 1-18.

[0353] EXAMPLE 4: Post-serum stress enzymatic activity of ETV:GCase structures

[0354] Because GCase is a lysosomal protein, it normally functions and is stable at an acidic pH. Its liability is that it is unstable and has a short half-life (ti / 2 of a few minutes) at physiological pH (e.g., pH 7.4) in biological fluids, such as serum. Accordingly, ETV:GCase fusion proteins containing engineered GCase amino acid sequences (Example 1) were incubated overnight in human or mouse serum at 37°C, and specific activity was measured to assess the serum stability of the fusion proteins. The specific activity of certain fusion proteins as also assessed after 72 hours of incubation in human or mouse serum at 37°C.

[0355] Results

[0356] ETV:GCase Fusion 1 generated in the presence of isofagomine was used as a comparator. Overnight incubation in serum diminished the specific activity by almost 80% (Table 3). Other ETV:GCase fusion proteins exhibited varying levels of specific activity postserum exposure. For example, ETV:GCase Fusions 8, 12, 18, and 30, generated in the absence of isofagomine (Example 2), exhibited high baseline levels of specific activity which were not sustained after overnight incubation in serum. In contrast, ETV:GCase Fusions 9, 15, 16, 26, 27, 28, 49, 53, 55, 57, 58, 66, and 67, generated in the absence of IFG, exhibited high baseline levels of specific activity that were relatively sustained or even improved after overnight serum exposure. Interestingly, ETV: GCase Fusions 16, 26, and 27 exhibited sustained specific activity after up to 72 hours of serum exposure. In addition, certain fusion proteins which exhibited baseline specific activity within range of or comparable to specific activity of Cerezyme® / imiglucerase (e.g., ETV:GCase Fusions 30-33, 47, 48, 59, and 61-63) illustrated relatively sustained or improved specific activity after overnight serum exposure.

[0357] The results indicate that certain fusion proteins, which exhibited high baseline specific activity without requiring isofagomine for stable expression are also able to withstand serum exposure at physiological temperature and pH without significantly negative impact to baseline specific activity.

[0358] Table 3. Baseline and Post-serum Exposure Specific Activity of ETV:GCase fusion proteins

[0359] ND = not detected

[0360] Analysis of pre- and post-serum exposure activity of ETV:GCase fusion proteins

[0361] The baseline and post-serum exposure enzymatic activity of ETV:GCase fusion proteins was measured using the method described in Example 2. Background subtracted values for each fusion protein were normalized to expression titer measured for the corresponding fusion protein. For serum stress, ETV: GCase fusion proteins were mixed 1 :5 with mouse serum (Rockland, Cat# D108-00-0050, or similar) or human serum (Rockland, Cat. #D119-00-0050) and incubated at 37°C for 18-24 hours or up to 72 hours prior to enzymatic activity measurement.

[0362] EXAMPLE 5: In vivo pharmacokinetics and pharmacodynamics of ETV:GCase fusion proteins

[0363] The pharmacokinetics and pharmacodynamics of ETV:GCase fusion proteins were evaluated in a mouse model with decreased GCase activity (D409V mice) that also carries the human transferrin receptor (TfR) apical domain.

[0364] Results

[0365] In this study, the exposure and pharmacodynamic responses of ETV:GCase fusion proteins (Example 1) were compared relative to each other in the serum, liver, and brain of D409V;TfRmu / huKI mice after a single intravenous (IV) dose of 40 mg / kg. All ETV:GCase fusion proteins administered to the mice exhibited human TfR apical domain binding affinity values ranging from about 400 to 500 nM.

[0366] Serum exposure is illustrated in FIG. 3 A (measurements out to 24 hours in 3 -month old mice) and Fig. 3B (measurements out to 7 days in 8-month old mice). As illustrated in FIG. 3 A, at a single dose of 40 mg / kg, serum exposure of ETV:GCase Fusion 1 was undetectable at 24 hours post-single dose. In contrast, serum concentrations of ETV: GCase Fusions 27 and 30 remained elevated (>100 nM) at 24-hours post single dose. As illustrated in FIG. 3B, in older mice, serum concentrations of ETV:GCase Fusions 30 and 31-33 remained elevated (>10 nM) at 24 hours post-single dose, and were detectable for up to 7 days post-single dose.

