WO2025181155A1 - Human beta-glucocerebrosidase binders and uses thereof - Google Patents

Abstract

This application relates to polypeptide agents and compositions specifically binding human beta-glucocerebrosidase (GCase, GBA1). Particularly, immunoglobulin single variable domains (ISVDs) are described which allosterically bind human GCase, thereby positively modulating GCase in its stability and/or catalytic activity. The invention relates further to vectors and nucleic acids encoding such ISVD-based modulators. Also encompassed are compositions, in particular pharmaceutical compositions, containing such ISVD modulators. The GCase-specific allosteric modulators, specifically the ISVDs and compositions described herein, may be used for prevention and/or treatment of GCase-related diseases, including Gaucher disease (GD) and Parkinson's disease (PD).

Description

HUMAN BETA-GLUCOCEREBROSIDASE BINDERS AND USES THEREOF

FIELD OF THE INVENTION

This application relates to polypeptide agents and compositions specifically binding human beta- glucocerebrosidase (GCase, GBA1). Particularly, immunoglobulin single variable domains (ISVDs) are described which allosterically bind human GCase, thereby positively modulating GCase in its stability and/or catalytic activity. The invention relates further to vectors and nucleic acids encoding such ISVD- based modulators. Also encompassed are compositions, in particular pharmaceutical compositions, containing such ISVD modulators. The GCase-specific allosteric modulators, specifically the ISVDs and compositions described herein, may be used for prevention and/or treatment of GCase-related diseases, including Gaucher disease (GD) and Parkinson's disease (PD).

BACKGROUND

Beta-glucocerebrosidase or beta-glucosidase (GCase, GBA1) is a 497-amino-acid membrane-associated protein with a 39-amino acid leader sequence and five glycosylation sites, classified in the glycoside hydrolase family GH30¹-². It contains three non-contiguous domains: a small three-stranded antiparallel P-sheet, an eight-stranded p-barrel and an (a/P)g TIM barrel catalytic domain³-⁴. GCase is a lysosomal enzyme that primarily cleaves the membrane lipid glucosylceramide (GlcCer) into glucose and ceramide, and glucosylsphingosine (GlcSph) into glucose and sphingosine⁵-⁶. Notably, GlcCer can be degraded by three different hydrolases: the lysosomal GBA1 and the non-lysosomal GBA2 and GBA3^7-9. However, despite the shared enzymatic activity, the three GBA enzymes show no structural or sequence homology¹⁰. Moreover, GCase catalyzes reversible transfer of a glucose moiety (transglucosylation) between glucosylated cholesterol (GlcChol) and GlcCer¹¹. Transglucosylation occurs during cholesterol accumulation in the lysosomes, for instance in Niemann-Pick disease type C¹¹. Additionally, GCase has the capability to break down synthetic p-xylosides¹².

The lysosomal GCase undergoes folding and processing in the endoplasmic reticulum (ER) and Golgi with the assistance of chaperone proteins, including calnexin and ca I reticu I i n¹³-¹⁴. Glycosylation plays a crucial role in the maturation of the catalytically active enzyme, particularly at the first site located at Asnl9¹⁵. When GCase is misfolded, it undergoes re-glycosylation to facilitate the refolding by chaperones. If correct folding is not possible (e.g., due to genetic mutations), immature GCase is retained within the ER by the quality control mechanisms, and eventually undergoes degradation by the VCP/26S proteasomal pathway¹⁶-¹⁷. The importance of glycosylation site occupancy for GCase activity is highlighted by the notion that all putative glycosylation sites are conserved between the human and murine enzyme¹⁸.

Biallelic mutations in the GBA1 gene cause Gaucher's disease (GD), the most common lysosomal storage disorder (LSD) affecting up to 1 in 60,000 live births¹⁹-²⁰. Over 495 GBA1 gene mutations leading to the production of defective GCase have been identified and linked to the disorder²¹. Mutations occurring within the p-structure, often referred to as the folding domain, typically hinder proper folding within the ER, leading to GCase degradation²². The best characterized example is the L444P substitution, which leads to diminished quantities of GCase within the lysosomes, despite its normal catalytic activity²³. Conversely, the N370S substitution (the most prevalent GCase mutation in Caucasians) leads to nearnormal levels of GCase within the lysosomes²⁴. However, the mutant enzyme displays abnormalities in its catalytic functionality both in vitro and in vivo²²'²⁵. Notably, the N370S mutation alters the capacity of the enzyme to interact with its physiological activators, including anionic phospholipids and saposin c (sap C)-containing membranes. Moreover, N370S Gaucher fibroblasts are characterized by excessive intracellular cholesterol accumulation²⁶.

Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder following Alzheimer's disease. Its occurrence in the general population amounts to 0.3%, rising to 1% among individuals aged over 60, and escalating further to 3% among those aged over 80²⁷. Interestingly, individuals carrying a homozygous mutation of GBA1, and thus affected by GD, face an increased risk of developing PD and typically experience an earlier onset of PD symptoms²⁸. There is evidence suggesting that mutant alleles linked to more severe forms of GD also carry a greater associated risk of parkinsonism²⁹. Moreover, the most common genetic abnormality associated with PD is a heterozygous GBA1 gene mutation (present in 5-8% of sporadic PD patients)³⁰. Clinically, patients carrying GBA1 mutations commonly exhibit an earlier onset of PD, associated with a more rapid advancement of motor impairment and cognitive deterioration, and demonstrate diminished survival rates³¹. This observation strongly implies that GCase plays a pivotal role in the pathophysiology of PD.

Both PD and GD involve the accumulation of a-synuclein (a-syn) in neurons³²-³³. A-syn is a presynaptic neuronal protein whose aggregation in Lewy bodies and Lewy neurites is considered the hallmark of PD's pathophysiology³⁴. Indeed, elevated levels of a-syn were observed in human dopaminergic midbrain neurons derived from induced pluripotent stem cells (iPSC) of patients with both GD and PD³⁵. Additionally, human midbrain organoids generated from embryonic stem cells lacking GBA1 expression also displayed increased levels of a-syn³⁶. Furthermore, elevated a-syn levels were detected in iPSC- derived neurons following the treatment with a GCase inhibitor conduritol-p-epoxide (CBE), as well as in iPSC-derived neurons from individuals with GBA1-PD^37-39. These studies suggest that even heterozygous GBA1 variants might contribute to an increased susceptibility to PD by influencing alterations in a-syn clearance. In this context, increasing GCase activity in the central nervous system (through restoration or overexpression) results in the removal of a-syn in both in vitro and in vivo models of GD and PD^40-42.

Overall, the primary mechanisms implicated in GCase-related toxicity in GD and PD patients involve: i. Aberrant trafficking of the mutant GCase protein, leading to its impaired folding and stability. This disruption promotes ER stress and damage⁴³-⁴⁴; ii. Diminished GCase activity, resulting in the buildup of GCase substrates within the lysosomes, further contributing to lysosomal dysfunction⁴⁵.

Currently, two treatments types are available for GD: enzyme replacement therapy (ERT) and substrate replacement therapy (SRT). ERT involves the use of recombinant GCase to lower GlcCer levels, effectively managing visceral and hematological complications in GD patients. It has demonstrated significant effectiveness in alleviating organ enlargement, anemia and skeletal manifestations⁴⁶. However, recombinant GCase cannot penetrate the blood-brain barrier, rendering it ineffective in reversing neurological impairments in patients with GD type II (GD2) and GD type III (GD3)⁴⁷ (further characterized in the Description of this application). Additionally, approaches based on introducing wild-type (WT) GCase are considered suboptimal because they do not eliminate the misfolded or aberrant protein from tissues. On the other hand, SRT functions by reducing GCase substrate levels, aiming to counteract the buildup of glycolipids. This approach seeks to restore a better balance in the activity of the deficient enzyme⁴⁸.

Several alternative treatment strategies targeting GCase in both PD and GD have been proposed. These strategies primarily aim to stabilize and/or activate the protein, or enhance its trafficking to the lysosomes. Initially, such approaches involved iminosugar-based GCase inhibitors, e.g., isofagomine (afegostat), which acted as pharmacological GCase chaperones binding to its active site⁴⁹. Subsequently, several non-inhibitory small molecules have been evaluated at various stages of pre-clinical or clinical development (e.g., Ambroxol, JZ-4109, NCGC607, BIA-28-6156/LTI-291)³⁵-⁵⁰-⁵².

Despite the array of various approaches, stabilizing mutant GCase and enhancing its trafficking and/or activity in patients still poses a significant challenge. Hence, there is a pressing need and considerable anticipation for new therapeutic modalities in this field. Specifically, drugs enhancing the stability of lysosomal GCase without impeding its enzymatic activity - such as by binding away from the GCase catalytic site - could prove particularly beneficial in the treatment of GD and/or PD. SUMMARY OF THE INVENTION

The present invention provides for novel molecules that bind human beta-glucocerebrosidase (GCase, GBA1) to support or improve its catalytic activity. This approach bypasses the effects of loss-of-function mutations in the GBA1 gene, which typically result in the accumulation of the GCase substrate glucosylceramide (GlcCer) within cells. Thus, the molecules disclosed in this invention may counteract the pathologies associated with mutated GCase, for instance Gaucher disease (GD) and/or Parkinson's disease (PD).

One aspect of this invention relates to modulator polypeptides that specifically bind GCase in an allosteric manner. Specifically, the modulator polypeptide may be an antibody, active antibody fragment, immunoglobulin single variable domain (ISVD), single domain antibody, or a VHH antibody (nanobody).

According to another aspect, the allosteric modulator polypeptide exhibits GCase-stabilizing properties by interacting with specific residues of the GCase enzyme (referred to herein as the protein defined in SEQ ID NO:81), said residues covering a novel binding site spanning GCase domain II and domain III. In a preferred embodiment, said allosteric modulator polypeptide specifically binds an epitope defined by GCase residues 77, 452 and 453 of domain II, and residues 78, 79, 162, 165, 166, 168, 169, 170, 171, 172, 173, 174, 221, 224, 225, 226, 227, 228, 272, 274, 275, Til , 306, 429 and 430 of domain III, wherein the residues are numbered as present in the GCase wild type sequence of SEQ ID NO: 81. Alternatively, the binding site may comprise the corresponding residues of a GCase homologue and/or mutant variant, such as a pathological mutant or a GCase mutant known to be less stable as compared to the WT GCase, or a mutant causative of GCase misfolding.

In further embodiments, the GCase-specific allosteric modulator of the invention comprises an ISVD comprising or consisting of an amino acid sequence that includes 4 framework regions (FR) and 3 CDRs, according to the formula: FR1-CDR1-FR2-CDR2-FR3-CDR-FR4, or any suitable fragment thereof.

In one embodiment, the allosteric modulator comprises or consists of an ISVD comprising the complementarity-determining-regions (CDRs) of the VHH molecules as present in SEQ ID NO: 1, 4, or 9, wherein the CDRs are annotated according to the annotation described herein, and as used in Table 1, i.e.,:

- the allosteric modulator defined by SEQ ID NO: 1 consists of CDR1 comprising SEQ ID NO: 21, and consists of CDR2 comprising SEQ ID NO: 22, and consists of CDR3 comprising SEQ ID NO: 23;

- the allosteric modulator defined by SEQ ID NO: 4 consists of CDR1 comprising SEQ ID NO: 30, and consists of CDR2 comprising SEQ ID NO: 31, and consists of CDR3 comprising SEQ ID NO: 32; - the allosteric modulator defined by SEQ ID NO: 9 consists of CDR1 comprising SEQ ID NO: 45, and consists of CDR2 comprising SEQ ID NO: 46, and consists of CDR3 comprising SEQ ID NO: 47;

- the allosteric modulator defined by SEQ ID NO: 97 consists of CDR1 comprising SEQ ID NO: 98, and consists of CDR2 comprising SEQ ID NO: 99, and consists of CDR3 comprising SEQ ID NO: 100; or according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering systems, as known in the art and as further referred to herein. Possibly, any one of said ISVDs contains a functionally modified version of any of those sequences, exhibiting at least 90% amino acid identity across the entire ISVD sequence, with non-identical amino acids potentially found in one or more framework residues, though with identical CDR sequences as provided in SEQ ID NO: 1, 4 or 9. It also encompasses a humanized variant derived from any of these sequences, as further described herein.

The present invention also encompasses allosteric polypeptide modulators targeting GCase, which, upon binding, elevate the catalytic activity of GCase. In a preferred embodiment, such polypeptides comprise or consist of ISVDs comprising the CDRs of SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16, 18, 19, 113, 117, 121, or 125, wherein the CDRs are annotated according to the annotation described herein, and as used in Table 1, or according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering systems, as known in the art and as further referred to herein.

In some embodiments, the allosteric polypeptide modulator targeting GCase comprises or consists of an ISVD wherein CDR1 may be selected from the group consisting of: i. SEQ ID NOs: 21, 24, 27 , 30, 33, 36, 42, 45, 48, 57, 66, 72, 75, 114, 118, 122, 126, or ii. CDR1 fragments of Nbs with the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16, 18, 19, 113, 117, 121, or 125 according to the annotation by MacCallum, AbM, Chothia, Kabat or IMGT numbering system; accordingly, wherein CDR2 may be selected from the group consisting of: i. SEQ ID NOs: 22, 25, 28, 31, 34, 37, 43, 46, 49, 58, 67, 73, 76, 115, 119, 123, 127, or ii. CDR2 fragments of Nbs with the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16, 18, 19, 113, 117, 121, or 125 according to the annotation by MacCallum, AbM, Chothia, Kabat or IMGT numbering system; following, wherein CDR3 may be selected from the group consisting of: i. SEQ ID NOs: 23, 26, 29, 32, 35, 38, 44, 47, 50, 59, 68, 74, 77, 116, 120, 124, 128, or ii. CDR3 fragments of Nbs with the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16, 18, 19, 113, 117, 121, or 125 according to the annotation by MacCallum, AbM, Chothia, Kabat or IMGT numbering system. According to another aspect, the GCase-specific allosteric modulator detailed here can be fused with a moiety. In one embodiment, the moiety is a targeting moiety, preferably one that specifically targets the endoplasmic reticulum (ER) or lysosome. In another embodiment, the moiety is a functional moiety, preferably a therapeutic moiety, a half-life-extending moiety, or a blood-brain barrier-crossing moiety.

The invention further pertains to allosteric GCase modulators which are multivalent and/or multispecific, and preferably contain at least two GCase-binding moieties. In the most preferred embodiment, these multivalent and/or multispecific modulators comprise or consist of a minimum of two GCase-specific ISVDs, which may be different or identical in sequence.

This application also relates to a nucleic acid molecule containing a polynucleotide sequence that encodes any one the allosteric modulator polypeptides described herewith, along with a vector that contains this nucleic acid molecule.

Also envisaged is a pharmaceutical composition comprising any one of the GCase-specific allosteric modulators according to this invention, or the nucleic acid molecule or the vector encoding any one of said modulators, as described herewith.

Furthermore, the invention relates to the above-described GCase-binding allosteric modulators, pharmaceutical composition(s), nucleic acid(s) and/or vector(s), for use as a medicine, for instance in the prevention or treatment of GCase-related disorders. One specific embodiment involves utilizing the GCase-binding allosteric modulator(s), pharmaceutical composition(s), nucleic acid(s), and/or vector(s) detailed herein as a therapeutic or preventive measure for GD and/or PD.

Objects of the present invention are presented in more detail in the following Description and Examples.

DESCRIPTION OF THE FIGURES

The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn in scale for illustrative purposes.

Figure 1. Overview of the selection and purification of a set of 20 nanobodies generated and characterized herein.

A. Selection conditions for each Nb.

B. Purification of the 20 Nbs and purity analysis using SDS-PAGE. Figure 2. Binding of the set of 20 nanobodies to wild-type and N370S GCase.

A. ELISA of 20 purified Nbs with GCase or N370S GCase coated onto the bottom of the ELISA wells. An irrelevant Nb was used as negative control, while the positive control involved the signal of Nbl7 directly coated onto the ELISA plate.

B. Representative bio-layer interferometry (BLI) traces of Nbl binding to wild-type GCase (upper panel) and fitting of the signal amplitudes (Req) on the Langmuir equation to determine the K_D values (lower panel).

C. Equilibrium dissociation constant (K_D) values of the 20 Nbs binding with GCase or N370S GCase. The values were determined by fitting of the signal amplitudes (Req) on the Langmuir equation.

Figure 3. 4-MU in vitro GCase activity assay in the presence of various nanobodies.

A. Schematic representation of the experimental procedures of the in vitro 4-MU GCase activity assay.

B. Velaglucerase activity assay in the presence of different Nbs revealed that a specific subset of Nbs significantly enhances in vitro GCase enzymatic activity. Isofagomine (IFG) was used as a negative control. n=6; data are represented as mean ±SEM; statistical analysis was performed using the One-Way Anova multiple comparison test; * p<0.05, ** p<0.01, *** pcO.001, **** pcO.0001

Figure 4. Impact of various nanobodies on the GCase thermal stability (T_m).

A. Thermal unfolding curves of GCase, obtained using the thermal shift assay at pH=7.0 (darker line) and pH=5.2 (lighter line) in the absence or presence of Nbl.

B. Overview of the outcomes of the thermal stability assay performed with the full set of 20 Nbs. The plotted variation of T_m (AT_m) signifies the difference between the T_m of GCase alone and GCase with a Nb. The covalent GCase inhibitor conduritol-p-epoxide (CBE) was used as positive control, and an irrelevant nanobody (Irr Nb) was used as a negative control.

Figure 5. Epitope mapping of selected nanobodies.

A. Illustration of the experimental design. Biotinylated Nbs were loaded on a streptavidin-coated sensor and then dipped into a solution of GCase in the presence or absence of various nonbiotinylated Nbs.

B. Results of the epitope mapping as outlined in panel 'A'. Blue curves correspond to the binding signal with GCase protein alone, and red curves correspond to the signal obtained for the same sensor dipped into a GCase-Nb solution. Figure 6. Comparison of the conformational changes between GCase bound to Nbl and unbound GCase (chain A derived from PDB 1OGS).

A. Superposition of GCase (darker color) from the GCase-Nbl complex on unbound GCase (lighter color). Nbl is indicated.

B. Close-up view of loopl (upper panel), Ioop2 (middle panel) and Ioop3 (lower panel). The arrow marks the active site, and the most important residues are indicated.

Figure 7. ER- and lysosome-targeted nanobodies show differential expression levels in the two compartments.

A. A representative western blot membrane of HEK293T cells transfected with a mock vector or five selected Nbs targeted to the ER, compared to untransfected (control) cells.

B. The relative quantification of the flag intensity band, representing the Nbs specifically targeted to the ER, when normalized to p-actin, demonstrated a variable yet consistent expression of all the distinct Nbs within this cellular compartment. n=9-ll; data are represented as mean ±SEM; statistical analysis was performed using the One- Way Anova multiple comparison test; ns = nonsignificant, * p<0.05

C. A representative western blot membrane of HEK293T cells transfected with a mock vector or five selected Nbs targeted to the lysosomes, compared to untransfected (control) cells.

D. The relative quantification of the flag intensity bands corresponding to the Nbs specifically targeted to the lysosomes, when normalized to p-actin, revealed a considerable variation in the expression levels among the different Nbs. Nb4 and Nb9 exhibited a notably higher expression compared to the other Nbs. n=ll; data are represented as mean ±SEM; statistical analysis was performed using the One- Way Anova multiple comparison test; ns = nonsignificant, **** p<0.0001

E. Representative confocal images of HEK293T cells transfected with Nbl-flag-IRES-EGFP targeted to the ER, stained for flag (yellow) and calnexin (magenta). EGFP was used as a marker for Nb- flag-expressing cells (scale bar: 10 pm).

F. Colocalization of ER-targeted Nbs in the ER in HEK293T cells overexpressing the selected Nbs was evaluated by calculating the Pearson's coefficient using the JACoP plugin of ImageJ. The results indicate that all the Nbs exhibit a certain level of colocalization with the ER-marker calnexin. However, a notable decrease in colocalization was observed for Nb4, Nb9, NblO, and Nbl6 compared to the mock transfection. n=4-28 cells in two independent experiments; data are represented as a violin plot and median values; statistical analysis was performed using the One-Way Anova multiple comparison test; ns = nonsignificant, ** pcO.Ol, **** pcO.OOOl

G. Representative confocal images of HEK293T cells transfected with Nb9-flag-IRES-EGFP targeted to the ER stained for flag (yellow) and LAMP2A (magenta). EGFP was used as a marker for Nb- flag-expressing cells (scale bar: 10 pm).

