Allosteric regulators of leucine-rich repeat kinase 2

Abstract

The present invention relates to binding agents for human leucine-rich repeat kinase 2 (LRRK2). More specifically, allosteric modulators of LRRK2 activity have been identified for targeting LRRK2 in human cells while leaving the subcellular localization of LRRK2 unaffected. Even more specifically, protein-binding agents for allosteric modulation of LRRK2 kinase activity are disclosed, comprising an immunoglobulin single variable domain (ISVD) that binds to human LRRK2 with nanomolar affinity. Thus, the present invention reveals means and methods for a novel approach to targeting LRRK2 by allosteric modulation of its activity, for use in treating LRRK2-related pathologies such as Parkinson's disease, as well as for use in detecting LRRK2 in vitro and in vivo, and for use as a diagnostic agent.

Description

[Technical Field]

[0001] The present invention relates to binding agents for human leucine-rich repeat kinase 2 (LRRK2). More specifically, allosteric modulators of LRRK2 activity have been identified for targeting LRRK2 in human cells while leaving the subcellular localization of LRRK2 unaffected. Even more specifically, protein-binding agents for allosteric modulation of LRRK2 kinase activity are disclosed, comprising an immunoglobulin single variable domain (ISVD) that binds to human LRRK2 with nanomolar affinity. Thus, the present invention reveals means and methods for a novel approach to targeting LRRK2 by allosteric modulation of its activity, for use in treating LRRK2-related pathologies such as Parkinson's disease, as well as for use in detecting LRRK2 in vitro and in vivo, and for use as a diagnostic agent. [Background technology]

[0002] Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease (PD), inherited in an autosomal dominant manner [62-64]. LRRK2 gene variants have also been associated with idiopathic forms of PD [65,66]. PD is a neurodegenerative movement disorder

[61] , the prevalence of which is expected to increase globally in the future [58-60], and there is a lack of therapeutic options except for symptomatic treatment. Furthermore, mutations in LRRK2 have been associated with other diseases, including chronic inflammatory conditions such as Crohn's disease [67-68]. LRRK2 is a large multidomain protein (285 kDa) belonging to the ROCO protein family. The enzymatic core of the protein consists of an active GTPase domain (Roc), a dimerization module (COR), and an active Ser / Thr protein kinase domain (KD) [13,16,69,70]. Several Rab GTPases have been identified as physiological substrates of the LRRK2 kinase [25-27]. Furthermore, this provides the autophosphorylation activity of LRRK2

[28] . The monomeric form of the LRRK2 protein occurs predominantly in the cytosol and has reduced kinase activity, while dimeric LRRK2 formed in the membrane exhibits higher kinase activity [19,20,71,72]. The most common pathogenic mutations in LRRK2 are clustered in the Roc-COR and kinase domains. Importantly, several PD mutations lead to decreased GTPase activity and / or increased kinase activity [16,50,73-78]. In particular, autophosphorylation of serine 1292

[28] and Rab protein phosphorylation

[25] are increased by pathogenic LRRK2 variants, particularly the most common G2019S mutation located in the kinase domain. These findings support the idea that LRRK2 mutations cause PD through a gain-of-function mechanism and suggest that modulation of LRRK2 protein function may be a promising drug target [47,79,80]. Because PD mutations result in increased LRRK2 kinase activity, drug development to date has primarily focused on developing kinase inhibitors that target the ATP-binding pocket. Several inhibitors that are selective for LRRK2 kinase activity have been identified.However, long-term inhibition of LRRK2 with these ATP-competitive inhibitors has been reported to lead to severe kidney abnormalities in rodents and the accumulation of lamellar bodies in type II alveolar epithelial cells of the lungs of non-human primates [46,47,81-83], suggesting that current LRRK2 kinase inhibitors will require further optimization and testing.

[0003] Although a high-resolution structure of the catalytic half of LRRK2 was recently published as a starting point for exploring alternative pathways and binding modes for therapeutic targeting of LRRK2

[36] , to date, no full-length human LRRK2 has been described, and only portions of the protein, such as the catalytic domain, have been revealed at atomic resolution. Current LRRK2-specific ATP-competitive inhibitors are primarily type I kinase inhibitors and are known to pose significant toxicity risks. This hurdle to the clinical development of such orthosteric kinase inhibitors indicates the need for a different approach based on allosteric modulation of LRRK2 kinase activity. To date, only one small compound, natural vitamin B12 and its derivatives, has been identified that can inhibit LRRK2 activity through a non-ATP-competitive mechanism

[43] . These vitamin B12 compounds appear to bind directly to LRRK2 via contact sites on the kinase domain and act as mixed allosteric inhibitors that can affect ATP binding to LRRK2 by blocking LRRK2 dimerization. The compounds have been shown to stretch a novel binding site in the LRRK2 kinase domain, involving contact with the kinase domain via the adenosyl moiety of the compounds, the bulky corrin ring of cobalamin, and the DMZ base, which may alter its conformation and dimerization status. It remains to be seen whether these vitamin B12 derivatives offer a new class of therapeutic agents with reduced toxicity and high specificity.