[0367] In the liver, exposure results were more varied at 7 days post-single dose. As illustrated in FIG. 4 A, in the younger (3 -month old) mice cohort, ETV:GCase Fusions 1 and 27 exhibited relatively similar concentration levels in the liver (about 6.6 nM and 4.4 nM, respectively), while ETV:GCase Fusion 30 concentration levels were about 5-fold higher (about 31.4 nM). As illustrated in FIG. 4B, in the older (8-month old) mice cohort, ETV:GCase Fusion 30 concentration levels were measured at about 56 nM, while concentration levels of ETV:GCase Fusions 31-33 were about 2-fold higher.

[0368] In the brain, within the younger (3-month old) mice cohort, ETV:GCase Fusion 1 was detected at very low concentration levels (<0.1 nM) at 7 days post-single dose, while ETV:GCase Fusions 27 and 30 were detected at higher concentrations (3.9 nM and 20.3 nM, respectively) (FIG. 5 A). Within the older (8-month old) mice cohort, ETV:GCase Fusions 30-33 were also detected at higher (>7 nM) concentrations (FIG. 5B).

[0369] The results illustrate that the ETV:GCase fusion protein structures containing engineered GCase sequences achieve various levels of improved brain uptake relative to an ETV:GCase structure containing the wild-type GCase sequence (ETV:GCase Fusion 1).

[0370] The pharmacodynamic response of ETV: GCase fusion proteins was assessed by measuring glucosylsphingosine (GlcSph) levels in the serum, liver, and brain of D409V;TfRmu / huKI mice after a single intravenous (IV) dose of 40 mg / kg.

[0371] As illustrated in FIG. 6, in the younger (3-month old) mice cohort, at a single dose of 40 mg / kg, ETV:GCase Fusion 1 produced a serum treatment efficiency of 43% in D409V;TfRmu / huKI mice relative to vehicle-treated mice. In contrast, serum treatment efficiencies of ETV: GCase Fusions 27 and 30 of 63% and 89%, respectively, were observed in D409V;TfRmu / huKI mice relative to vehicle-treated mice. The measured serum treatment efficiencies of ETV:GCase Fusions 30 and 31-33 were even higher in the older (8-month) mice cohort.

[0372] A similar trend in treatment efficiency was observed in the liver tissue of the treated mice. As illustrated in FIG. 7, in the younger (3-month old) mice cohort, at a single dose of 40 mg / kg, ETV:GCase Fusion 1 produced a liver treatment efficiency of 57% in D409V;TfRmu / huKI mice relative to vehicle-treated mice. In contrast, liver treatment efficiencies of ETV: GCase Fusions 27 and 30 of 74% and 95%, respectively, were observed in D409V;TfRmu / huKI mice relative to vehicle-treated mice. The measured liver treatment efficiencies of ETV: GCase Fusions 30 and 31-33 were also high (>94%) in the older (8-month) mice cohort. The same pattern in treatment efficiency was observed in the brain tissue of the treated mice. As illustrated in FIG. 8, in the younger (3 -month old) mice cohort, at a single dose of 40 mg / kg, ETV:GCase Fusion 1 produced a brain treatment efficiency of only 19% in D409V;TfRmu / huKI mice relative to vehicle-treated mice. In contrast, the brain treatment efficiencies of ETV:GCase Fusions 27 and 30 were 69% and 85%, respectively, in D409V;TfRmu / huKI mice relative to vehicle-treated mice. The measured brain treatment efficiencies of ETV:GCase Fusions 30 and 31-33 were also relatively high (>70%) in the older (8-month) mice cohort.