H. Localization of ER-targeted Nbs to the lysosomes in HEK293T cells overexpressing the selected Nbs was assessed by calculating the Pearson's coefficient using the JACoP plugin of ImageJ, based on colocalization between flag and LAMP2A staining. The results indicate that all Nbs exhibit some degree of colocalization with the lysosomal marker, with Nb9 showing a significantly higher colocalization compared to the Mock transfection. n=26-35 cells from 2 independent experiments; data are represented as violin plots with median values; statistical analysis was performed using the Shapiro-Wilk test for normality, followed by the Kruskal-Wallis test with multiple comparisons.

Figure 8. Expression of nanobodies has no impact on the ER or lysosome.

A. Representative western blot membranes showing GCase and calnexin levels in HEK293 cells overexpressing the selected ER-Nbs.

B. Representative western blot membranes showing GCase and LAMP1 levels in HEK293 cells overexpressing the selected lysosome-Nbs.

C. The quantification of calnexin expression levels using densitometry analysis revealed no discernible differences among control cells, mock-transfected cells, and cells expressing the Nbs. n=6-8; statistical analysis was performed using the One-Way Anova test with multiple comparisons

D. The densitometry analysis quantifying LAMP1 expression levels revealed no noticeable differences among control cells, mock-transfected cells, and cells expressing the Nbs. n=6-8; statistical analysis was performed using the One-Way Anova test with multiple comparisons.

Figure 9. Expression of nanobodies has no impact on GCase levels, while all lysosome-targeted Nbs ameliorate GCase activity in vitro.

A. Quantification of GCase expression levels in cells expressing the ER-targeted Nbs revealed no discernible differences compared to control cells. n=9-ll; One-Way Anova was used for statistical analysis B. 4-MU GCase enzymatic activity assay performed on lysates obtained from cells expressing the ER-targeted Nbs revealed no changes compared to controls for any of the selected Nbs. n=16; One-Way Anova was used for statistical analysis; ns = non-significant

C. Quantification of GCase expression levels in cells expressing the lysosome-targeted Nbs showed no differences compared to control cells. n=9-ll; One-Way Anova was used for statistical analysis

D. 4-MU GCase enzymatic activity assay performed on lysates obtained from cells expressing the lysosome-targeted Nbs showed increased GCase activity compared to controls for all of the selected Nbs. n=14-16; One-Way Anova was used for statistical analysis; ns = non significant; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001

Figure 10. Lysosomal GCase activity in HEK293T cells overexpressing ER- or lysosome-targeted nanobodies.

A. Flow cytometry-based lysosomal GCase activity assay conducted on live wild-type HEK293 cells expressing lysosome-targeted Nbl, Nb4, or Nb9. PFB-FDGIu was used as a substrate.

B. Flow cytometry-based lysosomal GCase activity assay conducted on live wild-type HEK293 cells expressing ER-targeted Nbl, Nb4, or Nb9. PFB-FDGIu was used as a substrate. n=5-6; data represented as mean ±SEM; One-Way Anova with multiple comparisons was used for statistical analysis; ns = non significant; * p<0.05, ** p<0.01

Figure 11. ER-Nbl and ER-Nb9 increase GCase trafficking to the lysosomes.

A. Representative western blot of lysates from Nb-transfected HEK293T cells treated with ENDO H and PNGase F to distinguish between ENDO H-resistant bands (post-ER, higher molecular weight) and ENDO H-sensitive bands (ER, lower molecular weight).

B. The ratio between the post-ER and ER fraction of GCase (ENDO H-resistant/ENDO H-sensitive) measured in cell lysates incubated with ENDO-H (the ER fraction) and PNGase F (as a reference for detection of the post-ER fraction) was increased for the ER-Nbl and ER-Nb9, compared to the control. n=5; data represented as mean ±SEM; Shapiro-Wilk test for Normality and Ordinary One Way Anova with multiple comparisons was used for the statistical analysis (DF Nbs = 3, DF residual = 10, F value 5.996). Figure 12. 4-MU in vitro GCase activity assay with the N370S mutant in the presence of selected Nbs.

A. N370S GCase activity assay performed in the presence of Nbl, Nb4, Nb9, NblO or Nbl6 showed that NblO and Nbl6 were able to significantly improve in vitro N370S GCase enzymatic activity. Isofagomine (IFG) was used as a negative control. n=6; data are represented as mean ±SEM; statistical analysis was performed using the One-Way Anova multiple comparison test

B. GCase activity assay performed on gut tissue lysates from hN370S GCase mice in the presence of Nbl, Nb4, Nb9, NblO or Nbl6 revealed that Nbl and Nbl6 were able to significantly enhance in vitro N370S GCase enzymatic activity. n = 4 tissues; 3 independent experiments were conducted, each with 3 technical replicates. Data represent the average value of technical replicates for each experiment in each biological sample. Statistical analysis was performed using a One-Way ANOVA with multiple comparisons (DF Nbs = 5, DF residual = 55, F value = 4.926)

C. Co-expression of the N370S GCase mutant with ER-Nb4 and ER-Nb9 in GBA1 knockdown HEK293T (11F clone) cells led to a 60-70% increase in lysosomal N370S GCase activity compared to ER-Mock transfected cells, while ER-Nbl and ER-Nbl6 showed no effect. n=4 in two independent experiments; data are represented as violin plots; statistical analysis was performed using an Ordinary One-Way ANOVA test with multiple comparisons, followed by the Shapiro-Wilk normality test (DF Nbs = 3, DF residual = 12, F = 8.352)

D. Co-expression of the N370S GCase mutant with lysosome-targeted Nbl6 in GBA1 knockdown HEK293T (11F clone) cells significantly increased lysosomal N370S GCase activity compared to lysosome-Mock transfected cells. n=6, in three independent experiments; data are represented as violin plots; statistical analysis was performed using an Ordinary One-Way ANOVA test with multiple comparisons, followed by the Shapiro-Wilk normality test (DF Nbs = 3, DF residual = 20, F = 0.5994).

Figure 13. Comparison of the GCase binding pockets among various established small molecules, GCase's natural ligands, and Nbl.

The binding of GCase and Nbl was compared in silico with the binding of GCase and several small molecules described in the prior-art, as well as with the binding of GCase and sap C and LIMP-2.

A. Nbl binds at the interface of GCase domain II and III, positioned on the opposite side of the GCase active site. GCase is shown in surface representation, with annotated domains I, II, and III. Nbl is shown in cartoon representation with annotated CDR1, CDR2, and CDR3.

Following, the binding of GCase with Nbl and the following small molecules was compared: B. JZ-5029 and isofagomine⁵¹;

C. NCGC00241607 (NCGC607)⁵³: a docking pose binding site 1 (BS1) is shown;

D. Compound 14⁵⁴;

E. Ambroxol⁵⁰: docking poses C2.1 and C2.2 are shown.

F. The Nbl epitope on GCase does not overlap with the presumed sap C and LIMP-2 binding pockets. Residues of GCase located on the apparent binding surfaces for sap C and LIMP-2 are depicted as spheres (on the right side). The position of the GCase active site is indicated with an arrow. Bound Nbl is visualized on the left side.

Figure 14. Nbl amino acid sequence and illustration of the different CDR annotations referred to in this application.

CDR annotations according to MacCallum, AbM, Chothia, Kabat and IMGT numbering systems are shown in colored boxes corresponding to the sequence of Nbl (SEQ. ID NO:1). For amino acid residue numbering of the VHH sequence, the Kabat numbering was applied.

Figure 15. ER- and lysosome-targeted Nbs are expressed differently in the two compartments.

A. A representative western blot membrane of HEK293T cells transfected with a mock vector or an indicated Nb targeted to the ER.

B. The relative quantification of the flag intensity band, representing the Nbs specifically targeted to the ER, when normalized to p-actin, demonstrated a variable yet consistent expression of all the distinct Nbs within this cellular compartment. n=9 in 6 independent experiments; data are represented as mean ±SEM, statistical analysis was performed using a Kruskal-Wallis multiple comparison test

C. A representative western blot membrane of HEK293T cells transfected with a mock vector or an indicated Nb targeted to the lysosome.

D. The relative quantification of the flag intensity band, representing nanobodies specifically targeted to the lysosome, normalized to -actin, revealed significant differences in the expression levels among the nanobodies. Notably, Nb4, Nb9, and Nbl6 exhibited the highest expression levels. n=9 in 6 independent experiments; data are represented as mean ±SEM, statistical analysis was performed using a Kruskal-Wallis multiple comparison test

E. Representative confocal images of HEK293T cells overexpressing the mock sequence, Nbl, Nb4, Nb9, or Nbl6, targeted to either the ER or lysosomes, and stained with an anti-FLAG antibody. The corresponding EGFP fluorescence signal is shown. Scale bar: 10 pm. Figure 16. Expression of the ER- or lysosome-targeted nanobodies does not induce ER or lysosomal stress in HEK293 cells, nor does it alter GCase expression levels.

A. Representative western blot of HEK293 cells overexpressing the selected ER-targeted nanobodies (ER-Nbs), showing GCase and calnexin levels.

B. Representative western blot of HEK293 cells overexpressing the selected lysosome-targeted nanobodies (lysosomes-Nbs), showing GCase and LAMP1 levels.

C. Quantification of GCase expression levels in cells expressing the ER-targeted Nbs shows no significant differences compared to Mock-transfected cells. n=9-ll, in 6 independent experiments; data are represented as violin plots, and statistical analysis was performed using One-Way ANOVA (DF Nbs = 3, DF residual = 40, F value = 1.542)

D. Quantification of GCase expression levels in cells expressing the lysosome-targeted Nbs shows no significant differences compared to Mock-transfected cells. n=9-ll, in 6 independent experiments; statistical analysis was performed using One-Way ANOVA (DF Nbs = 3, DF residual = 40, F value = 0.7367)

E. Calnexin expression levels quantified by densitometry analysis show no significant differences between Mock-transfected cells and Nb-expressing cells. n=6-8 in 6 independent experiments; statistical analysis was performed using One-Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 40, F value = 1.305)

F. LAMP1 expression levels remained unchanged across the different conditions used. n=6-8 in 6 independent experiments; data are represented as violin plots, and statistical analysis was performed using One-Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 28, F value = 0.6592).

Figure 17. Expression of ER-targeted Nb4, Nb9, and Nbl6, as well as lysosome-targeted Nbl6, increases lysosomal GCase activity in live cells, but does not impact lysosomal proteolytic activity.

A. A scheme outlining the lysosomal GCase activity assay, which was performed using the PFB- FDGIu substrate and flow cytometry on wild-type (WT) HEK293 live cells. The cells expressed Nbl, Nb4, Nb9, or Nbl6, targeted either to the lysosomes or the ER, as indicated in each experiment.

B. The expression of ER-Nb4, ER-Nb9, and ER-Nbl6 resulted in a significant increase in lysosomal GCase activity, approximately 15% higher compared to ER-Mock transfected cells. n=5 in 3 independent experiments; data are presented as violin plots; the Shapiro-Wilk test was used for normality assessment, and statistical analysis was performed using an Ordinary One- Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 16, F value = 5.451) C. Nbl6 targeted to the lysosomes improved GCase enzymatic activity. n=6 in 3 independent experiments; data are shown as violin plots; the Shapiro-Wilk test was applied for normality assessment, and statistical analysis was conducted using an Ordinary One- Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 20, F value = 1.909)

D. The DQ-BSA assay in HEK293T cells was performed as outlined in the scheme to evaluate lysosomal proteolytic activity following expression of the selected Nbs.

E. Lysosomal proteolytic activity in HEK293T cells overexpressing the selected ER-Nbs was expressed as the ratio between the DQ-BSA fluorescence mean of Nb-transfected cells and Mock-transfected cells. n=7-9 in 3 independent experiments; data are presented as violin plots; the Shapiro-Wilk test was used for normality assessment, and statistical analysis was performed using an Ordinary One- Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 31, F value = 1.218)

F. Lysosomal proteolytic activity in HEK293T cells overexpressing the selected lysosome-targeted Nbs was expressed as the ratio between the DQ-BSA fluorescence mean of Nb-transfected cells and Mock-transfected cells. n=8-9 in 3 independent experiments; data are presented as violin plots; the Shapiro-Wilk test was used for normality assessment, and statistical analysis was performed using an Ordinary One- Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 28, F value = 2.213).

Figure 18. ER-targeted Nbs and lysosome-targeted Nbs do not impair Cathepsin B activity in HEK293T cells.

Cathepsin B activity was assessed using the Magic Red assay in HEK293T cells overexpressing ER- targeted Nbs (A) or lysosome-targeted Nbs (B). The activity is expressed as the ratio of the Magic Red fluorescence mean in Nb-transfected cells to that in Mock-transfected cells. n=6 in three independent experiments; data are represented as violin plots; normality was evaluated using the Shapiro-Wilk test, and statistical analysis was performed using an Ordinary One-Way ANOVA with multiple comparisons (DF Nbs = 3, DF residual = 31, F value = 1.968)

The PFB-FDGIu assay was performed in HEK293T cells overexpressing ER-targeted Nbs (C) or lysosome-targeted Nbs (D) following a 24-hour treatment with the GCase inhibitor CBE (50 pM). No significant variations in GCase activity were observed between the CBE-treated groups.

Figure 19. The stabilizing Nbs protect GCase from Cathepsin L proteolysis in vitro.

A. In vitro cleavage of wild-type GCase by recombinant cathepsin L in either absence or presence of Nbl, Nb4 or Nb9. Samples were analyzed by SDS-PAGE. The intensities of the bands corresponding to full length GCase (indicated by a blue box, ± 62 kDa) were quantified (n=6). B. Graph showing the percentage of uncleaved GCase remaining after normalization, following treatment with Cathepsin L, in the absence or presence of Nbs. statistical analysis was performed using an Ordinary One WayAnova multiple comparison test in GraphPad Prism (ns: P > 0.05; *: P < 0.05; **_: p < 0.01; ***_: p < 0.001; ****: P < 0.0001).

Figure 20. Biolayer Interferometry (BLI) measurements to determine the equilibrium dissociation constants (K_D) of GCase binding to immobilized Nbs.

A. BLI sensorgrams were obtained by titrating increasing concentrations of GCase to site- specifically biotinylated Nbs that were trapped on a Streptavidin biosensor. The most representative dose-response curve for each Nb is shown. The sensorgrams were fitted on a 1:1 binding model (ForteBio Analysis Software) and the resulting Req values were subsequently plotted against the GCase concentration.

B. Equilibrium dissociation constant (K_D) values resulting from the set-up described in (A). Values are determined by fitting the signal amplitudes (Req) on the Langmuir equation. K_D values are represented as mean values ± standard deviations (n=3).

Figure 21: Fluorescence anisotropy titration curve to assess the binding of GCase to Nbl6.

Nbl6 was site-specifically labelled at its C-terminus with 5-TAMRA and titrated with increasing GCase concentrations. The anisotropy signal was plotted against the GCase concentration, and the resulting curve was fitted using a quadratic equation to determine the K_D value.

Figure 22. Crosslinking and LC-MS analysis of the GCase-Nbl and GCase-Nbl6 complexes.

A. SDS-PAGE analysis of the complexes formed between GCase and either Nbl or Nbl6 after crosslinking with disuccinimidyl suberate (DSS). Crosslinking of individual GCase and Nbl6 or GCase with an irrelevant Nb protein was used as a control. As a reference, the corresponding non-crosslinked samples were also analyzed on SDS-PAGE (left gel). The bands indicated with arrows were excised for LC-MS analysis.

B. Table summarizing the results of the LC-MS analysis of the bands excised from the SDS-PAGE shown in panel (A) (the arrows). The bands were excised, and the proteins were digested with trypsin according to the ProteaseMax manufacturer's protocol and analyzed using LC-MS/MS.

C. Zoom-in on the part of the gel (A) where the bands were excised.

Figure 23. Production and characterization of framework-optimized (humanized) Nanobodies.

A. Western blot analysis of framework-optimized Nbs expressed in E. coli. B. Binding of optimized/humanized nanobodies to wild-type GCase. Equilibrium dissociation constant (K_D) values of the full set of optimized Nbs vs original Nbs for GCase. Values are determined by fitting of the signal amplitudes (Req) on the Langmuir equation.

C. The effect of the framework-cleaned Nbs on the melting temperature (ATm) of GCase, in comparison to the effect of the "parental" Nbs, was evaluated. ATm represents the difference between the Tm of GCase and the Tm of GCase in the presence of the specified Nb.

D. Effect of the framework cleaned-up Nbs on the melting temperature (ATm) of GCase, in comparison to the effect of the "parental" Nbs. Overview of the results of the TSA assay for the full set of framework cleaned-up Nbs vs parental Nbs. The plotted variation of Tm (ATm) is the difference between the Tm of GCase and GCase in the presence of the specified Nb. The covalent inhibitor conduritol-p-epoxide (CBE) was used as a positive control, and an irrelevant nanobody (Irr Nb) as a negative control.

Figure 24: The GCase surface encompassing the potential binding region of various antibodies reported in the literature based on the epitope that was used for immunization.

The structure of GCase in complex with Nbl (PDB 9ENA) is shown in light grey, with GCase depicted in surface representation and Nbl in cartoon representation. The deduced binding region, based on the immunogen, which encompasses the epitope of various antibodies, is highlighted in dark grey. (A) Potential binding surface of sc-365745; (B) Potential binding surface of sc-32883 (shown in two orientations); (C) Potential binding surface of G4046; (D) Potential binding surface of G4171; (E) Potential binding surface of Ab55080; (F) Potential binding surface of TA325083 and (G) Potential binding surface of H00002629-IVI01.

DESCRIPTION

Reference will now be made in detail to representative embodiments of the invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or a group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, encompassing both its organization and method of operation, as well as its features and advantages, can be best comprehended by referring to the detailed description below when read in conjunction with the accompanying drawings. The aspects and advantages of the invention will become apparent and be clarified through reference to the embodiment(s) described hereinafter. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment; however, the may also refer to the same embodiment.

Definitions

Where an indefinite or definite article is used when referring to a singular noun, e.g., "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Furthermore, the terms "first", "second", "third" and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to a person skilled in the art of the present invention. For definitions and terms of the art, practitioners are particularly directed to "Molecular Cloning: A Laboratory Manual", 4^th edition (2012), by Sambrook J. and Green M.⁵⁵; "Advances in Protein Molecular and Structural Biology Methods", 1^st edition (2022) by Tripathi T. and Dubey V.K.⁵⁶; and "Current Protocols in Molecular Biology (Supplement 114), edited by Ausubel M. et al.⁵⁷. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g. in molecular biology, biochemistry, structural biology, and/or computational biology).

In describing and claiming the embodiments of the invention, the following terminology is used.

The term "allosteric modulator", as used herein, refers to a compound that binds to a site on a macromolecule that is distinct from the orthosteric site (i.e., the primary binding site or enzymatically active site of a protein or an enzyme). Allosteric modulators may influence the conformation of the target macromolecule and modify its features and/or activity, for instance its catalytic properties, binding affinity with its orthosteric ligand(s) and/or its signal transduction efficacy. For example, an allosteric modulator of GCase may bind GCase at an allosteric or regulatory site or epitope wherein said site is distinct from the orthosteric site leading to a change in GCase conformation and/or stability. As a result, interactive properties of GCase with respect to its substrate binding and/or catalytic activity may be modified in either a positive or negative manner.

The term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and to variants and synthetic analogues of the same, and does not refer to a specific molecule length. Thus, "peptide", "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. A "peptide" may also be referred to as a partial amino acid sequence derived from its original protein, for instance after enzymatic (e.g., tryptic) digestion. These terms apply to naturally-occurring amino acid polymers as well as to amino acid polymers in which one or more amino acid residue is a synthetic, non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid. The term "polypeptide" is also intended to refer to the products of post-translational modifications of the polypeptide, including (without limitation) acetylation, acylation, adenylation, alkylation, amidation, arginylation, beta-lysine addition, biotinylation, butyrylation, carbamylation, carbonylation, citrul lination, C-linked glycosylation, crotonylation, deamidation, diphthamide formation, eliminylation, ethanolamine phosphoglycerol attachment, farnesylation, flavinylation, formylation, gammacarboxylation, geranylgeranylation, glutarylation, glutathionylation, glypiation, hydroxylation, hypusine formation, iodination, ISGylation, isoaspartate formation, isopeptide bond formation, isoprenylation, lipoylation, malonylation, methylation, myristylation, neddylation, nitration, N-linked glycosylation, nucleotide addition, O-GIcNAcylation, O-linked glycosylation, oxidation, palmitoylation, PARylation, PEGylation, phosphate ester (O-linked) or phosphoramidate (N-linked) formation, phosphopantetheinylation, phosphorylation, polyglutamylation, polyglycylation, prenylation, propionylation, protein splicing, proteolytic cleavage, pupylation, pyroglutamate formation, racemization, retinylidene Schiff base formation, S-nitrosylation, stearoylation, sulfation, sumoylation and ubiquitination. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. As used herein, the term "glucocerebrosidase" or "GCase" refers to human lysosomal p- glucocerebrosidase (also named acid -glucosidase or D-glucosyl-N-acylsphingosine glucohydrolase), an enzyme naturally encoded by the human GBA1 gene. The GCase enzyme (EC 3.2.1.45) possesses glucosylceramidase activity that is needed to cleave, by hydrolysis, the beta-glucosidic linkages of glucocerebroside, an intermediate in glycolipid metabolism that is abundant in cell membranes. The GBA1 gene in humans can produce five alternatively spliced mRNAs which encode several distinct GCase isoforms. As used herein, the terms "glucocerebrosidase" or "GCase" refer to all possible isoforms of glucocerebrosidase encoded by the GBA1 gene. Furthermore, the terms "glucocerebrosidase" or "GCase" refer to the enzyme and any additional co-translational or post-translational modifications. The amino acid sequence of human GCase protein is represented by SEQ. ID NO: 81.