[0004] Therefore, there remains a need to find alternative LRRK2 modulators with high specificity and alternative modes of action to overcome clinical hurdles for therapeutic targeting and treatment of LRRK2-related disorders such as Parkinson's, among other diseases. Summary of the Invention

[0005] The present invention is based on a novel approach that targets multiple enzymatic functions and regulatory mechanisms of LRRK2 in an allosteric manner using compounds that bind outside the ATP pocket, thereby exploring the benefits of increased selectivity and lower toxicity [84, 85]. The present invention provides immunoglobulin single variable domain (ISVD) antibodies (specifically, VHHs or nanobodies, used interchangeably herein) that modulate the dynamics, regulation, and activity of the LRRK2 protein. ISVDs have been identified as allosteric modulators of human LRRK2 kinase activity. A wide range of VHH families that bind to different LRRK2 domains with varying affinities have been selected for their advantageous properties as potential therapeutic or diagnostic agents. Some of the VHHs of the present invention robustly inhibit both cellular and in vitro LRRK2 kinase activity, while others significantly increase cellular LRRK2 activity. Furthermore, LRRK2 inhibitor Nbs with full kinase inhibitory activity and specific inhibition of Rab substrate phosphorylation are discovered herein. Interestingly, a subset of Nbs inhibit kinase activity without directly binding to the kinase domain, acting as mixed (noncompetitive) inhibitors, demonstrating their allosteric regulatory role in kinase inhibition. Surprisingly, in contrast to currently available kinase inhibitors, Nbs do not induce the formation of LRRK2 filaments on cellular microtubules, and some Nbs even reverse this adverse side effect, indicating that Nbs offer a novel type of allosteric LRRK2-modulating and binding drug that acts quite differently from previously identified inhibitors, such as currently available ATP-competitive kinase inhibitors, thereby offering novel therapeutic opportunities in the fight against Parkinson's disease.

[0006] Thus, in a first aspect, the present invention relates to binding agents that specifically bind to human leucine-rich repeat kinase 2 (LRRK2), and whose intracellular binding keeps LRRK2 from associating with microtubules. The fact that a large panel of LRRK2-binding ISVDs acts allosterically via binding to different protein domains or at the interface of various domains, and does not involve direct binding to the kinase domain or, more specifically, to the catalytic site of the kinase domain, results in a surprising effect that, unlike classical ATP-competitive LRRK2 inhibitors or compounds targeting the GTP-binding site, does not induce LRRK2 filaments on microtubules, in contrast to orthosteric ATP-competitive LRRK2 binding agents. Furthermore, said LRRK2 binding agents have K values ​​in the nanomolar range and / or in the sub-200 nM range. D In a specific embodiment, the LRRK2 binding agents of the present invention affect cellular and / or in vitro LRRK2 kinase activity.

[0007] Particular embodiments herein relate to allosteric modulators of LRRK2, which structurally include small compounds, chemicals, proteins, peptides or peptidomimetics, or antibodies, antibody mimetics, single domain antibodies, or more specifically immunoglobulin single variable domains (ISVDs), nanobodies, or any active antibody fragments.

[0008] More specifically, allosteric binding agents of LRRK2, including the ISVDs described herein, that have LRRK2 inhibitory activity, may be delivered as tools or therapeutic proteins, as nucleic acids, or as vectors, and hold promise as novel therapeutic strategies for treating PD and / or other LRRK2-linked diseases (including inflammatory diseases, e.g., IBD, more specifically Crohn's disease) using mechanisms of action not previously described for LRRK2-related therapeutic agents.

[0009] One embodiment relates to an allosteric modulator of LRRK2 that specifically binds to LRRK2 in a non-ATP-competitive binding mode, i.e., at a binding site different from the ATP catalytic site. Preferably, the allosteric modulator of LRRK2 specifically binds to LRRK2 at a binding site composed of LRRK2 amino acids that do not exclusively or exclusively include amino acids present in the LRRK2 kinase catalytic site or kinase domain. Alternatively, the allosteric modulator of LRRK2 preferentially binds to LRRK2 at one or more binding sites in a protein domain different from the kinase domain, i.e., at one or more protein domains selected from the group consisting of the N-terminal domain (armadillo, ankyrin repeat, or LRR), Roc, COR, and WD40 domain. In another embodiment, a combination of binding to the kinase domain and any of the other domains described above is possible. In another embodiment, the Nb may bind to the kinase domain at a binding site different from the ATP binding site. In one embodiment, the LRRK2 binding agent that modulates LRRK2 activity may increase LRRK2 kinase activity compared to a control, or alternatively may decrease, inhibit, or block kinase activity compared to the absence or control of the binding agent. In another embodiment, both kinase and GTP activity may be modulated in opposite ways by the same binding agent (i.e., inhibiting the kinase increases the GTPase, and vice versa).

[0010] In one embodiment, the allosteric modulator of LRRK2 comprises an ISVD that specifically binds to human LRRK2 and comprises the structure of the formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 herein, which depicts four framework regions and three complementarity determining regions. In a specific embodiment, the ISVD for allosteric modulation of LRRK2 kinase activity comprises a CDR1 consisting of a sequence selected from the group of CDR1 sequences of ISVDs disclosed herein in SEQ ID NOs: 1-19 (the CDR regions are annotated as in Tables 3-4 or alternatively as illustrated in FIG. 10); a CDR2 consisting of a sequence selected from the group of CDR2 sequences of ISVDs disclosed herein in SEQ ID NOs: 1-19; and a CDR3 consisting of a sequence selected from the group of CDR3 sequences of ISVDs disclosed herein in SEQ ID NOs: 1-19. Thus, described herein are LRRK2-specific allosteric binding agents comprising an ISVD, wherein the CDR1, CDR2, and CDR3 regions are selected from the CDR1, CDR2, and CDR3 regions of a sequence selected from the group of sequences set forth in SEQ ID NOs: 1 to 19, and the CDR regions are annotated as known in the art according to Kabat, MacCallum, IMGT, AbM, or Chothia, as further defined herein. Alternatively, the LRRK2-binding agent comprises at least one ISVD: CDR1 comprises a sequence selected from the group of CDR1 sequences set forth in SEQ ID NOs: 23 to 41, CDR2 comprises a sequence selected from the group of CDR2 sequences set forth in SEQ ID NOs: 42 to 60, and CDR3 comprises a sequence selected from the group of CDR3 sequences set forth in SEQ ID NOs: 61 to 79.