[0373] The results illustrate that the ETV:GCase fusion protein structures containing engineered GCase sequences produce superior pharmacodynamic responses in the brain tissue of D409V;TfRmu / huKI mice relative to an ETV:GCase structure containing the wild-type GCase sequence (ETV:GCase Fusion 1).

[0374] Table 4 summarizes the experimental mouse in vivo results for the ETV:GCase fusion proteins.

[0375] Table 4. 7-day post-single dose results in D409V;TfRmu / huKI mice.

[0376] *Indicates that the mice were 3 months of age. **Indicates that the mice were 8 months of age. Experimental Methods

[0377] The disease mouse model used in this study is a mouse model that harbors a mutation (D409V) that results in significantly diminished GCase activity while also harboring the human TfR apical domain knocked into the murine TfR (referred to herein as “D409V; TfRmu / huKI” or “D409V; TfRmu / hu” mice). TfR”111 11LT< I mice were generated as described in International Patent Publication No. WO 2018 / 152285 using CRISPR / Cas9 technology to express human Tfrc apical domain within the murine Tfrc gene; the resulting chimeric TfR was expressed in vivo under the control of the endogenous promoter. D409V mice were obtained from The Jackson Laboratories / Michael J. Fox Foundation (MJFF) (J AX stock #019106). Briefly, TfRmu / huKI mice were bred to D409V mice to generate homozygous D409V mice in a TfRmu / huKI homozygous background. Mice used in this study were mixed sex and housed under a 12 hour light-dark cycle with ad libitum access to food (#25502, irradiated; LabDiet) and water.

[0378] D409V;TfRmu / huKI mice (3-4 month old or 7-8 month old, n=5 or 6 per group) were administered a single dose of ETV:GCase fusion protein (40 mg / kg) via intravenous injection, and pharmacokinetic and pharmacodynamic responses were assessed. Age-matched littermate TfRmu / huKI mice (non-disease mice) injected i.v. with vehicle were used as controls. In-life serum samples were collected at 15 minutes, 2 hours, and 24 hours post-single dose. All animals were sacrificed 7 days post single dose, at which point liver and brain tissue were collected and flash-frozen on dry ice.

[0379] Total GCase enzyme levels in serum, liver, and brain tissues were measured using an electrochemiluminescence immunoassay (ECLIA). A 384-well MSD Streptavidin-coated plate (Meso Scale Diagnostics L21SA) was blocked with Casein-PBS (ThermoFisher 37528). After subsequent washes, the plate was incubated with a monoclonal anti-GCase antibody. Following subsequent washes, total enzyme in the sample was immobilized via the GCase domain of the molecule. Bound total enzyme was detected by a ruthenylated monoclonal anti-GCase antibody via binding of the GCase domain. 2X Read Buffer T was added to the wells, and the plate was read on an MSD Sector Imager. Chemiluminescent signals that were generated were proportional to the concentration of bound total GCase in the standard curve and sample.

[0380] Serum, liver, and brain tissues were analyzed for GlcSph levels as follows. Briefly, for liver and brain tissues, 20 mg portions of tissue were cut and homogenized in methanol with internal standards using Qiagen TissueLyser for 2 x 30 sec sequences at 25 Hz. Lysates were transferred to individual 1.5-mL protein low-bind tubes and centrifuged at 4°C. The supernatant was transferred to a fresh plate and incubated at -20 °C for 1 hour. Serum samples were directly aliquoted into wells containing methanol with internal standards, incubated with shaking for 5 minutes at room temperature, and subsequently incubated at -20 °C for 1 hour. Afterwards the samples w...

Claims

1. CLAIMSWhat is claimed is:

1. A protein comprising: a. a fusion polypeptide comprising a first Fc polypeptide linked to a glucocerebrosidase (GCase) amino acid sequence; and b. a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45.

2. The protein of claim 1, which has a higher level of enzymatic activity as compared to a corresponding protein comprising a wild-type GCase amino acid sequence.

3. The protein of claim 2, wherein the protein has not been exposed to or contacted with isofagomine and the corresponding protein has been exposed to or contacted with isofagomine.