The term "epitope", as used herein, refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule, such as on human GCase. Said epitopes may comprise at least one amino acid that is essential for binding the binding agent, though preferably comprise at least 3 amino acids in a spatial conformation that is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, consists of at least 8, 9, or 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance, cryo-EM, or other structural techniques. A "conformational epitope" refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional (3D) conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3D conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides. These sequences come together upon the folding of different polypeptides and their association in a unique quaternary structure. Similarly, conformational epitopes may here also consist of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation that is unique to the quaternary structure.

The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. A person skilled in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein, especially for membrane proteins. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, beta-sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as acidity (pH), salt concentration, ionic strength, and osmolality of the surrounding solution, and interactions with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assays for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labeling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to "Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules" (1980) by Cantor C.R. and Schimmel P.R.⁵⁸, and "Proteins: Structures and Molecular Properties" (1992) by Creighton T.E.⁵⁹.

The term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule comprising an Ig domain, which specifically binds with an antigen, as well as multimers thereof. "Antibodies" can be intact immunoglobulins or immunoreactive portions of intact immunoglobulins. The term encompasses naturally, recombinantly, semi-synthetically or synthetically produced antibodies. Hence, for example, an antibody can be present in or be isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom; or an antibody can be recombinant, i.e., produced by a recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized.

The terms "active antibody fragment", "antibody fragment", "antigen-binding fragment", and "functional antibody fragment" refer to a portion of any antibody that by itself has a high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity. Non-limiting examples of "active antibody fragments" include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain. The term "active antibody fragment" is used in this application to refer to a protein or peptide comprising an Ig domain or an antigen-binding domain capable of specifically binding to human GCase protein.

The term "immunoglobulin (Ig) domain", or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") denotes an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively. The framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. It is the immunoglobulin variable domains (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site. Typical ly, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In light of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD, or IgE molecule, known in the art) or of a Fab fragment, an F(ab')2 fragment, an Fv fragment (such as a disulphide-linked Fv), or a scFv fragment, or a diabody (all known in the art) derived from such a conventional 4-chain antibody, binds to the respective epitope of an antigen through a pair of associated immunoglobulin domains, such as light and heavy chain variable domains (i.e., a VH-VL pair of immunoglobulin domains), which collectively bind to an epitope of the respective antigen.

An "immunoglobulin single variable domain" or "ISVD" is a protein with an amino acid sequence comprising four Framework regions (FRs) and three complementary determining regions (CDRs) according to the format: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Similar to a complete antibody, an ISVD possesses the ability to selectively bind to a specific antigen. ISVDs are significantly smaller than conventional antibodies, with a molecular weight ranging from 12-18 kDa. This is in contrast to typical antibodies (150-160 kDa), which consist of two heavy and two light protein chains, as well as smaller fragments like Fab fragments (approximately 50 kDa, composed of one light chain and half a heavy chain) and single-chain variable fragments (approximately 25 kDa, featuring two variable domains, one from a light and one from a heavy chain). An "immunoglobulin domain" of this invention refers to "immunoglobulin single variable domains" ("ISVDs"), equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This differentiates immunoglobulin single variable domains from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or a VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of a single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or an amino acid sequence that is suitable for use as a dAb or a nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a nanobody (as defined herein) or a suitable fragment thereof. For a general description of nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as W02008/020079.

"VHHs", also known as "VHH domains", "VHH antibody fragments", and "VHH antibodies", can be described as the antigen binding Ig (variable) domains of "heavy chain antibodies" (i.e., of antibodies devoid of light chains)⁶⁰. The term "VHH" distinguishes these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies ("VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies ("VL domains"). For a further description of VHHs and nanobodies, we refer to the review articles by Muyldermans S.⁶¹-⁶², as well as to the following patent applications: WO94/04678, WQ95/04079 and WO96/341Q3 of the Vrije Universiteit Brussel; WO94/25591, WO99/37681, WOOO/4Q968, WOOO/435Q7, WOOO/65Q57, W001/40310, WQQl/44301, EP1134231 and WO2/48193 of Unilever; WO97/49805, WOOl/21817, WQ03/035694, WQ03/054016 and WQ03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WQ03/050531 of Algonomics N.V. and Ablynx N.V.; W001/90190 by the National Research Council of Canada; W003/025020 (EP1433793) by the Institute of Antibodies; as well as WQ04/041867, WQ04/041862, WQ04/041865, WQ04/041863, WQ04/062551, WQ05/044858, WQ06/40153, WQ06/079372, WO06/122786, WO06/122787 and WO06/122825 by Ablynx N.V., and the further published patent applications by Ablynx N.V. As highlighted in these references, a nanobody (in particular VHH sequences and partially humanized nanobodies) can be characterized by the presence of one or more "hallmark residues" in one or more of the framework sequences. Different numbering schemes can be applied for the amino acid residues of an IVD. For example, numbering can be performed according to the AHo numbering scheme for all heavy (VH) and light chain variable domains (VL) introduced by Honegger A. and Pluckthun A.⁶³, as applied to VHH domains from camelids. Alternative methods for numbering the amino acid residues of VH domains, which can also be applied in an analogous manner to VHH domains, are known in the art. For example, the delineation of the FR and CDR sequences can be done by using the Kabat numbering system as applied to VHH domains from camelids in the article of Riechmann L. and Muyldermans S.⁶⁴. It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, and often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein. Determination of CDR regions may also be done according to different methods, such as the designation based on contact analysis and binding site topography as described in MacCallum et al.⁶⁵. Or alternatively, the annotation of CDRs may be done according to AbM (AbM is Oxford Molecular Ltd.'s antibody modelling package as described at http://www.bioinf.org.uk/abs/index.html⁶⁶), Chothia⁶⁷, Kabat⁶⁸, or IMGT⁶⁹. Said annotations further include delineation of CDRs and framework regions (FRs) in immunoglobulin-domain-containing proteins, and are known methods and systems to a skilled artisan who thus can apply these annotations onto any immunoglobulin protein sequences without undue burden. These annotations differ slightly, but each intend to comprise the regions of the loops involved in binding the target.

The terms "stabilizing GCase" or "to stabilize GCase" or "GCase-stabilizing" refer to the process of promoting the native folded conformation of GCase protein (N state) as opposed to its denatured (unfolded or extended) conformation (D state). Proteins are entities of soft matter that typically adopt a well-defined three-dimensional structure in the environments where they carry out their functions, such as a water solution or a cell membrane space. This structure, referred to as the native or folded state, is only marginally stable. Indeed, the free energy difference between the folded state and the ensemble of highly disordered unfolded conformations is typically just a few kilocalories per mole⁷⁰-⁷¹. For instance, GCase stability (AG0_D) can be defined as the decrease in the Gibbs free energy when denatured GCase folds into its native conformation under physiological conditions, that is, AG0D=G0D-G0_N, where G0_D and G0_N are Gibbs free energies of the D and N states in the absence of a denaturating agent, respectively. Furthermore, the thermal stability of a protein can be quantified using the Gibbs-Helmholtz equation, which relates the free energy difference between the folded and unfolded states as a function of temperature AGf/_u (T) to the temperature variation of the enthalpy (AHf/_u) and entropy of unfolding (ASf/_u)^70,72. The temperature dependence of these quantities is influenced by the specific heat (C_p), representing the energy required to raise the temperature of a solution by one unit mass. Experimental observations confirm that, for a given protein and at a particular temperature, the specific heat of the unfolded solution exceeds that of its folded counterpart. This difference, known as the specific heat of unfolding (AC_P), is considered nearly constant as it is only weakly temperaturedependent⁷³. When the characteristic thermodynamic parameters, AHf/_u and ASf/_u, are assessed at the melting temperature (T_m), where the populations of the folded and unfolded states are equal, the modified Gibbs-Helmholtz equation is expressed as follows: AGf/_u = AH_m (1 - T/T_m) + AC_P [(T - T_m) - Tln(T/T_m)]⁷³, wherein AH_m is the enthalpy of unfolding and AC_P is the heat of unfolding. The resulting curve resembles an inverted parabola, featuring a maximum point where the folded state is most stable. Two zeros on the curve correspond to temperatures where the populations of the unfolded and folded states are equal. The first zero indicates high-temperature melting, while the second, located at a lower temperature, predicts cold unfolding.

In a functional context, "GCase stability" relates to its ability to retain its function and/or activity over time. Protein stability can be influenced by proteolytic cleavage, loss of structural integrity of the three- dimensional folding, and/or general physiological protein turn-over. Protein stability can be measured by a wide range of processes known to the skilled artisan and include, without being limited to any particular method, immunological methods using antibodies binding to three dimensional epitopes, pulse-chase methods such as cycloheximide chase and functional assays measuring time-dependent decline of a protein's enzymatic activity. The thermal stability of a protein can be experimentally characterized using, for example, a technique called differential scanning calorimetry⁷⁴. This method allows for measuring the variation in specific heat of a protein solution as a function of temperature. From the resulting profile, it is possible to identify the unfolding temperature (T_m) and, ideally, to deduce the specific heat of unfolding (AC_P) and the enthalpy of unfolding (AH_m). Other approaches, either more or less direct, can also be employed to derive the thermodynamic parameters of the Gibbs-Helmholtz equation. For instance, the unfolding temperature can be derived by employing differential scanning fluorimetry (DSF) or thermal shift assays (TSA)⁷⁵. According to data reported in the literature, AH_m can vary between 50 and 100 kcal/mol, although smaller or higher values have been observed in specific cases. The specific heat of unfolding can range from a few tenths of kcal/(mol K) to a few kcal/(mol K)⁷³.

In the present application referring to "stabilizing GCase" using an allosteric modulator, the binding of said modulator may for instance induce a conformational change of the GCase protein or keep the GCase protein in its folded state, thereby stabilizing the GCase or, as interchangeably used herein, increasing the GCase protein stability, or increasing the GCase thermal stability, as compared to the GCase in the absence of said allosteric modulator. As a result of the allosteric modulator stabilizing GCase, the enzymatic activity of said GCase (wild-type/mutant/variant/homologue/isoform) protein may be retained and/or increased as compared to the same GCase protein in the absence of said allosteric modulator of the present invention. Hence, stabilization of GCase may refer to an increase in GCase thermal stability of at least 1, 3, 4, 5, 6, 7 , 8, 9, 10, or more °C in its melting temperature (T_m) as compared to a control GCase sample, depending on the pH in which the T_m of the GCase is measured; and/or may result in an increased GCase enzymatic activity upon allosteric modulator binding of at least 10% , or 20%, or 30%, or even more than 50 % as compared to the GCase protein not bound to said allosteric modulator.

VHHs or Nbs are frequently categorized into various sequence families or even superfamilies to cluster clonally related sequences originating from the same progenitor during B cell maturation⁷⁶. This classification is commonly established according to the CDR sequences of the Nbs. For example, each Nb family is typically defined as a cluster of clonally related sequences with a sequence identity threshold applied to the CDR3 region⁷⁷. Within a single VHH family defined herein, the CDR3 sequence is thus identical or very similar in amino acid composition, preferably with at least 80% identity, or at least 85% identity, or at least 90% identity in the CDR3 sequence, resulting in Nbs of the same family binding to the same binding site, having the same effect or functional impact.

As used herein, the term "binding site" or "binding pocket" relates to an area of a protein molecule that interacts or "binds" (i.e., contributes to the specificity and affinity of the ligand-protein binding) with another molecule (such as a compound, protein, peptide, antibody or nanobody, among others) or a part of another molecule. "A binding site" includes residues or atoms with which a ligand molecule interacts through various non-covalent interactions, including ionic interactions, electrostatic interactions, hydrophobic interactions, hydrogen bonding, or Van der Waals interactions. This region typically includes amino acid residues which are directly involved in binding and participate in non- covalent intermolecular interactions. This region may also include amino acid residues which are not directly involved in binding or participate in non-covalent intermolecular interactions, but which are merely interspersed between interacting amino acid residues, and/or provide a structural, spatial, energetic or other function. The term "pocket" includes, but is not limited to a cleft, channel or site. A "binding site" may include both the surface area of a protein as well as areas that are distant from the surface. The same, similar, or overlapping binding sites can have affinity to more than one ligand. The term "binding site" also refers to an area which determines an exclusion zone or competition zone of a component for two ligands with the same binding site. For example, according to the present invention, the binding site, which may be defined by K77, V78, K79, H162, L165, Q166, A168, Q169, R170, P171, V172, S173, L174, A221, K224, L225, Q226, F227, W228, T272, H274, N275, R277, H306, E429, G430, P452, D453 of SEQ ID NO:81, refers to a region of the GCase comprising amino acids K77, V78, K79, H162, L165, Q166, A168, Q169, R170, P171, V172, S173, L174, A221, K224, L225, Q226, F227, W228, T272, H274, N275, R277, H306, E429, G430, P452, D453, and/or mutants thereof as described herein, that cooperate to bind a GCase-specific allosteric modulator, as described herein. "Binding" can signify any interaction, be it direct or indirect. A direct interaction implies a contact between the binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. The interaction can be completely indirect, with the help of one or more bridging molecules, or partly indirect, where there is still a direct contact between the partners, which is stabilized by the additional interaction of one or more molecules. The term "specifically binding", as used herein, refers to a binding domain that recognizes a specific target without substantially recognizing or binding other molecules in a sample. Specific binding does not imply exclusive binding, but it indicates that proteins exhibit an increased affinity or preference for one or a few of their binders.

The term "affinity", as used herein, generally refers to the degree to which a ligand, chemical, protein or peptide binds to another (target) protein or peptide so as to shift the equilibrium of single protein monomers toward the presence of a complex formed by their binding.

The term "protein domain" refers to a distinct region of a specific protein, corresponding to a discrete tertiary structure. "Protein domains" fold into compact three-dimensional atomic structures that arrange alpha-helical and beta-sheet structure elements into tightly packed conformations of the polypeptide chain. "Protein domains" are high-level parts of proteins that either occur alone or together with partner domains on the same protein chain. All domains exhibit evolutionary conservation, and many either perform specific functions or contribute in a specific way to the function of their proteins. The Structural Classification of Proteins (SCOP)⁷⁸ and its extended version SCOPe⁷⁹ are popular taxonomy gold standards of domain structure. Domains may exist in a variety of biological contexts, where similar domains can be found in proteins with different functions.

The term "amino acid" encompasses all natural a-amino acids of the L or D series, each having the following "side chain": H for glycine, CH3 for alanine, CH(CH3)2 for valine, CH2CH(CH3)2 for leucine, CH(CH3)CH2CH3 for isoleucine, CH2OH for serine, CH(OH)CH3 for threonine, CH2SH for cysteine, CH2CH2SCH3 for methionine, CH2-(phenyl) for phenylalanine, CH2-(phenyl)-OH for tyrosine, CH2- (indole) for tryptophan, CH2COOH for aspartic acid, CH2C(O)(NH2) for asparagine, CH2CH2COOH for glutamic acid, CH2CH2C(O)NH2 for glutamine, CH2CH2CH2-N(H)C(NH2)NH for arginine, CH2-(imidazole) for histidine, CH2(CH2)3NH2 for lysine, and NH(CH2)3CHCOOH for proline. This includes the same side chains of amino acids with suitable protecting groups. Additionally, the term "amino acid" encompasses non-natural amino acids such as ornithine (Orn), norleucine (Nle), norvaline (NVa), p-alanine, L or D a- phenylglycine (Phg), diaminopropionic acid, diaminobutyric acid, aminohydroxybutyric acid, and other synthetic amino acids known in the field of peptide chemistry. Table 2 lists amino acid names and their abbreviations used in this application.

The term "homologue", as used herein, refers to a protein (amino acid) sequence that is distinct but exhibits a statistically significant degree of similarity with another protein (amino acid) sequence, indicating a common origin between the compared proteins. "Homologue" or "homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. Typically, two proteins are considered homologous if they share a minimum of 70% amino acid sequence identity. Percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the percentage of identity is calculated over a window that spans the entire length of the sequence in question.

A "mutation" or "variant" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid sequence or nucleotide sequence of a parental protein/fragment thereof or a parental gene/fragment thereof. In particular, the term "functional variant" as used herein, pertains to a GCase-specific allosteric modulator polypeptide, more particularly comprising or consisting of an ISVD, characterized by at least 90% amino acid identity with the polypeptides comprising or consisting of the ISVDs described by any of the seq ID NOs: 1, 4, or 9, that possesses one or more activities exhibited by the polypeptides described by any of the seq ID NOs: 1, 4, or 9.

The term "wild type" or "wild-type" or "WT" refers to a gene or a gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the "normal" form of the gene. In contrast, the term "modified", "mutant", "engineered" or "variant" refers to a gene or a gene product that displays modifications in sequence, in post-translational modifications and/or in functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. Mutational variations in the GBA1 gene can induce structural alterations in the GCase enzyme, resulting in the loss-of-function phenotype. The residual catalytic activity of GCase differs depending on the severity of the GBA1 variant. For severe variants like L444P and mild variants such as N370S (occurring in a bi-allelic form), GCase activity decreases by approximately 80-95% and 50-60%, respectively, in comparison to the activity of the normal (i.e., wild-type) enzyme²². Several mechanisms can contribute to the reduced GCase activity, including: loss of transcription/translation; ER stress and activation of the unfolded protein response (UPR) triggered by the misfolded GCase; failure of GCase to exit the Golgi; and/or loss of critical amino acids in the enzyme's catalytic domain⁸⁰-⁸¹. In this regard, the terms "GCase activity" or "GCase catalytic activity" or "GCase enzymatic activity" are understood as the ability of GCase (or GCase homologue or GCase mutant/variant) to hydrolyze glucosylceramide and glucosylsphingosine to glucose and either ceramide or sphingosine, respectively.

Immunoglobulin single variable domains (ISVDs) such as domain antibodies and Nanobody® (including VHH domains) can be subjected to humanization, i.e., increase in the degree of sequence identity with the closest human germline sequence. Specifically, humanized immunoglobulin single variable domains, like Nanobodies® (including VHH domains), can be ISVDs where at least one amino acid residue, especially at least one framework residue, is present and/or corresponds to a humanizing substitution. Potentially useful humanizing substitutions can be identified by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences. Following this comparison, one or more of the potentially useful humanizing substitutions (or combinations thereof) can be introduced into the VHH sequence. This can be done using any known method, as further described herein. The resulting humanized VHH sequences can then be tested for their affinity for the target, stability, ease and level of expression, and/or other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what was described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody® (including VHH domains), may be partially humanized or fully humanized.

Humanized immunoglobulin single variable domains, in particular nanobodies, may have several advantages, such as reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. "Humanized" refers to a mutated form of a molecule, in a way that minimizes or eliminates immunogenicity upon administration in human patients. The humanizing substitutions should be chosen such that the resulting humanized amino acid sequence and/or VHH still retains the favorable properties of the VHH, for instance the antigen-binding capacity. Based on the description provided herein, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favorable properties provided by the humanizing substitutions on the one hand and the favorable properties of naturally occurring VHH domains on the other hand. Such methods are known by the skilled addressee, and are further clarified in the examples provided herein. A human consensus sequence can serve as a target sequence for humanization, although various other methods are also recognized in the art. One alternative involves a method in which the skilled person aligns a number of human germline alleles, such as, for instance, but not limited to, the alignment of IGHV3 alleles, to use the alignment for identifying residues suitable for humanization in the target sequence. Additionally, a subset of human germline alleles that are most homologous to the target sequence can be aligned as a starting point to identify suitable humanization residues. Alternatively, the VHH is analyzed to identify its closest homologue in the human alleles and used for humanization construct design. A humanization technique applied to Camelidae VHHs may also be performed by a method comprising the replacement of specific amino acids, either alone or in combination. Said replacements may be selected based on what is described in the literature (e.g., known humanization efforts), as well as from human consensus sequences compared to the natural VHH sequences, or the human alleles most similar to the VHH sequence of interest. As can be seen from the data on the VHH entropy and VHH variability given in Tables A5-A8 of W008/020079, certain amino acid residues in the framework regions exhibit higher conservation between human and Camelidae species than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions. For instance, a human-like class of Camelidae single-domain antibodies contains the hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species. However, this loss in hydrophilicity can be compensated by introducing other substitutions at position 103, which replace the conserved tryptophan residue present in VH from double-chain antibodies. As such, peptides belonging to these two classes show a high amino acid sequence homology to human VH framework regions and said peptides might be administered to a human directly without expectation of an unwanted immune response therefrom, and without the burden of further humanization. Indeed, certain Camelidae VHH sequences exhibit a high sequence homology to human VH framework regions. Consequently, these VHH sequences might be administered directly to patients without the prospect of an immune response and without the added burden of humanization.