[0011] A further specific embodiment discloses the LRRK2-specific allosteric binding agent, which comprises an ISVD, which includes any of the sequences of SEQ ID NOS: 1-19, or variants thereof containing the same CDR sequences and FR region substitutions of SEQ ID NOS: 1-19, but with at least 85% sequence identity. Another embodiment relates to the LRRK2 allosteric modulator, which comprises an ISVD, which is a humanized variant of any of the sequences selected from the group of SEQ ID NOS: 1-19, or a homologue thereof having at least 85% identity thereto, whose CDR sequences are identical to those of SEQ ID NOS: 1-19. Another embodiment relates to the LRRK2 allosteric binding agent defined herein, which comprises an ISVD and inhibits or blocks cellular LRRK2 kinase activity and / or specifically prevents substrate phosphorylation mediated by cellular LRRK2. A specific embodiment relates to an agent that specifically prevents or inhibits Rab substrate phosphorylation. An alternative embodiment relates to the LRRK2 modulators defined herein that include an ISVD that increases cellular LRRK2 kinase activity relative to a control. A further embodiment relates to the allosteric modulators of LRRK2 described herein that include an ISVD that prevents LRRK2 association with cellular microtubules, particularly in the presence of an ATP-competitive LRRK2 kinase inhibitor compound.

[0012] Another embodiment of the present invention discloses a multispecific or multivalent binding agent as an allosteric modulator of LRRK2 activity, which comprises at least one LRRK2 allosteric modulator disclosed herein. Another embodiment discloses a multispecific or multivalent binding agent as an allosteric modulator of LRRK2 activity, which comprises at least two of the LRRK2 allosteric modulators disclosed herein. Another embodiment of the present invention discloses a multispecific binding agent as an allosteric modulator of LRRK2 activity, which comprises an LRRK2 allosteric modulator disclosed herein and an additional binding agent with a different target specificity, or alternatively, an additional LRRK2 binding agent. In a specific embodiment, the multispecific or multivalent LRRK2 allosteric binding agent comprises at least one ISVD that specifically binds to LRRK2 disclosed herein.

[0013] Another aspect of the present invention relates to nucleic acid molecules encoding the allosteric modulators of LRRK2 described herein. Other embodiments include vectors containing the nucleic acid molecules described herein, which may be cloning or expression vectors, and delivery vehicles such as viral, lentiviral, or adenoviral vectors.

[0014] A further aspect provides a pharmaceutical composition comprising an allosteric modulator of LRRK2 or a multispecific LRRK2 binding agent comprising an allosteric modulator of LRRK2 disclosed herein. Alternatively, a pharmaceutical composition is provided comprising the (multispecific) allosteric modulator of LRRK2 disclosed herein and an ATP-competitive LRRK2 kinase inhibitor compound. In a specific embodiment, a pharmaceutical composition is provided comprising the (multispecific) allosteric modulator of LRRK2 disclosed herein and an ATP-competitive LRRK2 kinase inhibitor compound that is a type I ATP-competitive kinase inhibitor.

[0015] Another aspect of the present invention relates to an allosteric modulator of LRRK2 disclosed herein, or a nucleic acid molecule or vector provided herein, or a pharmaceutical composition described herein, for use as a pharmaceutical. A specific embodiment relates to an allosteric modulator of LRRK2 disclosed herein, or a nucleic acid molecule or vector provided herein, or a pharmaceutical composition described herein, for use as a pharmaceutical, or more specifically for use such as treating a subject for treating a disorder related to LRRK2. Thus, another specific embodiment relates to an allosteric modulator of LRRK2 disclosed herein, or a nucleic acid molecule or vector provided herein, or a pharmaceutical composition described herein, for use in treating Parkinson's disease.

[0016] A further aspect relates to the LRRK2-specific binding agent or ISVD disclosed herein, or the nucleic acid molecule or vector encoding the LRRK2-binding agent provided herein, or the pharmaceutical composition described herein, for use in a diagnostic assay or for medical in vivo imaging.

[0017] The final aspect relates to an in vitro method for detecting human LRRK2 protein in a sample, more specifically in a biological sample. The method for detecting LRRK2 in a sample may include reacting the sample with an LRRK2-specific binding agent or ISVD as disclosed herein and detecting the localization and distribution of the LRRK2-specific ISVD in the biological sample bound to LRRK2. In the method, the LRRK2-binding agent or ISVD disclosed herein may include a detectable label or tag. Another embodiment relates to an in vitro method for detecting the presence, absence, or level of LRRK2 protein in a sample, the method comprising: contacting the sample with an LRRK2-specific binding agent or ISVD, optionally including a label, and detecting the presence, absence, or level of the interacting or bound LRRK2-specific ISVD at the LRRK2-binding site. Optionally, the sample is a body fluid such as cerebrospinal fluid, or a protein extract or cell lysate.