4. The protein of claim 1, which has an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®).

5. The protein of claim 4, which has not been exposed to or contacted with isofagomine.

6. The protein of any one of claims 1-5, which has at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its baseline level of enzymatic activity in the presence of, or after the fusion protein is contacted with, serum.

7. The protein of claim 6, wherein the serum exposure is at about 4°C.

8. The protein of claim 6, wherein the serum exposure is at about 37°C.

9. The protein of claim 6, wherein the serum exposure is at about 37°C, for about 4 to about 168 hours; about 4 to about 72 hours; about 4 to about 48 hours; about 4 to about 24 hours; or about 4 to about 12 hours.

10. The protein of any one of claims 2-9, wherein the enzymatic activity is measured by a fluorescence-based assay.

11. The protein of claim 10, wherein the enzymatic activity is measured by a fluorescencebased assay, and wherein the assay comprises an in vitro reaction with 4-MUG (4- Methylumbelliferyl P-D-glucopyranoside) substrate.

12. The protein of any one of claims 1-11, wherein at least two modifications of the two or more modifications are at positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451, wherein the positions and modifications are relative to SEQ ID NO:45.

13. The protein of claim 12, wherein the GCase amino acid sequence comprises a modification at three, four, five, six, seven, eight, nine, ten, eleven or twelve positions selected from the group consisting of: 189, 190, 191, 192, 233, 245, 316, 343, 345, 346, 347, 425, and 451.

14. The protein of claim 12, wherein the GCase amino acid sequence comprises a modification at two or three positions selected from the group consisting of: 233, 343, and 347.

15. The protein of claim 14, wherein the GCase amino acid sequence further comprises a modification at position 191 and / or 192.

16. The protein of claim 14 or 15, wherein the GCase amino acid sequence further comprises a modification at one, two, three, four, five, six, or seven positions selected from the group consisting of: 189, 190, 245, 316, 345, 346, 425, and 451.

17. The protein of claim 12, wherein the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten, eleven or twelve modifications selected from the group consisting of:(i) Arg, Glu, or Gin at position 189;(ii) Arg, Asp, Gly, His or Asn at position 190;(iii) Leu, Trp, Met, Tyr or Phe at position 191;(iv) Vai, Asp, Thr, Ala, Glu or He at position 192;(v) Gly at position 233;(vi) Lys at position 245;(vii) Ser at position 316;(viii) Ala, Asn or Thr at position 343;(ix) Phe at position 345;(x) Glu, Asn, Gin or Leu at position 346;(xi) Thr, Ser or Pro at position 347;(xii) Arg at position 425; and(xiii) Arg at position 451.

18. The protein of claim 17, wherein the GCase amino acid sequence comprises two or three modifications selected from the group consisting of:(i) Gly at position 233;(ii) Ala, Asn or Thr at position 343; and(iii) Thr, Ser or Pro at position 347.

19. The protein of claim 18, wherein the GCase amino acid sequence further comprises:(i) Leu, Trp, Met, Tyr or Phe at position 191; and / or(ii) Vai, Asp, Thr, Ala, Glu or He at position 192.

20. The protein of claim 18 or 19, wherein the GCase amino acid sequence further comprises one, two, three, four, five, six, or seven modifications selected from the group consisting of:(i) Arg, Glu, or Gin at position 189;(ii) Arg, Asp, Gly, His or Asn at position 190;(iii) Lys at position 245;(iv) Ser at position 316;(v) Phe at position 345;(vi) Glu, Asn, Gin or Leu at position 346;(vii) Arg at position 425; and(viii) Arg at position 451.

21. The protein of claim 17, wherein the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten or eleven modifications selected from the group consisting of:(i) His or Asn at position 190;(ii) Tyr or Phe at position 191;(iii) Glu or He at position 192;(iv) Gly at position 233;(v) Ser at position 316;(vi) Asn or Thr at position 343;(vii) Phe at position 345;(viii) Gin or Leu at position 346;(ix) Ser or Pro at position 347;(x) Arg at position 425; and(xi) Arg at position 451.