Suitable mutations, in particular substitutions, can be introduced during humanization to generate a polypeptide with reduced binding to pre-existing antibodies (e.g., reference is made to WO 2012/175741 and WO2015/173325), for instance in at least one of the positions: 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108. The amino acid sequences and/or VHH of the invention may be suitably humanized at any framework residue(s), such as at one or more hallmark residues (as defined herewith) or at one or more other framework residues (i.e. non-hallmark residues), or any suitable combination thereof. Depending on the host organism employed for expressing the amino acid sequence, VHH, or polypeptide of the invention, deletions and/or substitutions can be designed to remove one or more sites of posttranslational modification, such as glycosylation sites. This can be accomplished by those skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups, for example to allow site-specific PEGylation. In some cases, at least one of the typical Camelidae hallmark residues with hydrophilic characteristics at position 37, 44, 45 and/or 47 is replaced (see W02008/020079, Table A3). Another example of humanization includes substitution of residues in FR1, such as position 1, 5, 11, 14, 16, and/or 28; in FR3, such as positions 73, 74, 75, 76, 78, 79, 82b, 83, 84, 93 and/or 94; and in FR4, such as position 103, 104, 108 and/or 111 (see W02008/020079, Tables A5-A8; all numbering according to the Kabat numbering system). Humanization typically only concerns substitutions in the FR and not in the CDRs, as this could/would impact binding affinity to the target and/or potency.

The composition or polypeptide modulator(s) of the invention as described herein may appear in a "multivalent" or "multispecific" form and thus be formed by bonding, chemically or by recombinant DNA techniques, together two or more identical or different binding agents. Said multivalent forms may be formed by connecting the building block directly or via a linker, or by fusing it with an Fc domainencoding sequence. Non-limiting examples of multivalent constructs include "bivalent" constructs, "trivalent" constructs, "tetravalent" constructs, and so on. The immunoglobulin single variable domains comprised within a multivalent construct may be identical or different, preferably binding to the same or overlapping binding site. In a particular embodiment, the allosteric modulators of the invention are in a "multispecific" form and comprise at least two moieties specifically binding GCase, or comprise at least two GCase-specific ISVDs. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, and so on. To illustrate this further, any multivalent or multi-specific (as defined herein) ISVD of the invention may be suitably directed against two or more different epitopes on the same GCase antigen, or may be directed against two or more different antigens. For example, one of them may target human GCase, and one of them may serve as a half-life extension by binding to serum albumin or SpA, or another specified target. Multivalent or multispecific ISVDs of the invention may also have (or be engineered and/or selected for) increased avidity and/or improved selectivity for the desired GCase interaction, and/or for any other desired property or combination of desired properties that may be obtained by the use of such multivalent or multi-specific immunoglobulin single variable domains. Upon binding human GCase, said multi-specific binding agent or multivalent ISVD may have an additive or synergistic impact on the binding and/or therapeutic effect of GCase, such as by increasing its stability and/or catalytic activity. The multivalent or multispecific binders or building blocks may be fused directly or fused by a suitable linker, as to allow that the at least two different binding sites can be reached or bound simultaneously by the multispecific agent. Said humanized forms thereof, such as IgG humanized forms, include but are not limited to the IgG humanization variants known in the art, for instance to modulate Fc-mediated effector functions, including variants with for instance C-terminal deletion of lysine, alteration or truncation in the hinge region, LALA of SEQ ID NO: 82 (L234A and L235A) or LALAPG of SEQ ID NO: 83 (L234A, L235A, and P329G) mutations, among other substitutions in the IgG sequence.

In an alternative arrangement, an Fc fusion is designed by linking the C-terminus of a bivalent or bispecific binder, fused by a linker, to an Fc domain. This produces, upon expression in a host cell, a multivalent or multispecific antibody-type molecule through disulfide bridges in the hinge region of the Fc part.

The terms "patient" or "subject", used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, a rat or a non-human primate. Preferably, the subject is a human. In one embodiment, the subject is a subject with or suspected of having a disease or disorder, in particular a disease or disorder as disclosed herein, i.e., a "GCase-related disorder". However, it will be understood that the aforementioned terms do not imply that symptoms are present. The term "medicament", as used herein, refers to a substance/composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the invention, the terms "disease" or "disorder" refer to any pathological state, in particular to the diseases or disorders as defined herein.

"GCase-related disorders", as referred to here, encompass all conditions where mutated, aberrant, modified, destabilized, non-functional, and/or partially-functional GCase contributes to disease pathology. Additionally, the term includes disorders where GCase levels (including mutated GCase variants) are diminished or increased compared to a healthy state. A non-exhaustive list of GCase-related disorders and conditions comprises Gaucher disease (GD) types I (GDI), II (GD2), and III (GD3), Parkinson's disease, Lewy body dementia, dementia, amyotrophic lateral sclerosis, neuropathy, multiple system atrophy, progressive supranuclear palsy, a-synucleinopathies (synucleinopathies), Alzheimer's disease, other forms of neurodegeneration, various forms of neuroinflammation, other inflammatory disorders, gammopathy, Niemann-Pick disease, lipid storage disorders, osteopenia, osteoporosis, osteolysis, osteonecrosis, hypocalcemia, hypoparathyroidism, hypoadiponectinemia, splenomegaly, atherosclerosis, growth retardation, delayed puberty, insulin resistance, diabetes, gallstones, gaucheroma, liver fibrosis, multiple myeloma, chronic lymphatic leukemia, childhood acute lymphoblastic leukemia, liver cancer (including hepatocellular carcinoma) and other types of cancer.

The term "Gaucher's disease" or "GD" refers to a lysosomal storage disease, specifically a sphingolipidosis characterized by the accumulation of GCase substrates in cells of the macrophagemonocyte system and/or in other cells, e.g., in neurons. Gaucher's disease results from a hereditary deficiency of the enzyme glucocerebrosidase (GCase), caused by recessive mutations in the gene coding for this enzyme, as described throughout this application. Different mutations in the GBA1 gene determine the remaining activity of the enzyme and largely influence the disease phenotype. Pathologically, GD often stems from the buildup of glucocerebrosides within the lysosomes of macrophages, forming what are termed "Gaucher cells". These cells typically exhibit small, eccentrically localized nuclei, cytoplasm with distinctive striations, deregulated expression of cell surface markers, iron sequestration, abnormal secretion of inflammatory cytokines and impaired ability to infiltrate tissues⁸²-⁸³. Gaucher cells can be found throughout the body, but are particularly abundant in the liver, spleen, and bone marrow²⁰.

The genetic heterogeneity associated with GD contributes to the highly variable clinical manifestations of the condition that may involve various organs and tissues. Typical symptoms of GD include anemia, thrombocytopenia, enlargement of the liver and/or spleen and skeletal abnormalities (osteopenia, lytic lesions, pathological fractures, chronic bone pain, bone crisis, bone infarcts, osteonecrosis and skeletal deformities/⁸⁴. There are three distinct types of GD categorized based on the absence or presence and severity of neurological manifestations, i.e., type I (GDI), type II (GD2), and type III (GD3)²⁰. GDI primarily affects macrophages without directly involving the central nervous system; however, parkinsonian symptoms in GDI patients have also been reported⁸⁵. Patients with GDI typically exhibit a broad spectrum of symptoms, varying from asymptomatic cases to cases with childhood-onset disease. In contrast, GD2 represents an acute neuronopathic form characterized by poor prognosis, with survival typically restricted to the initial two-three years of life. This disease type is characterized by neurological impairments alongside visceral symptoms. Neurological manifestations commence with oculomotor abnormalities and progress towards the brainstem involvement. GD3 also involves neurological manifestations, albeit emerging later in life compared to GD2. Symptoms of GD3 include abnormal eye movements, ataxia, seizures, and dementia; affected individuals usually survive into their third or fourth decade of life⁸⁴. The term "Parkinson's disease or "PD", as used herein, is intended to encompass all types of Parkinson's disease. The disease is characterized by classical motor symptoms associated with the presence of Lewy bodies (abnormal protein aggregates inside the nerve cells) and the loss of dopaminergic neurons in the substantia nigra. These motor symptoms encompass bradykinesia, muscular rigidity, rest tremor, and postural and gait impairment, among others⁸⁶. Additionally, PD patients suffer from non-motor symptoms which occur due to the loss of neurons from dopaminergic, non-dopaminergic or a combination of both pathways. These non-motor symptoms comprise neurobehavioral changes, autonomic dysfunctions (including blood pressure abnormalities, gastrointestinal problems, urinary dysfunctions), sensory impairments and sleep disorders⁸⁷.

Notably, neurons affected by both PD and GD express oxidative stress markers and are characterized by compromised mitochondrial complex I (Cl) stability and function^88-90. In this regard, in vitro studies demonstrated that inhibiting GCase activity with conduritol B-epoxide (CBE) or suppressing mitochondrial Cl with rotenone yielded comparable outcomes, i.e., an increase in the secretion of dopamine and serotonin metabolites into the culture medium as compared to a control sample⁹¹. These findings imply that GCase and mitochondrial Cl participate in the same pathway related to the dopamine/serotonin deficiency seen in PD patients.

In this context, reduction in GCase activity has been associated with impaired functioning of the autophagy-lysosome system, leading to a decline in the turnover of a-syn dependent on autophagy⁹². Thus, PD is characterized by progressive accumulation of a-syn neurotoxic species (i.e., oligomers, protofibrils and fibrils) in the substantia nigra⁹³ and progressively in other brain regions. Moreover, a- syn inhibits the function of both mutated and non-mutated GCase in PD neurons, fostering a bidirectional cycle that significantly contributes to the progression of the disease⁹⁴. Mutated GCase also disrupts chaperone-mediated autophagy (CMA), resulting in the buildup of various CMA substrates, including a-syn. This leads to a-syn-associated death of dopaminergic neurons⁹⁵. Changes in GCase activity can also influence the ability of a-syn to aggregate by affecting the composition of lipid membranes⁹⁶. Studies have shown that fibroblasts derived from PD patients carrying the heterozygous L444P variant exhibit alterations in lipid composition compared to control individuals or PD patients without this variant. Specifically, an increase in short-chain (C34) sphingomyelin, ceramide, and hexosylceramide levels alongside a consistent decrease in long-chain (C42) sphingomyelin, ceramide, and hexosylceramide levels was observed in these fibroblasts⁹⁷. The elevated ratio of C34 to C42 sphingolipids correlated with decreased GCase activity. These observations suggest that mutant GCase protein can contribute to PD's pathogenesis by disrupting lysosomal homeostasis, enhancing endoplasmic reticulum stress, modulating lipid metabolism and fueling mitochondrial impairment⁸⁰. The terms "treatment" or "treating" or "to treat" all indicate a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a pathological sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, or states. Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness. Treatment may also refer to a prophylactic treatment which relates to a medication or a treatment designed and used to prevent a disease from occurring.

A "pharmaceutical composition" pertains to a mixture of one or more active molecules. It may additionally encompass buffered solutions and/or solutes, including pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, and other components known to those skilled in the art. Suitable conditions as used herein could also refer to suitable binding conditions, for instance when Nbs or test compounds are aimed to bind human GCase.

Detailed description

The present invention stems from the exploration of methods to enhance the stability and/or catalytic activity of GCase (GBA1), aiming to expand treatment possibilities for individuals affected by Gaucher disease (GD) and/or Parkinson's disease (PD). Specifically, it introduces novel polypeptides which allosterically bind GCase, thereby modulating its conformation, stability, trafficking and/or function. The polypeptide modulators described here interact with a binding pocket of GCase that is situated distantly from its catalytic site. Consequently, these polypeptides represent an innovative class of GCase stabilizers and/or enhancers capable of modulating GCase without adversely affecting its inherent enzymatic activity.

In one aspect, the disclosure provides allosteric modulator polypeptides with specificity towards human glucocerebrosidase (GCase), an enzyme encoded by the human GBA1 gene. In one embodiment, the GCase is a wild-type GCase.

The polypeptide or GCase binder or binding agent of the present invention further provides the functionality of "modulating" GCase, wherein the term "modulation" may refer to the positive or negative effect on the GCase activity as compared to wild-type GCase alone, in the absence of the polypeptide binder, or as compared to a control sample. Alternatively, "modulation" may refer to positive or negative influence on the GCase protein (thermal) stability, which may hence result in a modulation of its enzymatic activity as compared to a control sample. Alternatively, "modulation" may refer to a positive or negative impact on the binding affinity of GCase with its orthosteric ligand(s) and/or on associated signal transduction efficacy. In this context, "control" refers to a GCase protein with normal or wild-type activity, or a sample where there is no polypeptide binder present. The term "control" may also describe a sample containing another polypeptide(s) known not to specifically bind to GCase, and/or not to affect GCase functionality and/or stability. In a specific embodiment, when the polypeptide binder comprises an ISVD, the control may contain an ISVD that does not specifically bind to the GCase protein or an ISVD that binds to GCase but does not modulate its activity and/or stability.

The allosteric modulation, as defined earlier, pertains to allosteric interaction as a mode of binding. It involves conformational binding to a site on GCase that is different from the orthosteric site. This conformational binding induces and/or stabilizes or acts as a chaperone for a GCase protein conformation, leading to the modulation of GCase functionality.

In alternative embodiments, the GCase is a mutated form of the GCase protein, referred to as a "mutant" or a "variant". The term "mutant human GCase protein" specifically refers to a protein that differs in one or more amino acids in the GCase sequence compared to SEQ. ID NO:81. The function of the "mutant human GCase protein" may vary from the wild-type GCase function, such as in enzymatic activity, thermodynamic stability, protein localization, substrate interaction, or other protein-related aspects. Specifically, one embodiment relates to an allosteric modulator polypeptide specifically binding a human GCase pathological mutant known to be encoded by the human GBA1 gene of a subject with onset or symptoms of diseases, in particular GBAl-related diseases or GCase-related diseases, or in particular GD or PD. In a specific embodiment, the mutated form of GCase carries a N370S (N409S) mutation. In a specific embodiment, the mutated form of GCase carries a L444P (L483P) mutation. In a specific embodiment, the mutated form of GCase carries a E326K (E365K) mutation. In a specific embodiment, the mutated form of GCase carries a T369M mutation. In a specific embodiment, the mutated form of GCase carries a RecNcil mutation. In a specific embodiment, the mutated form of GCase carries a 84GG (L29Afs*18) mutation. In a specific embodiment, the mutated form of GCase carries a A359X mutation. In a specific embodiment, the mutated form of GCase carries a c.84dupG mutation. In a specific embodiment, the mutated form of GCase carries a D140H mutation. In a specific embodiment, the mutated form of GCase carries a D399G mutation. In a specific embodiment, the mutated form of GCase carries a D409H mutation. In a specific embodiment, the mutated form of GCase carries a D443N mutation. In a specific embodiment, the mutated form of GCase carries a E235V mutation. In a specific embodiment, the mutated form of GCase carries a E388K mutation. In a specific embodiment, the mutated form of GCase carries a G389V mutation. In a specific embodiment, the mutated form of GCase carries a H255Q. mutation. In a specific embodiment, the mutated form of GCase carries a I161N mutation. In a specific embodiment, the mutated form of GCase carries a K(-27)R mutation. In a specific embodiment, the mutated form of GCase carries a L105R mutation. In a specific embodiment, the mutated form of GCase carries a L324P mutation. In a specific embodiment, the mutated form of GCase carries a N188R;S196P;V191G mutation. In a specific embodiment, the mutated form of GCase carries a Q256S mutation. In a specific embodiment, the mutated form of GCase carries a R120Q. mutation. In a specific embodiment, the mutated form of GCase carries a R120W mutation. In a specific embodiment, the mutated form of GCase carries a R262H mutation. In a specific embodiment, the mutated form of GCase carries a R463C mutation. In a specific embodiment, the mutated form of GCase carries a R463C/N370S mutation. In a specific embodiment, the mutated form of GCase carries a R496H mutation. In a specific embodiment, the mutated form of GCase carries a W184R mutation. In a specific embodiment, the mutated form of GCase carries a W393R mutation. In other specific embodiments, the mutated form of GCase may carry any other mutation. In a specific embodiment, the mutated form of GCase may carry more than one mutation selected from the mutations listed above and/or from other mutations.

In a further specific embodiment, the GCase-specific allosteric modulator polypeptide may be an antibody, active antibody fragment, immunoglobulin single variable domain (ISVD), single domain antibody, or VHH specifically designed to bind to human GCase.

Another aspect of this invention refers to GCase-specific allosteric polypeptides with GCase-modulatory properties. In one embodiment, such a GCase-specific allosteric polypeptide stabilizes GCase in a cell as compared to a control without said modulator, i.e., it induces the folding of GCase into a native stable conformation, as described above, and binds GCase at a binding site located on domain II and domain III.

More specifically, the term "stabilizing" the GCase may also encompass an increase in GCase thermal stability compared to the non-stabilized form of GCase (e.g., in the absence of the allosteric binder described herewith). Even more specifically, the increase in thermal stability can be measured or determined as an increase in the melting temperature of the protein. In a further specific embodiment, the stabilizing polypeptide, through allosteric binding to GCase, increases the melting temperature (Tm) of GCase by at least 4°C at neutral pH and/or by at least 2°C at pH below 7, such as in lysosomal conditions, compared to a control sample. The determination of thermal stability can be performed as exemplified in the present application, or by methods known to the skilled person. Also, the stability can be assessed using methods well-known in the art, for instance dynamic light scattering⁹⁸, differential scanning calorimetry⁹⁹, differential scanning fluorimetry⁷⁵, pulse-chase method¹⁰⁰, bleach-chase method¹⁰¹, cycloheximide-chase method¹⁰², circular dichroism¹⁰³, fluorescence-based activity assays¹⁰⁴, and other methods. In a preferred embodiment, binding of the polypeptide disclosed herewith to GCase involves an epitope comprising the amino acid residues of K77, V78, K79, H162, L165, Q166, A168, Q169, R170, P171, V172, S173, L174, A221, K224, L225, Q226, F227, W228, T272, H274, N275, R277, H306, E429, G430, P452, D453 of SEQ ID NO: 81.

In an alternative embodiment, said stabilizing polypeptide binds to a GCase homologue or mutant or variant, wherein the binding involves an epitope comprising amino acids corresponding to K77, V78, K79, H162, L165, Q166, A168, Q169, R170, P171, V172, S173, L174, A221, K224, L225, Q226, F227, W228, T272, H274, N275, R277, H306, E429, G430, P452, D453 of SEQ ID NO: 81, where one or more of these amino acids may have been modified or mutated.

In yet another embodiment, the GCase-specific allosteric stabilizer polypeptide contains an ISVD with specificity towards human GCase and comprises the complementarity-determining-regions (CDRs) of the ISVDs of the following sequences, or their humanized/optimized variants: i. QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSAKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGQCTYYSSGYYRDLRWYDYWGQGTQVTVSS (SEQ ID NO: 01); or ii. QVQLVESGGGLVQPGGSLRLSCAASGNIFSINAMGWYRQAPGKERELVADITSGGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCNADLGSIRWSPLKGQYEYDYWGQGTQVTVSS (SEQ ID NO: 04); or iii. QVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAVGWFRQAPGKEREFVAHISWTGGNIYYADSVKGR FTISRDNAKNTVLLQMNSLKPEDTAVYYCAVRPEYSASYYYATQYNYWGQGTQVTVSS (SEQ ID NO: 09), with the CDRs annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering system.

In an alternative embodiment, the GCase-specific allosteric stabilizer polypeptide contains an ISVD with specificity towards human GCase which comprises the following CDRs, or humanized/optimized variants thereof: i. Polypeptide 1:

- CDR1: GFTLDYYAIG (SEQ ID NO: 21),

- CDR2: CISSSDGSTYYADSAKG (SEQ ID NO: 22),

- CDR3: DRGQCTYYSSGYYRDLRWYDY (SEQ ID NO: 23); or ii. Polypeptide 2:

- CDR1: GNIFSINAMG (SEQ ID NO: 30),

- CDR2: DITSGGSTNYADSVKG (SEQ ID NO: 31),

- CDR3: DLGSIRWSPLKGQYEYDY (SEQ ID NO: 32); or iii. Polypeptide 3:

- CDR1: GRTFSSYAVG (SEQ ID NO: 45),

- CDR2: HISWTGGNIYYADSVKG (SEQ ID NO: 46),

- CDR3: RPEYSASYYYATQYNY (SEQ ID NO: 47).