[0018] Finally, the binding agents disclosed herein may also be used in screening assays, as tools, or in drug discovery. [Brief explanation of the drawings]

[0019] The drawings described are only schematic and non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

[0020] [Figure 1]Mapping of domain specificity of purified nanobodies using ELISA. (A) Domain mapping of 42 purified Nbs using ELISA against full-length LRRK2 or either RocCOR, Roc, COR-B, or kinase-WD40 constructs. Negative controls with either no antigen coated on the ELISA plate or no Nb added are also included. Each ELISA signal is the average of three ELISA setups. (B) Schematic overview of the results obtained in (A). Note that two Nbs did not show any binding above background; therefore, they are not included in this scheme. [Figure 2]Effect of a selected set of 18 Nbs on the kinase activity of the LRRK2(G2019S) variant in HEK293T cells. (a-c) LRRK2(G2019S) and its effector Rab29 were overexpressed together with GFP-fused Nbs ("Fluobody") in HEK293T cells. A negative control ("No") in which no Nb was overexpressed is also included. Panels (a), (b), and (c) show three replicates of the experiment. In the row labeled "pLRRK2," LRRK2 pS1292 levels were determined by Western blot using a site-specific anti-pLRRK2(pS1292) antibody (Abcam, ab203181) (shown at different development times). In the row labeled "pRAB1O," endogenous pT72-Rab10 levels were determined by Western blot using the MJFF / Abcam antibody MJF-R21 (Abcam, ab230261) (shown at different color development times). The three lower rows contain controls for LRRK2 (rat anti-LRRK2, 24D8), Fluobody (rat anti-GFP), and Rab10 (rabbit anti-Rab10) expression levels. (d) Based on the results in panels (a)–(c), Nbs can be divided into four functional groups. Group 1: Nbs that inhibit both cellular LRRK2 autophosphorylation and Rab10 phosphorylation; Group 2: Nbs that inhibit only Rab10 phosphorylation; Group 3: Nbs that activate LRRK2 kinase activity; and Group 4: Nbs with no consistent / clear effect on LRRK2 kinase activity. [Figure 3] Mapping of the binding epitopes of Nbs on LRRK2 using crosslink mass spectrometry. Nbs are divided into four functional groups according to their effect on LRRK2 kinase activity in cellulo, as defined in Figure 2d. The observed crosslinks between Nbs and LRRK2 are indicated by lines, and the corresponding lysine residues on LRRK2 are indicated by their residue numbers. The domain specificities of Nbs, previously determined by ELISA, are given below each Nb for reference. [Figure 4-1]Modulation of in vitro kinase activity by LRRK2-targeting Nbs. (A) The effect of selected LRRK2-targeting Nbs on LRRK2 kinase activity, measured using the fluorescence-based PhosphoSens® protein kinase assay. The LRRK2-optimized AQT0615 peptide was used as a substrate at a fixed concentration of 10 μM in the presence of 1 mM or 0.1 mM ATP, respectively. The effect of different Nbs at a concentration of 25 μM on relative kinase activity is plotted compared to a control in which no Nb was added ("No Nb"). Additional negative (25 μM irrelevant control Nb) and positive (25 μM ATP-competitive LRRK2 inhibitor MLi-2) controls are included. (B) The effect of different Nbs (25 μM) on relative kinase activity is plotted compared to the "No Nb" control in the presence of either 500 μM GDP (top) or 500 μM GTPγS (bottom). A positive control of 0.2 μM of the ATP-competitive LRRK2 inhibitor MLi-2 is included. Each bar reflects the mean (±SD) of three independent measurements. [Figure 4-2]Modulation of in vitro kinase activity by LRRK2-targeting Nbs. (A) The effect of selected LRRK2-targeting Nbs on LRRK2 kinase activity, measured using the fluorescence-based PhosphoSens® protein kinase assay. The LRRK2-optimized AQT0615 peptide was used as a substrate at a fixed concentration of 10 μM in the presence of 1 mM or 0.1 mM ATP, respectively. The effect of different Nbs at a concentration of 25 μM on relative kinase activity is plotted compared to a control in which no Nb was added ("No Nb"). Additional negative (25 μM irrelevant control Nb) and positive (25 μM ATP-competitive LRRK2 inhibitor MLi-2) controls are included. (B) The effect of different Nbs (25 μM) on relative kinase activity is plotted compared to the "No Nb" control in the presence of either 500 μM GDP (top) or 500 μM GTPγS (bottom). A positive control of 0.2 μM of the ATP-competitive LRRK2 inhibitor MLi-2 is included. Each bar reflects the mean (±SD) of three independent measurements. [Figure 5] Effect of LRRK2-targeting nanobodies on LRRK2 cellular localization. HEK293 cells were cotransfected with the indicated GFP-Nb and mScarlet-LRRK2. As a positive control, mScarlet-LRRK2-transfected cells were treated with the pharmacological ATP-competitive inhibitor MLi-2 (1 μM MLi-2, 90 min treatment) and showed induction of LRRK2 translocation onto microtubules, seen as filamentous bundle-like structures and indicated by white arrows (upper left panel). In contrast, cells cotransfected with GFP-Nb and mScarlet-LRRK2 showed normal cytoplasmic distribution of LRRK2 without translocation to microtubules, similar to cells cotransfected with an irrelevant Nb. Scale bar 5 μm. [Figure 6]Effect of LRRK2-targeting nanobodies on MLi-2-induced microtubule translocation of LRRK2. HEK293 cells were cotransfected with the indicated GFP-Nb and mScarlett-LRRK2 and treated with the pharmacological ATP-competitive inhibitor MLi-2 (1 μM MLi-2, 90 min treatment). Cotransfection of mScarlett-LRRK2 with GFP alone (upper left panel) or an irrelevant Nb (lower left panel) demonstrated MLi-2-induced translocation of LRRK2 onto microtubules, seen as filamentous bundle-like structures and indicated by white arrows. Cotransfection with a subset of GFP-Nb inhibited MLi-2-induced LRRK2 translocation. Scale bar 5 μm. [Figure 7] SDS-PAGE analysis showing purified full-length and domain constructs of LRRK2. 