22. The protein of claim 21, wherein the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine, ten or eleven modifications selected from the group consisting of:(i) His at position 190;(ii) Tyr at position 191;(iii) Glu at position 192;(iv) Gly at position 233;(v) Ser at position 316;(vi) Asn at position 343;(vii) Phe at position 345;(viii) Gin at position 346;(ix) Ser at position 347;(x) Arg at position 425; and(xi) Arg at position 451.

23. The protein of claim 22, wherein the GCase amino acid sequence comprises:(i) His at position 190;(ii) Tyr at position 191;(iii) Glu at position 192;(iv) Gly at position 233;(v) Asn at position 343;(vi) Phe at position 345;(vii) Gin at position 346; and(viii) Ser at position 347.

24. The protein of claim 22, wherein the GCase amino acid sequence comprises:(i) His at position 190;(ii) Tyr at position 191;(iii) Glu at position 192;(iv) Gly at position 233;(v) Ser at position 316;(vi) Asn at position 343;(vii) Phe at position 345;(viii) Gin at position 346;(ix) Ser at position 347;(x) Arg at position 425; and(xi) Arg at position 451.

25. The protein of claim 21, wherein the GCase amino acid sequence comprises two, three, four, five, six, seven, eight, nine or ten modifications selected from the group consisting of:(i) Asn at position 190;(ii) Phe at position 191;(iii) He at position 192;(iv) Gly at position 233;(v) Ser at position 316;(vi) Thr at position 343;(vii) Leu at position 346;(viii) Pro at position 347;(ix) Arg at position 425; and(x) Arg at position 451.

26. The protein of claim 25, wherein the GCase amino acid sequence comprises:(i) Phe at position 191;(ii) He at position 192;(iii) Gly at position 233;(iv) Thr at position 343;(v) Leu at position 346; and(vi) Pro at position 347.

27. The protein of claim 25, wherein the GCase amino acid sequence comprises:(i) Phe at position 191;(ii) He at position 192;(iii) Gly at position 233;(iv) Thr at position 343; and(v) Pro at position 347.

28. The protein of claim 25, wherein the GCase amino acid sequence comprises:(i) Gly at position 233;(ii) Thr at position 343; and(iii) Pro at position 347.

29. The protein of claim 25, wherein the GCase amino acid sequence comprises:(i) Asn at position 190;(ii) Phe at position 191;(iii) lie at position 192;(iv) Gly at position 233;(v) Ser at position 316;(vi) Thr at position 343;(vii) Leu at position 346;(viii) Pro at position 347;(ix) Arg at position 425; and(x) Arg at position 451.

30. The protein of any one of claims 1-30, wherein the GCase amino acid sequence further comprises a modification at position 94, wherein the modification and position are relative to SEQ ID NO: 45.

31. The protein of any one of claims 1-13, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 47, wherein:(i) Xi is selected from the group consisting of: G, E, R, and Q;(ii) X2 is selected from the group consisting of: A, N, H, D, R, and G;(iii) X3 is selected from the group consisting of: Y, F, L, M, V, and W;(iv) X4 is selected from the group consisting of: V, I, T, A, E, N and D;(v) X5 is G;(vi) Xe is P or K;(vii) X7 is S or F;(viii) Xs is selected from the group consisting of: T, A, and N;(ix) X9 is selected from the group consisting of: F and S;(x) X10 is selected from the group consisting of: K, L, Q, N, and E;(xi) Xu is selected from the group consisting of: P, S, and T;(xii) X12 is R or K; and(xiii) X13 is R or H.

32. The protein of claim 31, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 47.

33. The protein of claim 31, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 96% identity to SEQ ID NO: 47.

34. The protein of claim 31, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 97% identity to SEQ ID NO: 47.

35. The protein of claim 31, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 47.

36. The protein of claim 31, wherein the GCase amino acid sequence comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 47.