In another embodiment, the GCase-specific allosteric stabilizer polypeptide contains ISVDs comprising any of the following sequences or sequences with at least 90 % amino acid identity, with changed amino acids located in one or more framework residues: i. QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSAKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGQCTYYSSGYYRDLRWYDYWGQGTQVTVSS (SEQ ID NO: 01); or ii. QVQLVESGGGLVQPGGSLRLSCAASGNIFSINAMGWYRQAPGKERELVADITSGGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCNADLGSIRWSPLKGQYEYDYWGQGTQVTVSS (SEQ ID NO: 04); or iii. QVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAVGWFRQAPGKEREFVAHISWTGGNIYYADSVKGR FTISRDNAKNTVLLQMNSLKPEDTAVYYCAVRPEYSASYYYATQYNYWGQGTQVTVSS (SEQ ID NO: 09).

Alternatively, the GCase-specific allosteric stabilizer polypeptide of the current embodiment has undergone humanization, as described above. Notably, all variants of the GCase-specific allosteric stabilizer polypeptide described in this embodiment are functional, i.e., show the ability to al losterically bind and stabilize GCase or GCase mutant or GCase homologue.

In another aspect, the present invention describes a GCase-specific allosteric modulator polypeptide which is able to increase intracellular GCase catalytic activity, understood as its ability to hydrolyze the beta-glycosidic linkages of glucocerebroside. Such a modulator contains an ISVD with specificity towards human GCase and comprises the complementarity-determining-regions (CDRs) of the ISVDs of the following sequences, or their humanized/optimized variants: i. QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTYYADSAKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCATDRGQCTYYSSGYYRDLRWYDYWGQGTQVTVSS (SEQ ID NO: 01); or ii. QVQLVESGGGLVQPGGSLRLSCAGSGFTFSSYAMSWVRQAPGKGLEWVSDISSDGGTTRYVESVKGR FTISRDNAKNTLYLQMNSLKPEDTAVYYCAKWSPGSGWFAQRDFEYWGQGTQVTVSS (SEQ ID NO: 02); or iii. QVQLVESGGGLVQPGGSLRLSCAASGFTFSMYGMSWVRQAPGKGPEWVSAISSGGEYTRYAHSVKG RFTISRDNAKNTLLLQM HSLKPEDTAVYYCAKWTPDSTWYRGHEYDYWGQGTQVTVSS (SEQ ID NO: 03); or iv. QVQLVESGGGLVQPGGSLRLSCAASGNIFSINAMGWYRQAPGKERELVADITSGGSTNYADSVKGRFT ISRDNAKNTVYLQMNSLKPEDTAVYYCNADLGSIRWSPLKGQYEYDYWGQGTQVTVSS (SEQ ID NO: 04); or v. QVQLVESGGGLVQAGGSLRLSCAASGRTFSRYSMGWFRQTPGKEREFVAAINWSGVNTHYADSVKG RFTISRDNAKNTVDLQMNSLKPEDTAVYFCASDDRPYNSVWTFDYWGQGTQVTVSS (SEQ ID NO: 05); or vi. QVQLVESGGGLVQPGGSLRLSCAASGSIFSINTMGWYRQAPGKEREMVAYIITFGSTNYADSVKGRFTI SGDNANNTMWLQMNSLKPEDTAVYYCYAAIRPTDSSTYTSYWGQGTQVTVSS (SEQ ID NO: 06); or vii. QVQLVESGGGLVQAGGSLQLSCAASGRTFEIYGMGWFRQAPGEERQFVAAIGRSGDVTYYADSVEGR FTISRSNSKNTVYLQMNSLKPEDAAVYYCAAQSSVYASLIYMSGYNNWGQGTQVTVSS (SEQ ID NO: 08); or viii. QVQLVESGGGLVQAGDSLRLSCAASGRTFSSYAVGWFRQAPGKEREFVAHISWTGGNIYYADSVKGR FTISRDNAKNTVLLQMNSLKPEDTAVYYCAVRPEYSASYYYATQYNYWGQGTQVTVSS (SEQ ID NO: 09); or ix. QVQLVESGGGLVQPGGSLRLSCAASGFTLDYYSIAWFRQAPGKEREGVSCISSSDGSTYYADSVKGRSTI SRDNAKNTVYLQMNSLKPEDTAVYYCAADDPLWRGGSEERSTWCQEYEYSYWGQGTQVTVSS (SEQ ID NO: 10); or x. QVQLVESGGGLVQAGGSLRLSCAASGSIFGINAMGWYRQAPGKQRELVAAITSGM NTNYADSVKGR FTISRDNAKNTVYLQMSDLKPEDTAVYYCSADIKTSAFRFRRTYWGKGTQVTVSS (SEQ ID NO: 13); or xi. QVQLVESGGGLVQAGGSLRLSCVASGFTFEDYTLGWFRQAPGKEREGVSCIRSSDGSTNYAASVKGRF TISRDNGKNMVYLQMGSLKPEDTAVYYCNADLRGDNYWGQGTQVTVSS (SEQ ID NO: 16); or xii. QVQLVESGGGLVQPGGSLTLSCVASGFSLEHYAMGWFRQAPGKEREGVSCISSRPGTYYYPSVAGRFTI SRGNAKNTVYLHMNNLKPEDTAVYYCAATAYESCWISKYDYWGRGTQVTVSS (SEQ ID NO: 18); or xiii. QVQLVESGGGLVQAGGSLRLSCVASRREIFSISDMGWYRQAPGQQRELVAAISRGNRTNYADSVKGR FTISRDNAKNALYLQMNSLKPEDTAVYYCTKSTLSFFDGRYDTRGQGTQVTVSS (SEQ ID NO: 19), with the CDRs annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering system.

In an alternative embodiment, the GCase-specific allosteric modulator polypeptide which increases GCase activity contains an ISVD with specificity towards human GCase which comprises the following CDRs, or their humanized/optimized variants: i. Polypeptide 1: - CDR1: GFTLDYYAIG (SEQ ID NO: 21),

- CDR2: CISSSDGSTYYADSAKG (SEQ ID NO: 22),

- CDR3: DRGQCTYYSSGYYRDLRWYDY (SEQ ID NO: 23); or ii. Polypeptide 2:

- CDR1: GFTFSSYAMS (SEQ ID NO: 24),

- CDR2: DISSDGGTTRYVESVKG (SEQ ID NO: 25),

- CDR3: WSPGSGWFAQRDFEY (SEQ ID NO: 26); or iii. Polypeptide 3:

- CDR1: GFTFSMYGMS (SEQ ID NO: 27);

- CDR2: AISSGGEYTRYAHSVKG (SEQ ID NO: 28),

- CDR3: WTPDSTWYRGHEYDY (SEQ ID NO: 29); or iv. Polypeptide 4:

- CDR1: GNIFSINAMG (SEQ ID NO: 30),

- CDR2: DITSGGSTNYADSVKG (SEQ ID NO: 31),

- CDR3: DLGSIRWSPLKGQYEYDY (SEQ ID NO: 32); or v. Polypeptide 5:

- CDR1: GRTFSRYSMG (SEQ ID NO: 33),

- CDR2: AINWSGVNTHYADSVKG (SEQ ID NO: 34),

- CDR3: DDRPYNSVWTFDY (SEQ ID NO: 35); or vi. Polypeptide 6:

- CDR1: GSIFSINTMG (SEQ ID NO: 36),

- CDR2: YIITFGSTNYADSVKG (SEQ ID NO: 37),

- CDR3: AIRPTDSSTYTSY (SEQ ID NO: 38); or vii. Polypeptide 7:

- CDR1: GRTFEIYGMG (SEQ ID NO: 42),

- CDR2: AIGRSGDVTYYADSVEG (SEQ ID NO: 43),

- CDR3: QSSVYASLIYMSGYNN (SEQ ID NO: 44); or viii. Polypeptide 8:

- CDR1: GRTFSSYAVG (SEQ ID NO: 45),

- CDR2: HISWTGGNIYYADSVKG (SEQ ID NO: 46),

- CDR3: RPEYSASYYYATQYNY (SEQ ID NO: 47); or ix. Polypeptide 9:

- CDR1: GFTLDYYSIA (SEQ ID NO: 48),

- CDR2: CISSSDGSTYYADSVKG (SEQ ID NO: 49), - CDR3: DDPLWRGGSEERSTWCQEYEYSY (SEQ ID NO: 50); or x. Polypeptide 10:

- CDR1: GSIFGINAMG (SEQ ID NO: 57),

- CDR2: AITSGMNTNYADSVKG (SEQ ID NO: 58),

- CDR3: DIKTSAFRFRRTY (SEQ ID NO: 5); or xi. Polypeptide 11:

- CDR1: GFTFEDYTLG (SEQ ID NO: 66),

- CDR2: CIRSSDGSTNYAASVKG (SEQ ID NO: 67),

- CDR3: DLRGDNY (SEQ ID NO: 68); or xii. Polypeptide 12:

- CDR1: GFSLEHYAMG (SEQ ID NO: 72),

- CDR2: CISSRPGTYYYPSVAG (SEQ ID NO: 73),

- CDR3: TAYESCWISKYDY (SEQ ID NO: 74); or xiii. Polypeptide 13:

- CDR1: RREIFSISDMG (SEQ ID NO: 75),

- CDR2: AISRGNRTNYADSVKG (SEQ ID NO: 76),

- CDR3: STLSFFDGRYDT (SEQ ID NO: 77).

In yet another aspect, the allosteric polypeptide GCase modulator of the invention may be coupled to a targeting moiety, or a functional moiety.

In one embodiment, the targeting moiety is an endoplasmic reticulum (ER)-targeting moiety. Nonlimiting examples of ER-targeting ligands include p-toluenesulfonyl group, dansyl group, and peptides such as KDEL peptide, pardaxin peptide, preproalbumin, and peptide encoded by the sequence MKWVTFLLLLFISAFSR (SEQ ID NO: 94). These ligands have the capability to recognize and bind to specific receptors or components of the ER, facilitating the delivery of molecules to the ER.

In another embodiment, the targeting moiety is a lysosome-targeting moiety. Non-limiting examples of lysosome-targeting molecules include mannose-6-phosphate receptor (M6PR), sortilin, folate receptor, ASPGR, IFITM3, molecules of the endosome/lysosome pathway (e.g., LIMP-1 , LIMP-2), and peptide encoded by the sequence KFERQKILDQRFFE (SEQ ID NO: 95).

In yet another embodiment, the GCase-specific allosteric polypeptide modulator described herein comprises an ISVD that is conjugated to a further functional moiety, wherein the functional moiety is a molecule or a component which performs an additional function for the binding polypeptides when used for a specific purpose. Said purpose may include therapeutic use, blood-brain barrier (BBB) crossing, halflife extension, and others. So the functional moiety conjugated to the GCase-specific ISVD of the binding agent may for instance comprise a therapeutic moiety, such as a biological or a neuronal target-specific drug, a half-life extension, a small-molecule compound, an enzyme, an antibody, a genome-editing component, such as a nuclease, a nucleic acid molecule, or a nanoparticle such as a liposome.

Another aspect of the invention relates the GCase-specific allosteric modulator, which is a multivalent or multispecific agent, as defined herein. In a specific embodiment, the multivalent or multispecific moieties of said binding agent may be directly linked or fused by a short spacer or linker. Alternatively, said multivalent or multispecific moieties of said GCase-specific allosteric modulator are present in the format of an Fc fusion or an antibody, or any other chimeric format as known to the skilled person and/or as described herein. Thus, another embodiment relates to said polypeptide binding agents that comprise one or more ISVDs (or variants or humanized forms thereof as described herein) specifically binding human GCase, wherein the at least one or more ISVD (or variant or humanized form thereof as described herein) are linked to another ISVD, or another moiety, by direct linking or by fusion via a spacer or linker, such as a peptide linker. In a preferred embodiment, the multivalent GCase-specific allosteric modulator comprises at least two moieties specifically binding GCase, or comprises at least two GCase-specific ISVDs.

In yet another aspect, the invention provides nucleic acid molecules such as isolated nucleic acids, (isolated) chimeric gene constructs, expression cassettes, recombinant vectors (such as expression or cloning vectors) comprising a nucleotide sequence, such a coding sequence, that encodes the GCase- specific binding agent as described herein.

A further aspect of the invention relates to a pharmaceutical composition comprising the GCase-specific allosteric modulator as described herein, the nucleic acid molecule or vector as described herein, the multivalent or multispecific binding agent as described herein, or the GCase-specific allosteric modulator comprising a functional moiety or a targeting moiety, as described herein, and/or optionally a further therapeutic agent, a carrier, excipient or diluent, as defined herein.

Another aspect of the invention describes medicaments or pharmaceutical compositions comprising the binding agent(s) and/or nucleic acid(s) encoding it/them, and/or vector(s) comprising the nucleic acid(s), as described herein. In particular, a pharmaceutical composition is a pharmaceutically acceptable composition; such compositions are in a particular embodiment further comprising a (pharmaceutically) suitable or acceptable carrier, diluent, stabilizer, etc.

Alternatively, the use of the binding agent or nucleic acid encoding it, as described herein, or the use of a pharmaceutical composition comprising the binding agent, nucleic acid encoding it, and/or a recombinant vector containing such nucleic acid, as described herein, is envisaged for medicinal purposes. In particular, the composition, binding agent or nucleic acid encoding it as described herein, or the medicament or pharmaceutical composition comprising a binding agent, nucleic acid encoding it, and/or a recombinant vector comprising such nucleic acid, as described herein, is envisioned for use in treating a subject with GCase-related disorders, as defined herewith. In a preferred embodiment, the GCase-specific allosteric modulator, and/or the nucleic acid encoding it, and/or a recombinant vector containing such nucleic acid, and/or the pharmaceutical composition comprising any of the above, may be used as a therapeutic and/or preventive treatment of Gaucher disease (GD) and/or Parkinson's disease (PD).

In any embodiment of this invention, the GCase-specific allosteric modulator may comprise an ISVD consisting of an amino acid sequence of any of the ISVD sequences described herein, wherein the amino acid residue at position 5 is a Valine (V) or alternatively, the amino acid residue at position 5 is a Glutamine (Q), applying the numbering as annotated herein, according to the Kabat numbering system.

In any embodiment of this invention, the GCase-specific allosteric modulator may comprise an ISVD with an optimized and/or humanized amino acid sequence, including alternative amino acid residues as those modified in SEQ ID NO: 97 compared to SEQ ID NO: 01; or those modified in SEQ ID NO: 101 compared to SEQ ID NO: 01; or those modified in SEQ ID NO: 105 compared to SEQ ID NO: 04; or those modified in SEQ ID NO: 109 compared to SEQ ID NO: 09; or those modified in SEQ ID NO: 113 compared to SEQ ID NO: 16; or those modified in SEQ ID NO: 117 compared to SEQ ID NO: 17; or those modified in SEQ ID NO: 121 compared to SEQ ID NO: 17; or those modified in SEQ ID NO: 125 compared to SEQ ID NO: 17.

It is important to understand that while specific embodiments, configurations, materials, and/or molecules have been discussed herein, various changes or modifications in form and detail can be made without deviating from the scope of this invention. The following examples are presented to better illustrate specific embodiments and should not be considered as limiting this application. The application is limited only by the claims.

Table 1. Amino acid sequences of polypeptides and nucleotide sequences of RNA molecules used in this application.

Table 2. Amino acid names and their abbreviations used throughout this application.

EXAMPLES Materials and methods

Immunization and Nb selection

A llama was immunized using a six-week protocol with weekly immunizations of GCase (Velaglucerase, VRPIV®, Takeda) in the presence of GERBU adjuvant, and blood was collected 4 days after the last injection. All animal vaccinations were performed in strict accordance with good practices and EU animal welfare legislation. The construction of immune libraries and Nb selection via phage display were performed using previously described protocols¹⁰⁵. In brief, starting from the blood collected from the llama after immunization, the open reading frames coding for the variable domains of the heavy-chain antibody repertoire were cloned in a pMESy4 phagemid vector (GenBank KF415192), resulting in an immune library of 1.6x1014 transformants. This Nb repertoire was expressed on the tip of filamentous phages after rescue with the VCSM13 helper phage. Four phage display selections (two rounds each) were performed using solid phase coating in a 96-well MaxiSorp NUNC-lmmuno ELISA plate (Thermo Fischer Scientific): (1) glycosylated GCase, (2) glycosylated GCase bound to CBE, (3) deglycosylated GCase, (4) deglycosylated GCase bound to CBE. For selections (2) and (4), proteins were incubated for 30 min on ice with 10 pM conduritol-p-epoxide (CBE). Solid phase coating of all proteins was performed in a coating buffer containing 100 mM NaHCOs at pH=8.2. Washing steps were performed with Mcllvaine buffer (100 mM NajHPC and 10 mM citric acid) at pH=5.4, and 0.4% milk was added to this buffer for the binding step. Several single colonies were selected after each round of phage display and sequence analysis was performed to classify the resulting Nb clones into sequence families based on their CDR3 sequences.

Nanobody cloning, expression and purification

After Nb selection, the Nb-coding open reading frames were re-cloned from the pMESy4 to the pHEN29 vector¹⁰⁶. Upon expression in an E. coli non-suppressor strain, both vectors yield proteins with an N- terminal pelB signal sequence to translocate the recombinant protein to the periplasm. Additionally, pMESy4 provides a C-terminal His6-tag and EPEA-tag (CaptureSelect™ C-tag), while pHEN29 provides a C -terminal LPETGG-His6-EPEA-tag that allows site-specific labelling of the proteins using Sortase chemistry¹⁰⁶. Expression of Nbs from the former plasmid was used for structural biology studies, while the latter was used for labelling of Nbs in BLI experiments.

The Nbs were expressed and purified as previously described¹⁰⁷. The Nb expression plasmids were transformed in E. coli WK6 (Su-) cells. Cells were grown at 37°C in Terrific Broth medium to an OD600 "'1.0. Protein expression was induced by adding 1 mM IPTG (isopropyl p-D-l-thiogalactopyranoside) followed by overnight incubation at 28°C. After harvesting, cells were lysed by osmotic shock to recover the periplasmic fraction. Nbs were purified via affinity chromatography on Ni²⁺-NTA Sepharose resin and were subsequently subjected to dialysis (50 mM HEPES pH=8.0 and 300 mM NaCI).

Deglycosylation of GCase

Velaglucerase (VRPIV®, Takeda) and Imiglucerase (Cerezyme®, Sanofi) were deglycosylated using PNGase F enzyme (New England Biolabs). The deglycosylation reaction was performed according to the recommendation from the manufacturer, using 0.5 U PNGase F / 1 pg glycosylated protein for 72 hours at 25°C. The deglycosylation reaction was confirmed by SDS-PAGE, and deglycosylated proteins were further purified using size exclusion chromatography (S75 10300 increase GL) in 10 mM MES pH=6.5, 100 mM NaCI, 1 mM DTT and 5% glycerol.

Enzyme-linked immunosorbent assays (ELISA)

GCase, deglycosylated GCase and the GCase N370S mutant were solid-phase coated on the bottom of a 96-well ELISA plates (Maxisorp Nunc-immuno plate, Thermo Fischer Scientific), using a concentration of 1 pg/mL protein in the coating buffer (100 mM NaHCO3 pH=8.2). All the washing, binding (0.4% milk) and blocking (4% milk) steps were performed using PBS supplemented with 0.05% Tween-20 (polysorbate). The binding of the Nbs to the coated protein was detected via their EPEA-tag using a 1:4000 CaptureSelectTM Biotin anti-C-tag conjugate (ThermoFischer Scientific) in combination with 1:1000 Streptavidin Alkaline Phosphatase (Promega). Color was developed by adding 100 pL of 3 mg/mL disodium 4-nitrophenyl phosphate solution (DNPP, Sigma Aldrich) dissolved in 100 mM Tris, 100 mM NaCI and 5 mM MgCL pH=9.5, and measured at 405 nm.