3 μg of each purified full-length LRRK2 and the domain constructs RocCOR, Roc, (MBP-)COR-B, and kinase-WD40 used in this study were loaded onto the gel. Markers: (Left) Spectra™ Multicolor High-Range Protein Ladder (Cat. No. 26625; Thermo Scientific™); (Right) PageRuler™ Prestained Protein Ladder, 10 to 180 kDa (Cat. No. 26616; Thermo Scientific™). [Figure 8]LRRK2 cross-linking. Prior to immunization, LRRK2 was cross-linked with the lysine-specific cross-linker DSS. Two cross-linking setups were performed, in which cross-linking was allowed to proceed to different levels (A and B). LRRK2 was purified in the presence of GDP (lanes 2 and 3) or GTPγS (lanes 4 and 5). In both gels, lanes 2 and 4 show LRRK2 before cross-linking, and lanes 3 and 5 show LRRK2 after DSS cross-linking. For immunization 2, a mixture of samples shown in lane 5 was used. For immunization 3, a mixture of samples shown in lane 3 was used. Markers: (A) Quad Color protein marker from Biozym; (B) Spectra™ Multicolor High Range Protein Ladder (Cat. No. 26625 Thermo Scientific™). [Figure 9] SDS-PAGE of the 42 purified nanobodies shown in Figure 1 and an irrelevant Nb used in this study. 2 μg of each nanobody was loaded on the gel (with the exception of CA16070, where 0.7 μg was loaded due to the poor expression level of this Nb). Marker: PageRuler™ prestained protein ladder, 10 to 180 kDa (Cat. No. 26616 Thermo Scientific™). [Figure 10] Amino acid sequence and CDR annotation of CA12610 Nb. Amino acid numbering is according to Kabat. As examples of different CDR annotations possible for the Nbs disclosed herein, regions corresponding to alternative CDR annotations (AbM, Chothia, Kabat, IMGT) are marked in gray compared to those currently used. Llama germline hallmark residues are bold / underlined. [Figure 11-1]Modulation of in vitro kinase activity by LRRK2-targeting Nbs. A–C, Dose-response curves (upper panels) for the inhibition of in vitro LRRK2 kinase activity by group 1 Nbs: Nb1 (A), Nb6 (B), and Nb23 (C). Here, Nb concentrations were varied in two-fold serial dilutions from 200 or 150 μM to 0.006 μM. The two lower panels show Michaelis-Menten curves obtained for LRRK2 at various concentrations of ATP and a fixed (subsaturating) concentration of the peptide substrate (AQT0615) and at various concentrations of Nb1 (A), Nb6 (B), and Nb23 (C), along with the corresponding linearization according to the Lineweaver-Burk method (double reciprocal plot). The Nb concentrations used are indicated below the plots. Each data point reflects the mean (±SD) of three independent measurements. IC50 (±SD) values ​​generated from a three-parameter logistic equation fit, and Ki app and α values ​​(±SD) generated from a global fit of the mixed inhibitory mechanism are indicated on the graph. [Figure 11-2] Modulation of in vitro kinase activity by LRRK2-targeting Nbs. A–C, Dose-response curves (upper panels) for the inhibition of in vitro LRRK2 kinase activity by group 1 Nbs: Nb1 (A), Nb6 (B), and Nb23 (C). Here, Nb concentrations were varied in two-fold serial dilutions from 200 or 150 μM to 0.006 μM. The two lower panels show Michaelis-Menten curves obtained for LRRK2 at various concentrations of ATP and a fixed (subsaturating) concentration of the peptide substrate (AQT0615) and at various concentrations of Nb1 (A), Nb6 (B), and Nb23 (C), along with the corresponding linearization according to the Lineweaver-Burk method (double reciprocal plot). The Nb concentrations used are indicated below the plots. Each data point reflects the mean (±SD) of three independent measurements. IC50 (±SD) values ​​generated from a three-parameter logistic equation fit, and Ki app and α values ​​(±SD) generated from a global fit of the mixed inhibitory mechanism are indicated on the graph. [Figure 11-3]Modulation of in vitro kinase activity by LRRK2-targeting Nbs. A–C, Dose-response curves (upper panels) for the inhibition of in vitro LRRK2 kinase activity by group 1 Nbs: Nb1 (A), Nb6 (B), and Nb23 (C). Here, Nb concentrations were varied in two-fold serial dilutions from 200 or 150 μM to 0.006 μM. The two lower panels show Michaelis-Menten curves obtained for LRRK2 at various concentrations of ATP and a fixed (subsaturating) concentration of the peptide substrate (AQT0615) and at various concentrations of Nb1 (A), Nb6 (B), and Nb23 (C), along with the corresponding linearization according to the Lineweaver-Burk method (double reciprocal plot). The Nb concentrations used are indicated below the plots. Each data point reflects the mean (±SD) of three independent measurements. IC50 (±SD) values ​​generated from a three-parameter logistic equation fit, and Ki app and α values ​​(±SD) generated from a global fit of the mixed inhibitory mechanism are indicated on the graph. [Figure 12] Nanobodies bind to and immunoprecipitate LRRK2. HEK293 cells were transiently co-transfected with GFP-tagged Nb and (S)trep-(F)lag-tagged LRRK2 constructs for 48 hours prior to lysis. Pull-down assays were performed by means of magnetic GFP-Trap beads. HEK293 cell lysates overexpressing SF-LRRK2 alone were used as a negative control. Nb and LRRK2 were detected by immunoblot. Unlike irrelevant Nb and the negative control, all tested Nb pull down LRRK2. Blots are representative of n=3. [Figure 13-1]Measurement of the affinity of Nbs for LRRK2 using microscale thermophoresis (MST). Binding isotherms obtained by titrating increasing concentrations of LRRK2 against fluorescently (m-TAMRA)-labeled Nbs and measuring the MST signal are shown. Nbs are classified into four functional groups as defined in Figure 2d: (a) Group 1 Nbs, (b) Group 2 Nbs, (c) Group 3 Nbs, and (d) Group 4 Nbs. In panel (e), two negative controls are shown. Here, LRRK2 was titrated against either free m-TAMRA or an irrelevant Nb labeled with m-TAMRA. All measurements were performed in the presence of 500 μM GDP, with the exception of Nb42, for which GTPγS was used. The corresponding equilibrium dissociation constants (Kd ± standard error) obtained by fitting a quadratic equation for binding are given (each data point is the mean of three independent measurements, and error bars represent the standard deviation; NB = no detectable MST binding signal). [Figure 13-2] Measurement of the affinity of Nbs for LRRK2 using microscale thermophoresis (MST). Binding isotherms obtained by titrating increasing concentrations of LRRK2 against fluorescently (m-TAMRA)-labeled Nbs and measuring the MST signal are shown. Nbs are classified into four functional groups as defined in Figure 2d: (a) Group 1 Nbs, (b) Group 2 Nbs, (c) Group 3 Nbs, and (d) Group 4 Nbs. In panel (e), two negative controls are shown. Here, LRRK2 was titrated against either free m-TAMRA or an irrelevant Nb labeled with m-TAMRA. All measurements were performed in the presence of 500 μM GDP, with the exception of Nb42, for which GTPγS was used. The corresponding equilibrium dissociation constants (Kd ± standard error) obtained by fitting a quadratic equation for binding are given (each data point is the mean of three independent measurements, and error bars represent the standard deviation; NB = no detectable MST binding signal). [Figure 13-3]Measurement of the affinity of Nbs for LRRK2 using microscale thermophoresis (MST). Binding isotherms obtained by titrating increasing concentrations of LRRK2 against fluorescently (m-TAMRA)-labeled Nbs and measuring the MST signal are shown. Nbs are classified into four functional groups as defined in Figure 2d: (a) Group 1 Nbs, (b) Group 2 Nbs, (c) Group 3 Nbs, and (d) Group 4 Nbs. In panel (e), two negative controls are shown. Here, LRRK2 was titrated against either free m-TAMRA or an irrelevant Nb labeled with m-TAMRA. All measurements were performed in the presence of 500 μM GDP, with the exception of Nb42, for which GTPγS was used. The corresponding equilibrium dissociation constants (Kd ± standard error) obtained by fitting a quadratic equation for binding are given (each data point is the mean of three independent measurements, and error bars represent the standard deviation; NB = no detectable MST binding signal). [Figure 14-1] Affinity measurement of Nbs for LRRK2 using biolayer interferometry (BLI). Binding isotherms obtained by titrating increasing concentrations of Nbs against LRRK2 trapped on a streptavidin biosensor via biotinylated Nb40 (or Nb42 in the case of Nb40 affinity measurement) and measuring the BLI signal are shown. Nbs were classified into four functional groups as defined in Figure 2d: (a) Group 1 Nbs, (b) Group 2 Nbs, (c) Group 3 Nbs, and (d) Group 4 Nbs. All measurements were performed in the presence of 500 μM GDP. The corresponding equilibrium dissociation constants (Kd ± standard error) obtained by fitting the Langmuir binding equation are given (each data point is the average of three independent measurements, and error bars represent the standard deviation). [Figure 14-2]Affinity measurement of Nbs for LRRK2 using biolayer interferometry (BLI). Binding isotherms obtained by titrating increasing concentrations of Nbs against LRRK2 trapped on a streptavidin biosensor via biotinylated Nb40 (or Nb42 in the case of Nb40 affinity measurement) and measuring the BLI signal are shown. Nbs were classified into four functional groups as defined in Figure 2d: (a) Group 1 Nbs, (b) Group 2 Nbs, (c) Group 3 Nbs, and (d) Group 4 Nbs. All measurements were performed in the presence of 500 μM GDP. The corresponding equilibrium dissociation constants (Kd ± standard error) obtained by fitting the Langmuir binding equation are given (each data point is the average of three independent measurements, and error bars represent the standard deviation). [Figure 15] Nanobodies bind to and immunoprecipitate endogenous LRRK2. Lysates from RAW264.7 cells were incubated with 1.5 μM purified His-tagged Nbs, and pull-down was performed using magnetic Dynabeads. LRRK2 was detected by immunoblotting, demonstrating that all tested Nbs immunoprecipitate LRRK2. Blots are representative of n=3. [Figure 16-1] Nanobodies colocalize with endogenous LRRK2. RAW264.7 cells were transfected with GFP-fused Nbs and treated with zymosan for 30 min. Recruitment of LRRK2 and Nbs to phagosomes was analyzed by immunofluorescence. a, Images of Nb38, Nb22, Nb23, Nb40, no Nb, or irrelevant (IRR) Nb treatment; b, Images of Nb36, Nb42, Nb17, Nb39, Nb1, and Nb6 treatment. Scale bar = 10 μm. [Figure 16-2]Nanobodies colocalize with endogenous LRRK2. RAW264.7 cells were transfected with GFP-fused Nbs and treated with zymosan for 30 min. Recruitment of LRRK2 and Nbs to phagosomes was analyzed by immunofluorescence. a, Images of Nb38, Nb22, Nb23, Nb40, no Nb, or irrelevant (IRR) Nb treatment; b, Images of Nb36, Nb42, Nb17, Nb39, Nb1, and Nb6 treatment. Scale bar = 10 μm. [Figure 17] Nanobodies bind to LRRK2 through a binding site distinct from previously described LRRK2 kinase inhibitors. Results of a competitive ELISA titration experiment assessing whether the ATP-competitive kinase inhibitor Mli-2 and the previously described non-ATP-competitive LRRK2 inhibitor 5'-deoxyadenosylcobalamin (AdoCbl = coenzyme B12) compete for the same binding site with Group 1 nanobodies: Nb1 (a), Nb6 (b), and Nb23 (c). LRRK2 was coated onto the wells of an ELISA plate, and the ELISA signal of a dilution series of each Nb (detected via their C-terminal EPEA tag) is plotted as a function of Nb concentration. The effect of the presence of a large excess of Mli-2 (1 μM), AdoCbl (250 μM), and the corresponding untagged Nb (Nb*, 9 μM) as a positive (+) control is also determined. A "no antigen" control, in which LRRK2 was not coated on the bottom side of the well, is also included. In contrast to the positive control, where addition of Nb* causes a clear rightward shift of the ELISA titration curve compared to the titration curve of Nb alone (“−control”), neither Mli-2 nor AdoCbl show a rightward shift of the curve. DETAILED DESCRIPTION OF THE INVENTION