37. The protein of claim 31, wherein the GCase amino acid sequence comprises SEQ ID NO: 47.

38. The protein of any one of claims 31-37, wherein:(i) Xi is G;(ii) X2 is A, N, or H;(iii) X3is V, Y or F;(iv) X4 is N, E or I;(v) X5 is G;(vi) Xe is P;(vii) X7 is S or F;(viii) Xs is N or T;(ix) X9 is F or S;(x) X10 is: K, L, or Q;(xi) Xu is P or S;(xii) X12 is R or K; and(xiii) X13 is R or H.

39. The protein of any one of claims 31-37, wherein:(i) Xi is G;(ii) X2is H;(iii) X3 is Y;(iv) X4 is E;(v) X5 is G;(vi) Xe is P;(vii) X7 is S or F;(viii) Xs is N;(ix) X9 is F;(x) X10 is Q;(xi) Xu is S;(xii) X12 is R or K; and(xiii) X13 is R or H.

40. The protein of any one of claims 31-37, wherein:(i) Xi is G;(ii) X2 is A, or N;(iii) X3 is F or V;(iv) X4 is I or N;(v) X5 is G;(vi) Xe is P;(vii) X7 is S or F;(viii) Xs is T;(ix) X9 is S;(x) X10 is K, or L;(xi) Xu is P;(xii) X12 is R or K; and(xiii) X13 is R or H.

41. The protein of any one of claims 31-36, wherein position 94 comprises a modification relative to SEQ ID NO: 47 (e.g., comprises Glu at position 94).

42. The protein of any one of claims 1-41, wherein the second Fc polypeptide comprises at the following positions, according to EU numbering:(i) Thr at position 386;(ii) Glu at position 387;(iii) Trp at position 388;(vi) Glu at position 416; and(v) Phe at position 421.

43. The protein of claim 42, wherein the second Fc polypeptide further comprises one or more of the specified residues as follows, wherein the positions are according to EU numbering:(i) Glu, Trp, or Leu at position 380;(ii) Phe or Tyr at position 384;(iii) Ser, Ala, Vai or Asn at position 389;(iv) Asn or Ser at position 390;(v) Thr or Ser at position 413; and(vi) Glu or Ser at position 415.

44. The protein of any one of claims 1-41, wherein the second Fc polypeptide comprises at the following positions, according to EU numbering:(i) Glu, Trp, or Leu at position 380;(ii) Phe or Tyr at position 384;(iii) Thr at position 386;(iv) Glu at position 387;(v) Trp at position 388;(vi) Ser, Ala, Vai or Asn at position 389;(vii) Asn or Ser at position 390;(viii) Thr or Ser at position 413;(ix) Glu or Ser at position 415;(x) Glu at position 416; and(xi) Phe at position 421.

45. The protein of claim 44, wherein the second Fc polypeptide comprises at the following positions, according to EU numbering:(i) Glu, Trp, or Leu at position 380;(ii) Tyr at position 384;(iii) Thr at position 386;(iv) Glu at position 387;(v) Trp at position 388;(vi) Ser or Ala at position 389;(vii) Asn or Ser at position 390;(viii) Thr at position 413;(ix) Glu at position 415;(x) Glu at position 416; and(xi) Phe at position 421.

46. The protein of claim 45, wherein the second Fc polypeptide comprises at the following positions, according to EU numbering:(i) Glu at position 380;(ii) Tyr at position 384;(iii) Thr at position 386;(iv) Glu at position 387;(v) Trp at position 388;(vi) Ala at position 389;(vii) Asn at position 390;(viii) Thr at position 413;(ix) Glu at position 415;(x) Glu at position 416; and(xi) Phe at position 421.

47. The protein of any one of claims 1-46, wherein the first Fc polypeptide is linked to the GCase amino acid sequence by a peptide bond or by a polypeptide linker.

48. The protein of claim 47, wherein the first Fc polypeptide is linked to the GCase amino acid sequence by a polypeptide linker, and wherein the polypeptide linker is a flexible polypeptide linker.