Biolayer Interferometry (BLI)

Prior to BLI experiments, Nbs expressed and purified from the pHEN29 plasmid marked with a C-terminal LPETGG-His6-EPEA tag (SEQ ID NO: 84) were site-specifically labelled at their C-terminus using Sortase- mediated exchange with a biotin-labelled GGGYK (SEQ ID NO: 85) peptide (GenicBio). GCase was randomly labelled on lysine residues using the EZ-Link™ Sulfo-NHS-LC-Biotin kit (Thermo Fischer Scientific), according to the manufacturers' recommendations. BLI measurements were performed using an Octet Red96 (ForteBio, Inc.) system in PBS pH=7.5, 0.01% Tween-20 supplemented with 0.1% BSA. Either biotinylated GCase or biotinylated Nbs were loaded onto streptavidin-coated (SA) biosensors at a concentration of 1 pg/mL, and the binding of a concentration gradient of unlabelled Nbs or GCase, respectively, was assessed. The association/dissociation traces were fitted with a 1:1 binding model using either the local, partial or global (full) options (implemented in the ForteBio Analysis Software). The resulting Req values were subsequently plotted against the Nb concentration and used to derive the KD values from the corresponding dose-response curves fitted on a Langmuir model. The figures were generated using GraphPad PrismlO.

Thermal Shift assays (TSA)

Thermal unfolding of GCase was followed by thermal shift assays using SYPRO orange fluorescence as a dye and a CFX connect real-time PCR system (Bio-Rad)⁷⁵. 0.25 mg/mL (4pM) GCase was incubated with 20 pM of the different Nbs for 30 min on ice and combined with 5x SYPRO Orange protein Gel Stain (ThermoFischer Scientific) for a total volume of 25 pL. The temperature was increased from 20°C to 80°C at l°C/min steps. All measurements were performed in triplicates and the melting temperatures were determined by fitting the first derivatives of the data with a Boltzmann sigmoidal equation (GraphPad Prism). ATm (°C) values signify the differences in melting temperature between GCase and GCase incubated with a specific Nb.

Structure determination and analysis

Imiglucerase (Cerezyme®) was partially deglycosylated prior to crystallization as previously described⁴. Crystals of this protein in complex with Nbl were obtained by co-crystallization using the sitting-drop vapor diffusion method at 277K. GCase (Imiglucerase) and Nbl proteins were mixed to obtain a concentration of 10 mg/mL and 3.5 mg/mL, respectively (1:1.2 molar ratio). Crystals were obtained in a crystallization solution of 1.6 M magnesium sulfate heptahydrate, 0.1 M MES pH=6.5. Crystals were cryoprotected in mother liquor supplemented with 25% glycerol.

Data were collected at 100K at the Proxima 2 beamline of the Soleil synchrotron (X = 0.9801 A), to a resolution of 1.7 A. Diffraction data were integrated and scaled with autoPROC¹⁰⁸, using the default pipeline including XDS, Truncate, Aimless and STARANISO¹⁰⁹. Crystal belonged to the tetragonal space group 1422. The structure was solved using the molecular replacement method based on PDB 2J25 for GCase and 7A17 (chain B) for Nbl, and refined with the phenix.refine interface (Phenix version 1.20.1)¹¹⁰ alternated with manual building in Coot¹¹¹. All structural figures were produced with PyMOL (The PyMOL Molecular Graphics System, Version 2.3.3, Schrodinger, LLC.).

Cell culture, plasmids and transfection

Human embryonic kidney cells (HEK293T) and human fibroblast cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich), supplemented with fetal bovine serum (FBS, 10% Sigma Aldrich) and 100 U/mL penicillin, and 100 pg/mL streptomycin (Sigma Aldrich) at 37°C and 5% CO2. For transient transfection, polyethyleneimine (PEI) was used as a transfection reagent and HEK293T cells were incubated with DNA:PEI (1:2) in OPTIMEM (Life Technologies) for two hours, before media change. After 48 h, activity assays, western blot analysis or imaging were performed.

Animals

Gbal-/- hN370S mice were purchased from the Jackson laboratories¹¹². Mouse genotyping was performed with WONDER Taq Hot START (Euroclone) using the following primers: 5'-TCCTCACCTCCTCAGATGCT-3' (mutant forward; SEQ ID NO: 86); 5'-ACCCTCGGGTTTTAAGCTG-3' (mutant reverse; SEQ ID NO: 87); 5'-CTCTGCAGTTGTGGTCGTGT-3' (wild-type forward; SEQ ID NO: 88); 5'-GTCCATGCTAAGCCCAGGT-3' (wild-type reverse; SEQ ID NO: 89); 5'-CTGTCCCTGTATGCCTCT GG-3' (internal positive control forward; SEQ ID NO: 90); 5'-AGATGGAGAAAGGACTAGGCTACA-3' (internal positive control reverse; SEQ ID NO: 91); 5'-CAGCCATGATGCTTACCCTAC-3' (transgene reverse; SEQ ID NO: 92); 5'-GCTAACCATGTTCATGCCTTC-3' (transgene forward; SEQ ID NO: 93).

Animals experiments were conducted according to the Italian Ministry of Health and the approval by the Ethical Committee of the University of Padova (authorization number: D2784.N.QHV).

Primary striatal astrocytes

Mouse primary astrocytes were obtained from postnatal animals between days 0 and 2. Brains were dissected from the skull and placed in a dish, containing cold Phosphate buffered saline (PBS, Sigma Aldrich). Olfactory bulbs and cortices were removed under an optic microscope and the striatum was transferred to a separate dish containing cold PBS. Tissue dissection was performed in fresh DMEM supplemented with 10% of FBS and 100 U/mL penicillin, and 100 pg/mL streptomycin. The supernatant was transferred to a new tube and centrifuged (400xg, 10 min) and the pellet was washed with 10 mL of DMEM. Cells were seeded at a density of 5x106 cells/10 mL medium in cell culture flasks maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 pg/mL streptomycin at 37 °C in a controlled 5% CO2 environment. The culture medium was changed after 7 days and again after additional 3-4 days. Independent experiments were carried out using cells obtained from different pups.

Measurement of GCase activity in live cells

A selective lysosomal GCase substrate, 5-(Pentafluorobenzoylamino) Fluorescein Di- -D- Glucopyranoside (PFB-FDGIu, ThermoFisher Scientific) was used to evaluate GCase activity in live cells. HEK293T cells were cultured in a 24-well plate (150000 cells/well) and transfected with mCherry plasmids (1 pg DNA/well). After 48 h, PFB-FDGIu (50 pg/mL) was added for 6h and the nPFB-FDGIu fluorescence was measured by BD FACSAria™ III Cell Sorter (Xex 492 nm and Xem 516 nm). Nontransfected cells were employed as controls and treated with ambroxol (50 pM) or conduritol- -epoxide (50 pM) for a duration of 24 hours. Ambroxol is recognized as a GCase activator, while conduritol-p- epoxide serves as an inhibitor of GCase.

Measurement of GCase activity in cell or tissue lysates

GCase activity was measured in HEK293T transfected with different ER- or lysosomal targeting Nbs by using the 4-methylumbelliferyl-p-D-glucopyranoside (4-MU) assay. Cells were cultured in a 12-well plate (300000 cells/well) and transfected with eGFP plasmids (2 DNA pg/well). After 48h, cells were lysed in RIPA buffer supplemented with protease inhibitors (Roche) and protein was quantified by Pierce® Bicinchoninic acid (BCA) Protein Assay Kit assay. Tissues were isolated from 8-month-old Gbal-/- hN370S transgenic mice and stored at -80°C. Then, tissues were lysed in RIPA buffer (ratio 1:4) supplemented with protease inhibitors (Roche), and protein was quantified by BCA Protein Assay Kit assay.

Cell lysate samples were prepared in citrate phosphate buffer pH=4.5 (0.1 M Citric Acid, 0.2 M NajHPC ) (20 pL, 2 pg of protein) and incubated with the 4-MU substrate (3 mM in citrate phosphate buffer with 0.2% taurocholate) for 90 min at 37°C. Tissue lysates were prepared in the same manner but they were preincubated with Nbs (2.5 pM) for 30' at 37 °C before initiating the incubation with the substrate. The reaction was stopped by adding 240 pL of stop buffer (0.2 M NaOH, 0.2 M Glycine pH=10). Fluorescence was measured in a fluorescence microplate reader (Victor X3, Perkin Elmer). The assay was performed three times.

Western blot analysis

HEK293T cells were cultured in a 12-well plate (300000 cells/well) and transfected with eGFP plasmids (2 pg DNA/well). After 48h, cells were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (Roche). Lysates were centrifuged at 20000 x g at 4°C. Protein concentration was determined using the Pierce® BCA Protein Assay Kit. Equal protein quantities were loaded onto gradient gels with a composition of 4-20% Tris-MOPS-SDS (sodium dodecyl sulfate), provided by GenScript. The resolved proteins were then transferred to PVDF membranes (BioRad), through a semi-dry Trans Blot™ TurboTM Transfer System (BioRad). PVDF membranes were blocked in Tris-buffered saline containing 0.1% Tween™ (TBS-T) and 5% non-fat dry milk for 1 h and then incubated overnight at 4°C with primary antibodies diluted in TBS-T plus 5% non-fat milk. The following primary antibodies were used: mouse anti- p-actin (1:10000, A2066 Sigma-Aldrich), rabbit anti-calnexin (1:1000, ab22595 Abeam), mouse anti- LAMP1 (1:400, sc-20011 Santa Cruz Biotechnology), anti-FLAG HRP (1:1000, A8592 Sigma-Aldrich), rabbit anti-GBAl (1:1000, G4171 Sigma-Aldrich). After incubation with horse-radish peroxidase (HRP)- conjugated secondary antibodies (goat anti-rabbit-HRP and goat anti-mouse-HRP, Sigma-Aldrich) at room temperature for 1 h, immunoreactive proteins were visualized using Immobilon® Classico Western HRP Substrate (Millipore) or Immobilon® Forte Western HRP Substrate (Millipore) by Imager CHEMI Premium detector (VWR). The densitometric analysis of the detected bands was performed using the IMAGE J software.

Lysosomal proteolytic activity

HEK293T cells were plated in a 24-well plate and transfected with the selected ER- or lysosomal targeting Nbs (1 pg/well). Lysosomal protease activity was evaluated using the DQ-Red BSA dye, a fluorogenic substrate for proteases that is hydrolyzed in acidic, hydrolase-active endo-lysosomes to smaller protein fluorescent peptides¹¹³. Cells were incubated with DQ-Red BSA (10 pg/mL) for 30 minutes. Then, fresh medium was added and fluorescence was measured after 2h by BD LSR Fortessa™ X-20 Cell Analyzer (X_ex 590 nm and X_em 620 nm). Chloroquine (CO. 50 pM), a lysosomotropic agent that blocks endosomal acidification, was used as a positive control.

ENDO H and PNGase F treatment of cell lysates

HEK293T cells were transfected with ER-targeting Nbl, Nb4 and Nb9 (500000 cells/well, 3 pg DNA/well) and then lysed in RIPA buffer, as described above. 30 pg of proteins from lysed cells were digested with 500 units of endoglycosidase H (ENDO H, Promega) and 10 units of Peptide N-glycosidase F (PNGase F, Promega) enzymes according to manufacturer's instructions, after which they were used for immunoblotting. Non-transfected cells subjected to all digestion steps without enzymes were used as a positive control.

Immunofluorescence image analysis

HEK293T cells were cultured in 24-well (50000 cells/well) and transfected with ER- and lysosomal targeting Nbs (1 pg DNA/well). Cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 20 min, permeabilized with PBS-0.1% Triton™ X-100 for 20 minutes and blocked with PBS-5% fetal bovine serum (FBS) for 1 h. Primary and secondary antibodies were prepared in a blocking solution (1:200 in PBS-5% FBS). The following antibodies were used: anti-FLAG antibody (F7425, Sigma-Aldrich) together with the anti-calnexin (ab22595, Sigma-Aldrich), goat anti-rabbit Alexa Fluor 633 (A21071, ThermoFisher Scientific) and goat anti-mouse Alexa Fluor™ 568 (A11004, ThermoFisher Scientific).

Nuclei were stained with Hoechst 33258 pentahydrate (bis-benzimide) (Invitrogen, 1:10000 in H2O). The images were captured utilizing a 63x magnification objective on a Zeiss LSM700 laser scanning confocal microscope. The co-localization of ER-targeting Nbs in the ER of transfected cells was evaluated by calculating the Pearson coefficient using a colocalization plugin (Jacop) of Image J software.

N370S GCase purification

Recombinant GCase N370S mutant was produced using 293T Freestyle cells (Thermo Fisher, cat. no. R79007) grown in suspension in 293 Freestyle medium (Thermo Fisher). Cells were grown up to 10^A6 cells/ml at 37°C and 8% CO2 in agitation at 125 rpm, then transfected using lpg of pCMV_GBA_N370S_His and 3pg of PEI per 10^A6 cells in OPTIMEM (Life Technologies). 12 hours after transfection, 3.5mM valproic acid dissolved in water was added to the cell suspension and incubation was continued for 96 hours. Then, the medium containing the secreted N370S GCase protein was collected and clarified by centrifugation at 500 x g for 20 min at 4°C. The collected supernatant was filtered at 4°C with a 0.22 pm filter. The medium was then loaded on a His-select Nickel affinity column (Millipore) equilibrated in 50 mM Na^PC , 300 mM NaCI, 10 mM imidazole pH=8. The column was then washed with 50mM NaHjPC , 300 mM NaCI, 30 mM imidazole pH=8 and the protein was eluted with 50mM NaHjPC , 300 M NaCI, 30 mM imidazole pH=8. Buffer exchange was performed via PD-10 desalting column (GE Healthcare) to 50mM MES pH=5.0 for experiments and storage. Protein quality was confirmed by SDS-PAGE and western blot analysis.

Measurement of the activity of recombinant GCase

GCase activity was measured on the commercially available enzyme (Velaglucerase) and on recombinant N370S GCase. Briefly, protein samples were dissolved in citrate phosphate buffer pH=4.5 (0.1 M citric acid, 0.2 M NajHPC ) (20 pL, 2 pg of protein) and re-incubated with Nbs (2.5 pM) for 30' at 37 °C. Following, incubation with the 4-MU substrate (3 mM in citrate phosphate buffer with 0.2% taurodeoxycholate, Sigma-Aldrich) for 90 min at 37°C was performed. The reaction was stopped by adding 240 pL of stop buffer (0.2 M NaOH, 0.2 M glycine pH=10). Fluorescence was measured in a fluorescence microplate reader (Victor X3, Perkin Elmer). The assay was performed in a triplicate or quadruplicate.

Statistical analysis

Data were gathered from a minimum of four independent experiments. Statistical analysis and graphical representation were performed using GraphPad Prism Software Inc. (Version 8), employing a one-way ANOVA with Tukey's multiple comparison test. Statistical significance was considered at a P-value <0.05.

Nanobody cloning into vectors for expression in mammalian cells

For the expression of Nbs in mammalian cells, two different plasmid backbones were used, pRP[Exp]- Hygro-CMV for transfection and pLV[Exp]-Puro-EFlA for lentivirus production; both were customized and purchased from VectorBuilder Inc.

Plasmids were designed for each Nb to be expressed with a 3xFLAG epitope at the C-terminus and a sequence targeting the endoplasmic reticulum (ER) or the lysosome (LYSO). Furthermore, the expression of a fluorescent protein (EGFP or mCherry) via IRES was included in order to easily identify transfected/transduced cells. VectorBuilder provided plasmids with a Stuffer ORF sequence (amino acid 2-83 of E. coll beta-galactosidase) flanked by Nhel and BstBI restriction sites.

The ER targeting sequence was selected from the pool of known eukaryotic proteins with established ER-targeting capabilities and well-understood transport mechanisms. In particular, MKWVTFLLLLFISAFSR (SEQ ID NO: 94) is the preproalbumin signal peptide and was inserted upstream of the Nhel site in the ER plasmid series.

Targeting of proteins to the lysosome has a more complex mechanism and a single and well-defined targeting sequence is still lacking. In the study conducted by Fan et al. in 2014¹¹⁴, the authors showcased the feasibility of directing a protein of interest to the lysosomes. This was achieved by fusing the protein at its C-terminus with a sequence derived from the combination of three distinct signal sequences recognized for chaperone-mediated autophagy (CMA-targeting motif, CTM). Hence, in the current application, an identical sequence, namely KFERQKILDQRFFE (SEQ. ID NO: 95), was employed as a signal peptide for lysosomal targeting. The sequence was included in the frame following the 3XFLAG in the LYSO plasmid series.

The Nbs sequences of interest were uniformly amplified through PCR using primers designed based on conserved regions at both the N-terminus and C-terminus. These primers were designed to incorporate Nhel and BstBI restriction sites. The plasmids were all digested with these endonucleases to eliminate the Stuffer ORF and ligated with the sequences of interest. The original plasmids with Stuffer ORF were used as transfection controls.

List of plasmids i. pRP[Exp]-Hygro-CMV>{ER-ORF_Stuffer/3xFLAG}:IRES:EGFP (VectorBuilder Inc.); ii. pRP[Exp]-Hygro-CMV>{ER-ORF_Stuffer/3xFLAG}:IRES:mCherry (VectorBuilder Inc.); iii. pRP[Exp]-Hygro-CMV>{ORF_Stuffer/3xFLAG-LYSO}:IRES:EGFP (VectorBuilder Inc.); iv. pRP[Exp]-Hygro-CMV>{ORF_Stuffer/3xFLAG-LYSO}:IRES:mCherry (VectorBuilder Inc.); v. pRP[Exp]-Hygro-CMV>{ER-ORF_NbXX/3xFLAG}:IRES:EGFP; vi. pRP[Exp]-Hygro-CMV>{ER-ORF_NbXX/3xFLAG}:IRES:mCherry; vii. pRP[Exp]-Hygro-CMV>{ORF_NbXX /3xFLAG-LYSO}:IRES:EGFP; viii. pRP[Exp]-Hygro-CMV>{ORF_NbXX/3xFLAG-LYSO}:IRES:mCherry; ix. pLV[Exp]-Puro-EFlA>{ER-ORF_Stuffer/3xFLAG}:IRES:mCherry (VectorBuilder Inc.); x. pLV[Exp]-Puro-EFlA>{ORF_Stuffer/3xFLAG-LYSO}:IRES:mCherry (VectorBuilder Inc.); xi. pLV[Exp]-Puro-EFlA>{ER-ORF_NbXX/3xFLAG}:IRES:mCherry; xii. pLV[Exp]-Puro-EFlA>{ORF_NbXX/3xFLAG-LYSO}:IRES:mCherry.

EXAMPLE 1. Generation of GCase-targeting nanobodies.

In order to generate nanobodies (Nbs) that specifically bind to human lysosomal GCase, a llama was immunized with a commercially available form of GCase (Velaglucerase, VPRIV®)¹¹⁵. To maximize the chances of obtaining a large repertoire of Nbs which would preferentially bind GCase irrespective of its glycosylation pattern and outside of its active site pocket, various phage display selection strategies were used in parallel. To this end, GCase underwent initial deglycosylation using Peptide:N-glycosidase F (PNGase F) and/or was subjected to interaction with its covalent inhibitor conduritol-p-epoxide (CBE)¹¹⁶, resulting in the following combinations that were subsequently used in two rounds of phage display panning: i. glycosylated GCase, ii. glycosylated GCase bound to CBE, ill. deglycosylated GCase, iv. deglycosylated GCase bound to CBE.

Sequencing of the Nb open reading frames resulting from these 4 selection strategies provided 38 unique sequences. These were then organized into 20 sequence families, wherein members belonging to the same sequence family exhibit more than 80% sequence identity in their complementary determining regions 3 (CDR3). One representative of each family was re-cloned into a pHEN29 vector and subsequently expressed in the periplasm of E. coli as a C-terminally LPETGG-Hisg-EPEA-tagged protein. The respective 20 Nbs were purified to achieve complete homogeneity (Figure 1A-B).

The binding of the purified Nbs to GCase (Velaglucerase) was first confirmed via ELISA, in which binding was defined as a fluorescent signal at least 3-fold higher than the GCase background signal and 3-fold higher than the signal produced by an irrelevant Nb. Using this approach, the following 11 out of the 20 tested Nbs were found to bind to GCase: Nbl, Nb2, Nb3, Nb4, Nb6, Nb7, Nb8, Nb9, NblO, Nbl6, Nbl7 (Figure 2A). Notably, while no binding signal was observed for Nbl2 when glycosylated GCase was used, a clear binding signal was detected with deglycosylated GCase. Likewise, a significantly stronger ELISA signal was observed for NblO when utilizing the deglycosylated GCase. These observations are aligned with the fact that both Nbs resulted from the phage display panning on deglycosylated GCase.