[0021] While the present invention will be described with respect to particular embodiments and with reference to certain drawings, the present invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. It should, of course, be understood that not necessarily all aspects or advantages can be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the invention can be embodied or practiced in a manner that achieves or optimizes one advantage or group of advantages taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein. The present invention, both as to organization and method of operation, together with its features and advantages, can best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will be apparent or elucidated with reference to the embodiment(s) hereinafter described herein. Reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the 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.

[0022] definition Where an indefinite or definite article is used when referring to a singular noun, such as "a," "an," or "the," this includes a plural of that noun unless something else is specifically stated. Where the term "comprises" 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 claims are not necessarily used to describe a sequential or chronological order but are used to distinguish between similar elements. It is to be understood that terms so used are interchangeable in appropriate circumstances and that the embodiments of the invention described herein may operate 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 have to one skilled in the art of the invention. For definitions and terminology of the art, practitioners should refer to, inter alia, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, structural biology, and / or computational biology).

[0023] The terms "protein," "polypeptide," and "peptide" are further used interchangeably herein to refer to a polymer of amino acid residues and variants and synthetic analogs thereof. A "peptide" can also be referred to as a partial amino acid sequence derived from its original protein, for example, after trypsin digestion. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, e.g., chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. The term also encompasses post-translational modifications of the polypeptide, such as glycosylation, phosphorylation, and acetylation. Based on the amino acid sequence and modifications, the atomic or molecular mass or weight of a polypeptide is expressed in (kilo)daltons (kDa). By "isolated" or "purified" is meant a material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated polypeptide" or "purified polypeptide" refers to a polypeptide that has been purified from molecules that naturally occur next to it, e.g., an antibody or Nanobody identified and disclosed herein that has been removed from molecules that are present in a sample or mixture, such as the production host, adjacent to said polypeptide. Isolated proteins or peptides can be produced by amino acid chemical synthesis, or can be produced by recombinant production or by purification from a complex sample.

[0024] Protein "homologue" and "homologues" encompass peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the native protein and that have similar biological and functional activity as the native protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical on an amino acid-by-amino acid basis over a comparison window. Therefore, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met, also designated herein by single-letter code) occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. As used herein, a "substitution" or "mutation" or "variant" results from the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides, respectively, compared to the amino acid or nucleotide sequence of a parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conservative amino acid substitutions that have substantially no effect on the activity of the protein.