49. The protein of claim 48, wherein the flexible polypeptide linker is a glycine-rich linker.

50. The protein of claim 49, wherein the polypeptide linker is GS, G4S (SEQ ID NO: 152) or (G4S)2(SEQ ID NO: 151).

51. The protein of any one of claims 1-50, wherein the N-terminus of the first Fc polypeptide is linked to the GCase amino acid sequence.

52. The protein of any one of claims 1-51, which comprises a single GCase amino acid sequence.

53. The protein of any one of claims 1-52, wherein the second Fc polypeptide forms an Fc dimer with the first Fc polypeptide.

54. The protein of any one of claims 1-53, wherein the first Fc polypeptide and the second Fc polypeptide each contain modifications that promote heterodimerization.

55. The protein of claim 54, wherein one of the Fc polypeptides has a T366W substitution and the other Fc polypeptide has T366S, L368A, and Y407V substitutions, according to EU numbering.

56. The protein of claim 55, wherein the first Fc polypeptide contains the T366S, L368A, and Y407V substitutions and the second Fc polypeptide contains the T366W substitution.

57. The protein of claim 56, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 9-16 and 21-24; and the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 29-36 and 41-44.

58. The protein of any one of claims 1-57, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises a native FcRn binding site.

59. The protein of any one of claims 1-58, wherein the first Fc polypeptide and the second Fc polypeptide do not have effector function.

60. The protein of any one of claims 1-58, wherein the first Fc polypeptide and / or the second Fc polypeptide includes a modification that reduces effector function.

61. The protein of claim 60, wherein the modification that reduces effector function is the substitutions of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329, according to EU numbering.

62. The protein of claim 61, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11-16, and 21-24.

63. The protein of claim 62, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 11, 12, 21, and 22.

64. The protein of claim 62, wherein the first Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 15, 16, 23, and 24.

65. The protein of claim 61, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78- 105, 108-111, 120-121, 126-135, and 144-149.

66. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 94 or 95.

67. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 98 or 99.

68. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 100 or 101.

69. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 102 or 103.

70. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 104 or 105.

71. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 134 or 135.

72. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 146 or 147.

73. The protein of claim 65, wherein the fusion polypeptide comprises an amino acid sequence having at least 95% or 100% identity to SEQ ID NO: 148 or 149.

74. The protein of any one of claims 61-73, wherein the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31-36 and 41-44.

75. The protein of claim 74, wherein the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 31, 32, 41 and 42.

76. The protein of claim 74, wherein the second Fc polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs:35, 36, 43 and 44.

77. The protein of claim 1, wherein the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 64-67, 72-75, 78-105, 108-111, 120-121, 126-135, and 144-149; and wherein the second Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 31-36 and 41-44.

78. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 94 or 95; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

79. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 98 or 99; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

80. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 100 or 101; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

81. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 102 or 103; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

82. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 104 or 105; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

83. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 134 or 135; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

84. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 146 or 147; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

85. The protein of claim 77, wherein the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 148 or 149; and wherein the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or 44.

86. The protein of any one of claims 1-85, wherein the first Fc polypeptide is not modified to bind to a blood-brain barrier (BBB) receptor and the second Fc polypeptide is modified to specifically bind to a TfR.

87. The protein of any one of claims 1-86, wherein the protein binds to a TfR with an affinity of from about 100 nM to about 500 nM.

88. The protein of any one of claims 1-87, wherein the second Fc polypeptide binds to the apical domain of the TfR.

89. The protein of any one of claims 1-88, wherein the binding of the protein to the TfR does not substantially inhibit binding of transferrin to the TfR.

90. The protein of any one of claims 1-89, wherein the protein does not include an immunoglobulin heavy and / or light chain variable region sequence or an antigen-binding portion thereof.

91. A fusion polypeptide comprising an Fc polypeptide that is linked to a glucocerebrosidase (GCase) amino acid sequence, wherein the Fc polypeptide comprises a sequence having at least 90% identity to SEQ ID NO: 12 and contains one or more modifications that promote its heterodimerization to another Fc polypeptide, and wherein the GCase amino acid sequence comprises two or more modifications as compared to SEQ ID NO:45.