Next, biolayer interferometry (BLI) was used to determine the binding affinities (K_D) of the Nbs for (glycosylated) GCase (Figure 2B-C). To this end, randomly biotinylated GCase was immobilized on a streptavidin biosensor and titrated with increasing amounts of each of the Nbs, after which the equilibrium signals were plotted against the Nb concentrations and fitted on a Langmuir equation. Overall, the obtained K_D values were in good agreement with the outcomes of ELISA. K_D values in the sub-micromolar range were obtained for 9 of the Nbs, with Nbl, Nb8 and Nbl7 showing the highest affinities (K_D <5 nM). Nb5 and Nbl8 were characterized by low-affinity binding (K_D in pM-range), while binding of NblO and Nbl2 could only be observed when using deglycosylated GCase. No clear binding signals in BLI were obtained for Nbll, Nbl3, Nbl4, Nbl5, Nbl6, Nbl9 and Nb20, while binding of Nbl6 was evidently observed in ELISA.

EXAMPLE 2. The influence of the generated nanobodies on the activity of GCase in vitro.

A classical 4-MU-based assay was used to characterize the impact of the 20 Nbs from EXAMPLE 1 on the activity of wild type GCase. The aim of this approach was to evaluate if any of the Nbs were able to increase the GCase activity in vitro, either by slowing down the time-dependent unfolding of the enzyme or by activating it in an allosteric manner. To this end, GCase (Velaglucerase) was incubated for 30 minutes at 37°C in the presence of each Nb to facilitate complex formation (if any). Subsequently, the 4- MU substrate was introduced, and the reaction mixture was incubated for 90 minutes. After this period, the reaction was halted, and the measurement of fluorogenic products was conducted (Figure 3A). Using this approach, nanobodies able to increase the activity of the wild type (WT) GCase by more than 2-fold (Nb3, Nb5, Nb6, Nbl6, Nbl8) or by 1.5 -2 fold (Nbl, Nb2, Nb4, Nb8, Nb9, NblO Nbl3 and Nbl9) were identified. The other Nbs had no significant effect on the enzymatic activity of GCase (Figure 3B). Isofagomine was used as a control and was able to reduce the GCase activity by about 70%. To understand if the presence of other proteins or cofactors would change the outcome of these measurements, the 4-MU activity assay was performed on cell lysates expressing wild type GCase, following the same experimental setup as described above. Notably, only NblO and Nbl6 were able to increase GCase activity by >2 fold, while Nb9, Nbl3 and Nbl8 improved GCase activity by 1.5-2 fold. Hence, Nb9, NblO, Nbl3, Nbl6 and Nbl8 were able to increase the enzymatic activity of GCase in both tested conditions.

EXAMPLE 3. The impact of the generated nanobodies on GCase thermal stability.

Pathogenic mutations in the GBA1 gene can lead to decreased cellular GCase activity by affecting the protein stability leading to its unfolding in the ER. The development of molecular chaperones that increase GCase protein stability, and thereby facilitate its correct trafficking through the ER toward the lysosome, is therefore regarded as a valid therapeutic strategy^117-119. Hence, fluorescence-based thermal shift assay (TSA)¹²⁰ was employed to assess the influence of the 20 Nbs obtained in EXAMPLE 1 on GCase thermal stability. First, the melting temperature (T_m) of GCase was determined at pH=7.0 and pH=5.2 (cytosolic and lysosomal pH, respectively), yielding corresponding T_m values of 50.8°C and 58.0°C. Screening of the GCase stability in the presence of the full set of Nbs identified three Nbs with the ability to significantly stabilize GCase at cytosolic pH, including Nbl (yielding an increase in T_m of 7°C), Nb4 (yielding an increase in T_m of 4°C) and Nb9 (yielding an increase in T_m of 4°C) (Figure 4A). These stabilizing effects were less pronounced at lysosomal pH, with Nbl and Nb9 providing an increase in T_m of 3°C and 2°C, respectively. Nb4 was not able to provide any additional stabilization at lysosomal pH. Notably, a covalent active-site binding GCase inhibitor CBE increased the enzyme stability of about 10°C (Figure 4B); however, this compound is known in the art to inhibit the catalytic activity of GCase¹²¹.

EXAMPLE 4. Identification of the hot-spot epitope for GCase stability.

Identification and comparison of the binding regions of Nbl, Nb4, and Nb9 on GCase were conducted, as these Nbs, originating from different sequence families, demonstrated the ability to stabilize the GCase fold, as shown in EXAMPLE 3. To this end, BLI-based epitope mapping of these 3 Nbs was performed. Nbl7 was also included in the analysis as a high-affinity but non-stabilizing and nonactivating Nb control. A pairwise competition-binding experiment was conducted, wherein each of the Nbs was sequentially biotinylated and immobilized on the BLI sensor. The remaining Nbs were introduced in surplus to GCase in solution to evaluate their impact on GCase binding. The analysis revealed competition among Nbl, Nb4, and Nb9, but not Nbl7, for GCase binding (Figure 5A-B). This observation implies that all the stabilizing Nbs bind to the same or a closely overlapping GCase epitope.

EXAMPLE 5. The structure of GCase bound with Nbl reveals the protein stabilization mechanism.

The complex of GCase-Nbl was crystalized to identify and characterize the stability hotspot in GCase, as well as to unravel the mechanism by which nanobodies can stabilize GCase. To this end, a deglycosylated form of imiglucerase (Cerezyme®) was used as a source of GCase, which was mixed in a 1:1.2 ratio with Nbl before initiating crystallizations¹²². Diffraction-quality crystals were obtained using 1.6 M magnesium sulfate heptahydrate and 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) (pH=6.5) as the crystallization solution. Diffraction data were collected and the crystal structure was refined to 1.7 A resolution. The refined structure revealed one molecule of GCase bound to one molecule of Nbl in the asymmetric unit (Figure 6A). As previously described, the structure of GCase displays a globular fold formed by 3 non-contiguous domains: i. domain I (residues 1-29 and 383-414) is a small three-stranded antiparallel -sheet, ii. domain II (residues 30-77 and 431-497) forms an eight-stranded p-barrel, and ill. domain III (residues 78-382 and 415-430) adopts an (P/a)g triose-phosphate isomerase barrel, which contains the active site and the two catalytic residues E235 and E340³-⁴-¹²³.

During the refinement, sugar moieties were added on residues N19 and N270, which are well-known and thoroughly characterized GCase glycosylation sites¹²³. Additionally, two magnesium ions were also included in the GCase structure, one of which is located in the active site and directly interacts with the two catalytic glutamate residues.

The crystal structure revealed that Nbl binds to GCase on the opposite side of the active site and interacts with the protein at the interface between domain II and III (Figure 6B). Specifically, the following GCase epitope residues were identified to be involved in Nbl binding: residues 77, 452 and 453 of domain II, and residues 78, 79, 162, 165, 166, 168, 169, 170, 171, 172, 173, 174, 221, 224, 225, 226, 227 , 228, il, 27 , 275, 277, 306, 429 and 430 of domain III, wherein the residues are numbered as present in the GCase wild type sequence of SEQ. ID NO: 81.

The observation that the binding site of Nbl is far from the GCase active pocket was in alignment with the finding that this Nb does not negatively interfere with the GCase catalytic activity (EXAMPLE 2). Furthermore, the majority of GCase interaction residues on Nbl are located on CDR2 and CDR3. CDR1 has only 2 residues interacting with GCase domain II, with the main chain carbonyl of its residue Asp32 forming an H-bond with GCase residue Lys77. CDR2 forms interactions with residues located on GCase domains II and III. In particular, Ser55 forms an H-bond with Lys77 of domain II, while Thr60, Tyr61, Tyr62 and Asp64 form multiple salt bridges and/or H-bonds with residues Thr272, His274, Asn275 and Arg277 from domain III. Finally, the majority of interactions are mediated by the very long CDR3 loop that partially folds into a short stretch of an a-helix. Residues from CDR3 extensively interact with GCase domain III, with the side chain of Gln226 of GCase forming multiple hydrogen bonds with the main chain atoms of CDR3. The binding mode of Nbl at the interface of two GCase domains, as described herein, possibly explains the stabilizing effect of this Nb exerted by keeping these two GCase domains in tight proximity.

Moreover, superposition of the structure of the GCase-Nbl complex on the structure of an unbound GCase (chain A of PDB 1OGS) revealed no major conformational changes in GCase induced by the Nbl binding (root-mean-square deviation of all atoms = 0.260 A; root-mean-square deviation of C_a atoms = 0.229 A)⁴. Only minor conformational changes were observed in the loops surrounding the entry to the GCase active site pocket, in particular in loop 1 (residues 311-319), loop 2 (residues 345-349) and loop 3 (residues 394-399). Notably, the structure of Nbl-GCase complex shows GCase in a state that resembles the "active state", with loop 1 adopting a nearly helical conformation, the side chain of residue Asp315 pointing in the direction of residue Asn370, and the bulky side chains of Trp348 and Arg395 oriented away from the active site^3,124'¹²⁵.

EXAMPLE 6. Nanobodies expressed in cellular models can be either directed to the ER or to the lysosomes, without altering any of these two intracellular compartments.

Based on the data described in previous EXAMPLES, the following Nbs were selected for their significant ability to bind, activate and/or stabilize wild type GCase: Nbl, Nb4, Nb9, NblO and Nbl6. In order to test the ability of these Nbs to impact GCase functionality in cell models, a set of plasmids for the expression of these five Nbs in mammalian cultures was designed. These plasmid-derived Nbs were fused with a 3xFLAG® protein (Sigma-Aldrich), and the vectors bi-cistronically co-expressed either eGFP or mCherry markers, which enabled quantification of the Nbs and visualization of the transfected cells. Moreover, a targeting strategy was implemented to deliver the Nbs specifically to the intracellular compartments in which they may be beneficial, i.e., the ER or the lysosomes, using well-characterized signaling peptides¹¹⁴-¹²⁶. Expression vectors encoding for fragments with a non-relevant and non-functional protein (mock) and/or untransfected cells were used as controls. First, the five lysosome-targeted Nbs and the five ER-targeted Nbs were expressed in HEK293T cells. The expression levels of the Nbs were assessed by western blot, while their intracellular localization was evaluated by immunocytochemistry and confocal imaging (Figure 7). Western blot analysis revealed that various Nbs were characterized by different expression levels, which may be ascribed to differences in their stability, structure and/or sequences (Figure 7A-D). Lysosomal-targeted Nbs, particularly Nbl and NblO, showed a reduced expression level compared to the same Nbs targeted to the ER and to the other lysosome-targeted Nbs. Notably, when high expression levels were reached, the cells transfected with lysosome-targeted Nbs appeared more round-shaped compared with the un-transfected or with the ER- Nbs transfected cells, while lower expression levels had no impact on cell morphology. The colocalization between ER-targeted Nbs and the ER marker calnexin was quantified using the Pearson correlation coefficient, whereby positive correlation values ranging between 0 and 1 indicate colocalization (Figure 7E-F). The analysis revealed the average values of Pearson coefficients between 0.2 and 0.4. The partial colocalization (i.e., Pearson coefficient <1) implies that ER-targeted Nbs might have the capability to exit the ER, potentially via binding to GCase, which is subsequently transported to the lysosomes. Likewise, localization of ER-targeted Nbs to the lysosomes in this cellular model (HEK293T cells overexpressing the selected Nbs) was evaluated by calculating the Pearson's coefficient based on the colocalization between the flag and LAMP2A staining. The analysis showed that all Nbs exhibited some degree of colocalization with the lysosomal marker, with Nb9 displaying a significantly higher colocalization compared to the Mock transfection (Figure 7G-H).

Following, the effects of the overexpression of the ER- or lysosome-targeted Nbs were assessed. The expression level of calnexin, a Ca²⁺-binding lectin chaperone induced during ER stress¹²⁷, was measured to evaluate the state of the ER (Figure 8A). Furthermore, the expression level of lysosomal-associated membrane protein 1 (LAMP1), a major component of the lysosomal membrane¹²⁸, was measured to characterize the state of the lysosomes (Figure 8B). Notably, none of the markers was affected by the presence of Nbs (Figure 8C-D), suggesting the lack of ER-stress and lysosome enlargement during ERand lysosome-targeted Nb expression, respectively.

Confocal imaging revealed that Nbl6, similar to Nb9, exhibits reduced colocalization with calnexin, an ER marker. However, unlike ER-Nb9, the colocalization of ER-Nbl6 with the lysosomal marker LAMP1 remained unchanged. This supports the notion that while ER-Nbl6 can exit the ER, it is likely less stable than ER-Nb9 upon reaching the lysosomal compartment, where it may be degraded after exerting its function on GCase activity. EXAMPLE 7. A subset of lysosome-targeted nanobodies overexpressed in HEK293T cells enhance GCase activity in cell lysates.

To assess the impact of the different Nbs on GCase functionality, the levels and the activity of the enzyme in lysates from Nb-overexpressing HEK293T cells (as described in EXAMPLE 6) were measured. Concerning the ER-targeted Nbs, the expression level of GCase remained unchanged (Figure 9A). This outcome correlated with the absence of noteworthy differences observed in cells transfected with ER- targeted Nbs regarding GCase activity, as measured by the 4-MU assay (Figure 9B). None of the lysosomal-targeted Nbs affected GCase protein levels, mirroring the outcomes observed with the ER- targeted Nbs (Figure 9C). However, the lysosomal-targeted Nbs demonstrated the ability to enhance GCase activity (Figure 9D).

EXAMPLE 8. ER-targeted Nbl and Nb9 demonstrate the capacity to improve lysosomal GCase activity in wild-type cells.

To evaluate the impact of Nb expression on GCase functionality in vitro, the lysosomal GCase activity in live cells was investigated by flow-cytometry with the use of a fluorogenic substrate 5- (Pentafluorobenzoylamino)Fluorescein Di-P-D-Glucopyranoside (PFB-FDGIu). PFB-FDGIu was previously used to asses GCase activity in peripheral blood monocytes from GD patients and to monitor ERT efficacy in such subjects¹²⁹. It was also employed in cellular assays monitoring GCase activity in live neurons¹³⁰.

Experiments described in the current Example involved cells expressing lysosome- or ER- targeted Nbl, Nb4 or Nb9, together with an mCherry reporter. The Nbs directed towards the lysosomes did not trigger any alteration in the enzyme's lysosomal activity (Figure 10A). However, ER-targeted Nb4 and Nb9 significantly boosted endogenous lysosomal GCase activity, indicating that these Nbs hold promise as effective stabilizers of lysosomal GCase (Figure 10B).

EXAMPLE 9. ER-targeted Nbl and Nb9 improve GCase trafficking.

The ENDO H and PNGase F assay was performed to assess the ability of ER-targeted Nbl, Nb4 and Nb9 to improve the lysosomal GCase trafficking. The ENDO H enzyme eliminates immature glycans that have not completed the final processing in the Golgi apparatus. In contrast, PNGase F functions by removing the complex oligosaccharides attached to a protein upon its arrival in the Golgi, after which the protein is transported to the lysosomes. Therefore, the susceptibility of GCase to ENDO H or PNGase F serves as an indicator of the quantity of protein located in the ER or beyond the ER (post-ER), respectively. Assessing the ratio between ENDO H-resistant GCase and ENDO H-sensitive GCase allowed for the evaluation of the influence of the Nbs on the trafficking of GCase to the lysosomal compartment. Indeed, western blot analysis revealed that Nbl and Nb9 significantly increased the post-ER/ER GCase ratio in HEK293T cells, suggesting that they were able to promote the correct folding and trafficking of the protein through the ER and the Golgi (Figure 11A-B).

EXAMPLE 10. A subset of nanobodies binds the GCase N370S mutant and restores its enzymatic activity in vitro.

The N370S mutant is the most common GCase variant associated with PD and GD. The mutation leads to a significant reduction in the catalytic activity of GCase, despite increasing the enzyme's stability⁴³-¹³¹. Moreover, this mutant appears to undergo misprocessing in the ER, causing ER stress in iPSC-derived dopaminergic neurons⁴³. Unsurprisingly, significantly increased a-syn levels were detected in cholinergic neurons obtained from neural crest stem cells of PD patients carrying the heterozygous N370S mutation³⁹. Additionally, the retention of N370S in the ER was observed in fibroblasts from PD patients¹³².

Binding of the Nbs to the N370S and their impact on the mutant's activity were assessed using a purified recombinant GCase-N370S protein. First, the binding of all 20 Nbs (as described in Example 1) with the mutant protein was tested in ELISA. By employing a threshold criterion of at least 3-fold higher than the GCase background and 3-fold higher than the signal of the non-relevant Nb, strong binding was identified for the following six nanobodies: Nbl, Nb6, Nb8, NblO, Nbl6, and Nbl7 (Figure 2A). Then, the BLI assay was used to determine the binding affinities (K_D) of Nbl, Nb4, Nb9, NblO, Nbl6 and Nbl7. In this approach, the Nbs were site-specifically biotinylated at their C-terminus, captured on a streptavidin sensor and titrated with increasing concentrations of the N370S mutant. No binding with WT GCase was observed for Nbl6. The remaining Nbs demonstrated binding to the N370S mutant with affinities similar to those found for the WT GCase, except for Nbl, which exhibited an approximately 5-fold lower affinity for the mutant. On the other hand, NblO displayed a 5-fold higher affinity compared to the WT.

Next, the impact of Nb binding on GCase-N370S mutant activity in vitro was determined in a classical 4- MU assay using Nbl, Nb4, Nb9, NblO and Nbl6. A significant enzymatic activity increase was observed for NblO and Nbl6 (Figure 12A). Following, the impact of these Nbs on gut tissue lysates derived from a mouse model carrying the human N370S mutation was assessed to evaluate their therapeutic potential within a more physiologically relevant setting. Gbal ^/_ hN370S mice were used, which express the human GBA1 gene in a null murine Gbal background¹³³. In this approach, also NblO and Nbl6 exhibited a notable capability to restore the catalytic activity of the mutant GCase (Figure 12B).

Following, the impact of selected Nbs on N370S mutant GCase activity was evaluated in live cells through flow cytometry using the fluorogenic substrate PFB-FDGIu, as previously described¹³⁴. Here, GCase activity was evaluated in GBA1 knockdown HEK293T cells (clone 11F) overexpressing the N370S GCase mutant. Co-expression of the N370S GCase mutant with ER-Nb4 and ER-Nb9 in this model resulted in a 60-70% increase in lysosomal N370S GCase activity compared to ER-mock transfected cells, whereas ER- Nbl and ER-Nbl6 had no effect on the mutant (Figure 12C). On the other hand, co-expression of the N370S GCase mutant with lysosome-targeted Nbl6 significantly enhanced lysosomal N370S GCase activity compared to lysosome-mock transfected cells (Figure 12D).

EXAMPLE 11. Comparison of the GCase binding pockets among various established small molecules, GCase's natural ligands, and Nbl.

The GCase binding sites of several small molecules known in the art were compared in silica with the GCase binding of Nbl. To this end, either experimental structures of GCase in a complex with these compounds or results of molecular docking simulations, as described in literature, were used. The PyMOL Molecular Graphics System (version 2.3.3, Schrodinger, LLC) was utilized to generate all visualizations.

As detailed in Example 5, Nbl binds GCase distantly from the enzyme's active site, i.e., at the interface of domain II and III (Figure 6B). This interaction mode minimizes the probability of competition for the binding site between stabilizing Nbs that compete for the GCase-epitope of Nbl and GCase substrates. Moreover, the stabilizing effect of such Nbs may stem precisely from this interaction encompassing domain II and III, thereby maintaining a tight connection between both domains. The structural analysis revealed that the stabilizing Nbs competing for the GCase-epitope of Nbl bind to a genuine allosteric pocket, situated at a considerable distance from the GCase active site, which proves to be crucial for GCase stability (Figure 13A).

In contrast, in silica modeling of a noniminosugar GCase binder JZ-5029⁵¹ revealed that the molecule's binding position overlaps with the enzyme's active site, as well as with the binding site of GCase's inhibitor isofagomine (Figure 13B), suggesting that JZ-5029 might act as a competitive inhibitor of GCase. This possibility is further highlighted by the observation that the molecule was shown to inhibit GCase in vitro by binding to an epitope proximal to the enzyme's active site. This observation suggests that JZ-5029 would need to be dissociated from the complex upon the enzyme's arrival to the lysosomes, in order to avoid potential competition with the GCase substrate (i.e., GlcCer).

Yet another small-molecule chaperone of GCase, NCGC00241607 (NCGC607), is characterized by 6 allosteric binding sites on the GCase surface³⁵-⁵³. However, molecular docking simulations revealed that its most energetically favorable binding site (BS1) consists of residues located nearby to the active site of the enzyme (Figure 13C).

Next, compound 14 (member of a new class of pyrrolo[2,3-b]pyrazine molecules) described in a 2021 study⁵⁴ emerges as a genuine allosteric binder of GCase, enhancing its enzymatic activity without directly binding to the enzyme's active site. However, its binding site also localizes in close proximity to the enzyme's catalytic region (Figure 13D). Compound 14 was also shown to induce GCase dimerization upon binding. The crystals of GCase with compound 14 belong to the space group P212121 and diffract to a resolution of 1.85 A. The crystal's asymmetric unit comprises four GCase monomers. Analysis using the Proteins, Interfaces, Structures and Assemblies (PISA) software¹³⁵-¹³⁶ suggested that these monomers assemble into a pair of dimers, with the active sites oriented toward each other and situated at the dimer interface. Nevertheless, the connection between GCase activation and induction of dimerization remains speculative⁵⁴.

Furthermore, the GCase chaperone and mild inhibitor Ambroxol¹¹⁹-¹³⁷ either directly binds to the enzyme's active site or engages with GCase allosterically in close vicinity to its catalytic region (Figure 13E). Interestingly, the binding of Ambroxol to GCase is highly pH-dependent. The small molecule chaperone exhibits its strongest binding affinity at the neutral pH of the ER, where it aids in the folding process, and shows the weakest binding at the acidic pH of the lysosome, where its presence is no longer required¹¹⁹-¹³⁸.

Finally, the GCase epitope for the binding of the stabilizing Nbs competing for the GCase epitope of Nbl differs from the epitope for binding LIMP-2 and saposin C (sap C). These proteins serve as natural molecular chaperones for the import of GCase into the lysosome⁸⁰ and as activators of GCase in the lysosome¹²⁴, respectively. Interestingly, mutations in LIMP-2, along with sap C deficiency, have been explored as genetic modifiers in GD and in synucleinopathies¹³⁹-¹⁴⁰. Here, computational and experimental methods revealed that both proteins bind in the vicinity of the GCase active site pocket, in locations that do not overlap with the epitope involved in Nbl binding (Figure 13F). This suggests that any molecular chaperone binding to the new allosteric pocket described herein, including the stabilizing Nbs competing for the GCase epitope of Nbl, would likely not interfere with the binding of either sap c or LIMP-2.

EXAMPLE 12. ER-targeted Nb4, Nb9, and Nbl6, as well as lysosome-targeted Nbl6, enhance lysosomal GCase activity in live cells.

Given the positive effect of Nbl6 on N370S GCase activity in the 4-MU assay (Figure 12A-B), as well as the beneficial impact of lysosome-targeted Nbl6 on N370S GCase activity in GBA1 knockdown HEK293T cells (Figure 12D), we further investigated the effect of Nbl6 on lysosomal GCase activity in live WT HEK293T cells. Nbl6 was cloned into a bicistronic plasmid with a C-terminal 3xFLAG epitope and a sequence targeting either the endoplasmic reticulum (ER) or the lysosome (LYSO), along with a fluorescent protein (EGFP or mCherry). Expression of the nanobodies in the targeted compartments was confirmed in HEK293T cells using western blot and confocal imaging (Figure 15A-E). Furthermore, none of the ER- or lysosome-targeted nanobodies from the current Example, including Nbl6, affected GCase expression or altered calnexin or LAMP1 levels, confirming the lack of ER-stress and lysosome enlargement during ER- and lysosome-targeted Nb expression (Figure 16).

The impact of Nbl6 on GCase activity was evaluated in live cells through flow cytometry using the fluorogenic substrate PFB-FDGIu, as previously described¹³⁴ (Figure 17A). In this setting, both ER- and lysosome-targeted Nbl6 enhanced GCase activity in live cells (Figure 17B and 17C). Consistent with the in vitro data, which demonstrated that Nbl6 acts as a GCase activator (Figure 12), this nanobody is the only lysosome-targeted Nb that significantly increases GCase activity in live cells, compared to lyso-Nbl, Nb4, and Nb9. Interestingly, ER-Nbl6 also boosted GCase activity in live cells, suggesting its ability to bind GCase in the ER and facilitate its transport to the lysosomes, where it enhances GCase enzymatic activity. This further supports the notion that ER targeting could be the most relevant therapeutic strategy for GCase-related disorders.

Following, no impact of ER-Nbl6 or Iysosome-Nbl6 was observed on lysosomal proteolytic activity, as evaluated by flow cytometry using the DQ-BSA assay (Figure 17D-F).

EXAMPLE 13. ER- or lysosome-targeted Nbs exert a GCase-specific effect.

The impairment of GCase function and the subsequent substrate accumulation can influence the expression and activity of other lysosomal enzymes. One such enzyme is cathepsin B, a cysteine protease that interacts with GCase in the context of lysosomal function and diseases like Gaucher disease and Parkinson's disease. Recent studies have identified a novel role for cathepsin B in mediating the cleavage of prosaposin to generate saposin C, a lysosomal coactivator of GCase¹⁴¹. Furthermore, inhibition of cathepsin B has been shown to impair autophagy, reduce GCase activity, and promote lysosomal content accumulation¹⁴².

Hence, to assess whether the Nbs of this invention influence other lysosomal enzymes, we investigated cathepsin B activity in HEK293T cells overexpressing ER- or lysosome-targeted Nbs. Briefly, HEK293T cells were plated in a 24-well plate (150,000 cells/well) and transfected with ER- or lysosome-targeted Mock, Nbl, Nb4, Nb9, and Nbl6 constructs (DNA:PEI ratio 1:2). After 48 hours, cells were incubated with the Magic Red substrate (1:13,000) for 30 minutes. This non-cytotoxic substrate produces red fluorescence upon cleavage by active cathepsin B¹⁴³. Fluorescence was measured using the BD LSR Fortessa™ X-20 Cell Analyzer (Xex 590 nm, Xem 620 nm).

As shown in the Figure 18A-B, no statistically significant differences in fluorescence values were observed, indicating that the Nbs did not affect cathepsin B activity. These results align with the previously performed DQ-BSA assay in live HEK293 cells overexpressing selected Nbs (Figure 17D-F), which demonstrated that none of the tested Nbs impacted lysosomal proteolytic activity. This further supports the notion that these Nbs specifically modulate GCase activity without broadly affecting lysosomal function.

To assess potential off-target hydrolysis and its contribution to GCase activity, we performed the PFB- FDGIu assay (as described in Figure 17) on wild-type HEK293T live cells overexpressing the selected ERand lysosome-targeted Nbs following a 24-hour treatment with the GCase inhibitor CBE¹¹⁶ (50 pM). As shown in Figure 18C-D, cells transfected with different Nbs and treated with CBE did not exhibit significant variations in fluorescence signal. This suggests that off-target hydrolysis contributes equally to the fluorescence signal across all conditions, confirming that the observed differences are solely due to the effect of the Nbs on GCase function. These findings further reinforce the specificity of Nbs for GCase.

EXAMPLE 14. Nanobody-induced protection against Cathepsin L proteolysis.

To determine whether the stabilization of the GCase fold by Nbl, Nb4, or Nb9 provides protection against proteolytic degradation by cathepsins, GCase was incubated with cathepsin L— a cysteine protease known to regulate GCase in lysosomes— either alone or in the presence of each Nb. Consistent with previous findings¹⁴⁴, under the experimental conditions used, cathepsin L degraded more than 50% of GCase. However, co-incubation with Nbl or Nb9 resulted in a small but significant reduction in proteolysis (Figure 19A-B). A similar trend was observed for Nb4, though the effect did not reach statistical significance. These results confirm that the increase in thermal stability conferred by the stabilizing Nbs also protects GCase from proteolytic degradation, likely by preventing its unfolding or reinforcing its folded conformation.

EXAMPLE 15. Reverse BLI.

To verify that the observed binding affinities of the Nbs to GCase are not affected by GCase immobilization on the sensor surface (Figure 2B-C), we conducted a complementary BLI experiment. In this setup, increasing concentrations of GCase in solution were titrated against Nbs immobilized on streptavidin sensors via a site-specifical ly biotinylated C-terminus. With the exception of Nb5 and Nbl2, the obtained K_D values were comparable between both experimental approaches, whether GCase was immobilized (Figure 2B-C) or in solution (Figure 20). However, Nb5 and Nbl2 exhibited significantly higher affinities in the latter setup, suggesting that their binding epitopes may be partially shielded or undergo structural alterations when GCase is immobilized. EXAMPLE 16. Evaluation of Nbl6 binding to GCase and determination of K_D using fluorescence anisotropy.

Remarkably, while a clear signal was observed for Nbl6 binding to both wild-type GCase and GCase N30S in ELISA (Figure 2A), no binding was detected in BLI (Figure 2B, Figure 20B). To further confirm Nbl6 binding to GCase using an alternative method, we employed fluorescence anisotropy. First, we site- specifically labeled Nbl6 at its C-terminus with 5-TAMRA through Sortase-mediated exchange with a 5- TAMRA-coupled GGGYK (SEQ ID NO: 96) peptide. The labeled Nbl6 was then titrated with increasing concentrations of GCase, and the fluorescence anisotropy signal was measured and plotted as a function of GCase concentration (Figure 21). The resulting binding isotherm was fitted to a quadratic equation, yielding a K_D value of approximately 450 pM. While this result confirms that Nbl6 binds to GCase, the low affinity could explain the lack of signal observed in the previous BLI experiment.

EXAMPLE 17. Nbl6-GCase crosslinking.

To further validate the binding of Nbl6 to GCase in vitro, a crosslinking experiment was performed. GCase (20 pM) was mixed with either Nbl6, Nbl (as a positive control), or an irrelevant Nb (as a negative control) at a 1:5 ratio (100 pM Nbs). The protein mixtures were incubated on ice for 30 minutes, followed by the addition of the amine-reactive crosslinker disuccinimidyl suberate (DSS) at a final concentration of 1 mM. After an additional 30-minute incubation at room temperature, the reaction was quenched with 2 pL of 1 M Tris (pH 7.5), and the samples were analyzed via SDS-PAGE. While crosslinking was observed between GCase molecules both in the absence of any nanobody (lane 1) and in the presence of the irrelevant Nb (lane 5), several additional bands appeared specifically in the presence of Nbl (lane 4) and Nbl6 (lane 2) (Figure 22A). To confirm the presence of crosslinked GCase-Nbl/Nbl6 complexes, the corresponding bands were excised from the gel and analyzed by mass spectrometry. LC-MS analysis (Figure 22B) identified: (i) band 1 containing GCase and Nbl6, and (ii) band 2 containing GCase and Nbl. These results confirm that Nbl6 binds to GCase and forms a complex, aligning with the fluorescence polarization data and further supporting the interaction between the two proteins.

EXAMPLE 18. Production and characterization of humanized nanobodies.

As described herewith, ISVDs such as domain antibodies and nanobodies, can be subjected to humanization, i.e., increase in the degree of sequence identity with the closest human germline sequence, wherein preferably the CDRs remain identical and framework residues are potentially substituted. Hence, we produced humanized variants of several lead Nbs, namely: Nbl_01 (SEQ ID NO: 97), Nbl_02 (SEQ ID NO: 101), Nb4_01 (SEQ ID NO: 105), Nb9_01 (SEQ ID NO: 109), Nbl6_01 (SEQ ID NO: 113), Nbl7_01 (SEQ ID NO: 117), Nbl7_02 (SEQ ID NO: 121), Nbl7_03 (SEQ ID NO: 125). All the humanized Nbs were successfully expressed in E. coli and could be purified efficiently (Figure 23A). Similar to the parental Nbs, the affinity of the humanized Nbs for wild-type GCase was assessed using BLL The Nbs were biotinylated at their C-terminus using Sortase-mediated chemistry, then immobilized on a Streptavidin sensor and titrated with increasing concentrations of GCase. All experimental conditions were identical to those used in the binding experiments with the parental Nbs (see previous BLI Examples). As shown in Figure 23B, several humanized variants exhibited significantly increased affinities for GCase, offering a clear advantage for future therapeutic applications, for instance in the treatment of GCase-related disorders, including Parkinson's diseases and Gaucher's disease. Notably, the GCase stabilizing Nbs (Nbl, Nb4, Nb9) demonstrated affinity increases upon their humanizations, ranging from approximately 100-fold (Nbl_02 versus Nbl) to 15-fold (Nb9_01 versus Nb9). Remarkably, the binding of Nbl_02 to wild-type GCase resulted in a K_D value of 26 pM, highlighting its exceptional affinity.

Next, the influence of the humanized Nbs on GCase thermal stability was evaluated using a fluorescencebased thermal shift assay (TSA), and compared to the effects of their parental counterparts at pH 7.0 and 5.2. The difference in melting temperature (AT_m) of GCase, in the presence and absence of the various Nbs, was determined. As shown in Figure 23C-D, similar to the parental Nbs, all humanized versions of Nbl, Nb4, and Nb9 exhibited a stabilizing effect on GCase. Notably, this stabilizing effect was more pronounced for Nbl_02 and Nb4_01, compared to the parental Nbl and Nb4, respectively. In contrast, as observed with the parental Nbs, no significant effect on T_m, or a destabilizing effect, was observed for the optimized variants of Nbl6 and Nbl.

EXAMPLE 19. Assessment of probable binding regions of various anti-GCase antibodies reported in the literature.

Potential binding regions of various anti-GCase antibodies reported in the literature were visualized based on the epitopes used for immunizations and compared with the binding site of Nbl of the current invention. The following prior art antibodies were included in the analysis: i. sc-365745: Mouse monoclonal IgGl raised again amino acids 67-95 near the N-terminus of p- glucosidase of human origin; ii. sc-32883: Rabbit polyclonal antibody raised against amino acids 237-536 mapping at the C- terminus of -glucosidase of human origin; ill. G4046: Rabbit polyclonal antibody raised against a specific peptide corresponding to amino acids 83-100 of -glucosidase of human origin; iv. G4171: Rabbit polyclonal antibody raised against a specific peptide corresponding to amino acids 517-536 at the C-terminus of p-glucosidase of human origin; v. Ab55080: Mouse monoclonal lgG2a antibody raised against a recombinant fragment corresponding to amino acids 100-250 of human GCase; vi. TA325083: Rabbit polyclonal antibody raised against a synthetic peptide corresponding to amino acids 337-365 at the C-terminus of p-glucosidase of human origin; vii. H00002629-M01: Mouse monoclonal antibody raised against amino acids 146-235 of recombinant p-glucosidase.

As shown in Figure 24, all of the included antibodies known in the art and compared herein for binding to GCase appear to bind to epitopes on GCase that are distinct from the epitope targeted by Nbl. This further supports the notion that the GCase-activating and/or GCase-stabilizing Nbs of the current invention specifically bind a unique binding site on GCase, thereby locking a GCase conformation that results in an allosteric effect on its activity, as observed for these Nbs.

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Claims

1. An allosteric modulator polypeptide specifically binding human glucocerebrosidase (GCase).

2. The GCase-specific allosteric modulator polypeptide of claim 1, comprising an antibody, active antibody fragment, immunoglobulin single variable domain (ISVD), single domain antibody, or VHH which specifically binds the human GCase.

3. A GCase-specific allosteric modulator which stabilizes GCase, and wherein said modulator specifically binds GCase at a binding site on domain II and domain III, preferably comprising the amino acid residues K77, V78, K79, H162, L165, Q166, A168, Q169, R170, P171, V172, S173, L174, A221, K224, L225, Q226, F227, W228, T272, H274, N275, R277, H306, E429, G430, P452, D453 of SEQ ID NO:81, or preferably at a binding site comprising the corresponding amino acids thereof in a GCase homologue and/or mutant protein.

4. The GCase-specific allosteric modulator of claim 3, wherein said modulator comprises an ISVD specifically binding human GCase and comprising the complementarity-determining-regions (CDRs) as present in SEQ ID NO: 1, 4, 9 or 97, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering system, or comprising an ISVD comprising a sequence wherein:

CDR1 comprises SEQ ID NO: 21, CDR2 comprises SEQ ID NO: 22, and CDR3 comprises SEQ ID NO: 23;

CDR1 comprises SEQ ID NO: 30, CDR2 comprises SEQ ID NO: 31, and CDR3 comprises SEQ ID NO: 32;

CDR1 comprises SEQ ID NO: 45, CDR2 comprises SEQ ID NO: 46, and CDR3 comprises SEQ ID NO: 47;

CDR1 comprises SEQ ID NO: 98, CDR2 comprises SEQ ID NO: 99, and CDR3 comprises SEQ ID NO: 100.

5. The GCase-specific allosteric modulator of claims 3 or 4, wherein said ISVD comprises any one of SEQ ID NOs: 1, 4, 9, 97, or a functional variant of any one thereof with at least 90 % amino acid identity over the full length of the ISVD sequence wherein the non-identical amino acids are located in one or more framework residues, or a humanized variant of any one thereof.

6. The GCase-specific allosteric modulator of any one of claims 1 to 5, which increases the GCase activity in a cell as compared to a control without said modulator, and wherein said modulator comprises an ISVD specifically binding the human GCase comprising the CDRs of SEQ ID NO: 1, 2, 3, 4, 5, 6, 8, 9, 10, 13, 16, 18, 19, 113, 117, 121, or 125, wherein the CDRs are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia numbering system, or comprising an ISVD comprising a sequence wherein:

CDR1 comprises SEQ ID NO: 21, CDR2 comprises SEQ ID NO: 22, and CDR3 comprises SEQ ID NO: 23;

CDR1 comprises SEQ ID NO: 24, CDR2 comprises SEQ ID NO: 25, and CDR3 comprises SEQ ID NO: 26;

CDR1 comprises SEQ ID NO: 27 , CDR2 comprises SEQ ID NO: 28, and CDR3 comprises SEQ ID NO: 29;

CDR1 comprises SEQ ID NO: 30, CDR2 comprises SEQ ID NO: 31, and CDR3 comprises SEQ ID NO: 32;

CDR1 comprises SEQ ID NO: 33, CDR2 comprises SEQ ID NO: 34, and CDR3 comprises SEQ ID NO: 35;

CDR1 comprises SEQ ID NO: 36, CDR2 comprises SEQ ID NO: 37, and CDR3 comprises SEQ ID NO: 38;

CDR1 comprises SEQ ID NO: 42, CDR2 comprises SEQ ID NO: 43, and CDR3 comprises SEQ ID NO: 44;

CDR1 comprises SEQ ID NO: 45, CDR2 comprises SEQ ID NO: 46, and CDR3 comprises SEQ ID NO: 47;

CDR1 comprises SEQ ID NO: 48, CDR2 comprises SEQ ID NO: 49, and CDR3 comprises SEQ ID NO: 50;

CDR1 comprises SEQ ID NO: 57, CDR2 comprises SEQ ID NO: 58, and CDR3 comprises SEQ ID NO: 59;

CDR1 comprises SEQ ID NO: 66, CDR2 comprises SEQ ID NO: 67, and CDR3 comprises SEQ ID NO: 68;

CDR1 comprises SEQ ID NO: 72, CDR2 comprises SEQ ID NO: 73, and CDR3 comprises SEQ ID NO: 74;

CDR1 comprises SEQ ID NO: 75, CDR2 comprises SEQ ID NO: 76, and CDR3 comprises SEQ ID NO: 77;

CDR1 comprises SEQ ID NO: 114, CDR2 comprises SEQ ID NO: 115, and CDR3 comprises SEQ ID NO: 116;

CDR1 comprises SEQ ID NO: 118, CDR2 comprises SEQ ID NO: 119, and CDR3 comprises

SEQ ID NO: 120;

CDR1 comprises SEQ ID NO: 122, CDR2 comprises SEQ ID NO: 123, and CDR3 comprises SEQ ID NO: 124; CDR1 comprises SEQ. ID NO: 126, CDR2 comprises SEQ ID NO: 127, and CDR3 comprises

SEQ ID NO: 128.

7. The GCase-specific allosteric modulator of any one of claims 1 to 6, further comprising a targeting moiety, preferably an endoplasmic reticulum (ER)-targeting or a lysosome-targeting moiety.

8. The GCase-specific allosteric modulator of any one of claims 1 to 7, which comprises a further functional moiety, preferably a therapeutic moiety, a half-life-extending moiety, or a blood-brain barrier (BBB)-crossing moiety.

9. The GCase-specific allosteric modulator of any one of claims 1 to 8, which is a multivalent or multispecific modulator, preferably comprising at least two moieties specifically binding GCase, or comprising at least two GCase-specific ISVDs.

10. A nucleic acid molecule encoding a human GCase-specific allosteric modulator polypeptide according to any of the preceding claims.

11. A vector comprising the nucleic acid molecule according to claim 10.

12. A pharmaceutical composition comprising the GCase-specific allosteric modulator of any one of claims 1 to 9, the nucleic acid molecule of claim 10, or the vector of claim 11.

13. The GCase-specific allosteric modulator of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 12, for use as a medicament.

14. The GCase-specific allosteric modulator of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 12, for use in a therapeutic or preventive treatment of GCase-related disorders.

15. The GCase-specific allosteric modulator of any one of claims 1 to 9, the nucleic acid molecule of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 12, for use in a therapeutic or preventive treatment of Gaucher disease (GD) and/or Parkinson's disease (PD).