92. The fusion polypeptide of claim 91, wherein the Fc polypeptide is linked to the GCase amino acid sequence by a peptide bond or by a polypeptide linker.

93. The fusion polypeptide of claim 92, which comprises from N- to C-terminus: the GCase amino acid sequence; a polypeptide linker; and the Fc polypeptide.

94. The fusion polypeptide of any one of claims 91-93, wherein the Fc polypeptide comprises T366S, L368A, and Y407V substitutions, according to EU numbering.

95. The fusion polypeptide of any one of claims 91-94, wherein the Fc polypeptide comprises substitutions of Ala at position 234 and Ala at position 235; Ala at position 234, Ala at position 235 and Gly at position 329; or Ala at position 234, Ala at position 235 and Ser at position 329, according to EU numbering.

96. The fusion polypeptide of claim 95, wherein the polypeptide comprises an amino acid sequence having at least 95% or 100% identity to any one of SEQ ID NOs: 64-67, 72-75, 78- 105, 108-111, 120-121, 126-135, and 144-149.

97. A protein comprising the polypeptide of any one of claims 91-96 and the other Fc polypeptide of claim 91.

98. A pharmaceutical composition comprising the protein or polypeptide of any one of claims 1-97 and a pharmaceutically acceptable excipient.

99. A polynucleotide comprising a nucleic acid sequence encoding the fusion polypeptide of any one of claims 91-96.

100. A vector comprising the polynucleotide of claim 99.

101. A host cell comprising the polynucleotide of claim 99 or the vector of claim 100.

102. The host cell of claim 101, further comprising a polynucleotide comprising a nucleic acid sequence encoding the other Fc polypeptide of claim 91.

103. A pair of polynucleotides comprising nucleic acid sequences encoding the fusion polypeptide and the second Fc polypeptide of any one of claims 1-90.

104. One or more vectors comprising the pair of polynucleotides of claim 103.

105. A host cell comprising the pair of polynucleotides of claim 103.

106. A method for producing a polypeptide, comprising culturing the host cell of claim 101 under conditions in which the polynucleotide is expressed.

107. A method for producing a protein, comprising culturing the host cell of claim 105 under conditions in which the pair of polynucleotides is expressed.

108. The method of claim 106 or 107, wherein the host cell is cultured in the absence of isofagomine during protein production.

109. A protein produced by the method of claim 108.

110. A protein produced by cell expression in the absence of isofagomine, wherein the protein comprises: a. a fusion polypeptide comprising a first Fc polypeptide linked to a modified glucocerebrosidase (GCase) amino acid sequence; andb. a second Fc polypeptide that comprises a sequence having at least 90% identity to SEQ ID NO: 42 and that is capable of specifically binding to a transferrin receptor (TfR); and wherein the protein has 1) a higher level of enzymatic activity as compared to a corresponding protein comprising a wild-type GCase amino acid sequence; and / or 2) an equivalent or a higher level of enzymatic activity as compared to imiglucerase (CEREZYME®).

111. A method of treating a GCase deficiency in a patient in need thereof, the method comprising administering the protein of any one of claims 1-90 to the patient.

112. The protein as described in any one of claims 1-90 for use in treating a GCase deficiency in a patient in need thereof.

113. The use of a protein as described in any one of claims 1-90 in the preparation of a medicament for treating a GCase deficiency in a patient in need thereof.

114. A method of decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency, the method comprising administering a protein as described in any one of claims 1-90 to the patient.

115. A protein as described in any one of claims 1-90 for use in decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency.

116. The use of a protein as described in any one of claims 1-90 in the preparation of a medicament for decreasing the accumulation of a toxic metabolic product in a patient having a GCase deficiency.

117. The method, protein, or use of any one of claims 114-116, wherein the toxic metabolic product comprises glucosylceramide or glucosylsphingosine.

Images