[0025] "Bind" refers to any interaction, whether direct or indirect. A direct interaction implies contact between binding partners. An indirect interaction refers to any interaction in which the interacting partners interact in a complex of more than two molecules. An interaction can be completely indirect with the aid of one or more bridging molecules, or partially indirect, where there is still direct contact between the partners stabilized by the additional interaction of one or more molecules. As used herein, the term "specifically bind" refers to a binding domain that recognizes a specific target but does not substantially recognize or bind other molecules in a sample. Specific binding does not imply exclusive binding. However, specific binding does mean that a protein has a specific, increased affinity or preference for one or several of its binding factors. As used herein, the term "affinity" generally refers to the degree to which a ligand, chemical, protein, or peptide binds to another (target) protein or peptide in such a way as to shift the equilibrium of a single protein monomer toward the presence of a complex formed by their binding. Affinity is the strength of binding of a single molecule to its ligand. It is typically the equilibrium dissociation constant (K D ), which is used to assess and rank the strength of bimolecular interactions. Binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentrations of the reactants. At equilibrium, the rate of [antibody][antigen] complex formation equals the rate of dissociation into its components [antibody] + [antigen]. Measurement of the reaction rate constant is the equilibrium or affinity constant (1 / K D ) can be used to determine K D The smaller the value, the greater the affinity of the antibody for its target. The rate constants for both directions of the reaction are named: the association rate constant (K on ), which is the "on rate" (K), a constant used to characterize how quickly an antibody binds to its target. on ) is the part of the reaction used to calculate the dissociation rate constant (K off) is the "off rate" (K), a constant used to characterize how quickly an antibody dissociates from its target. off ) is the part of the reaction used to calculate the off-rate (K). In the measurements presented herein, the flatter the slope, the slower the off-rate or the stronger the antibody binding. Conversely, a steeper decline indicates a faster off-rate and weaker antibody binding. The ratio of the experimentally measured off- and on-rates (K off / K on ) is K D and K for measuring on and off rates. D Several determination methods for calculating σ are known to those skilled in the art (see below and examples). It is therefore considered a value that is independent of the assay used, taking into account standard error. As used herein, the term "protein complex" or "complex" or "assembled protein(s)" refers to a group of two or more associated macromolecules, whereby at least one of the macromolecules is a protein. As used herein, a protein complex typically refers to an association of macromolecules that can form under physiological conditions. The individual members of a protein complex are linked by non-covalent interactions.

[0026] A "binding agent" refers to a molecule capable of binding to another molecule, preferably specifically, recognizing a defined binding site, pocket, or epitope. A binding agent can be of any nature or type, regardless of its origin. A binding agent can be chemically synthesized, naturally occurring, recombinantly produced (and purified), or engineered and synthetically produced. Thus, a binding agent can be, inter alia, a small molecule, a chemical, a peptide, a polypeptide, an antibody, or a derivative of any of these, such as a peptidomimetic, an antibody mimetic, an active fragment, or a chemical derivative. The term "binding pocket" or "binding site" refers to a region of a molecule or molecular complex that, as a result of its shape and charge, favorably associates with another chemical entity, compound, protein, peptide, antibody, or Nb. The term "pocket" includes, but is not limited to, a cleft, a channel, or a site. The term "portion of a binding pocket / site" refers to fewer than all of the amino acid residues that define the binding pocket or binding site. For example, the residues may be key residues that play a role in ligand binding, or may be residues that are spatially related and define the three-dimensional compartment of the binding pocket. The residues may be contiguous or discontinuous in the primary sequence. For antibody-related molecules, the term "epitope" is also used interchangeably herein to describe a binding site. "Epitope" refers to an antigenic determinant of a polypeptide that constitutes a binding site or binding pocket on a target molecule, such as the LRRK2 protein, more specifically, a binding pocket on the LRRK2 domain accessible to an ISVD or VHH. An epitope may comprise three 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, at least 8, 9, or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a "conformational epitope" refers to an epitope that comprises amino acids in a spatial conformation that is unique to a folded, three-dimensional conformation of a polypeptide.Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that are together in the folded structure of a protein. However, a conformational epitope can also consist of a linear sequence of amino acids that adopts a conformation that is unique to the folded three-dimensional conformation of a polypeptide (and that is not present in the denatured state). In a protein complex, a conformational epitope consists of amino acids that are discontinuous in the linear sequence of one or more polypeptides, which are brought together by the folding of different folded polypeptides and their association in a unique quaternary structure. The term "conformation" or "conformational state" of a protein generally refers to the range of structures that a protein can adopt at any moment in time. Therefore, a conformational epitope can include amino acid interactions from different protein domains of the LRRK2 protein. Those skilled in the art will recognize that determinants of conformation or conformational state include the primary structure of a protein, as reflected in the amino acid sequence of the protein (including modified amino acids), and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features, such as protein secondary structure (e.g., α-helix, β-sheet, among others), tertiary structure (e.g., the three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions of the polypeptide chain with other protein subunits). Post-translational and other modifications of the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or attachment of hydrophobic groups, among others, can affect protein conformation. Furthermore, environmental factors, such as the pH, salt concentration, ionic strength, and osmolality of the surrounding solution, among others, as well as interactions with other proteins and cofactors, can affect protein conformation. The conformational state of a protein can be determined either by 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, see Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0027] As used herein, the terms "antibody," "antibody fragment," and "active antibody fragment" refer to a protein containing an immunoglobulin (Ig) domain or antigen-binding domain capable of specifically binding to an antigen, in this case, the LRRK2 protein. An "antibody" can further be an intact immunoglobulin derived from a natural or recombinant source, or an immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of an immunoglobulin molecule. The term "active antibody fragment" refers to any antibody or antibody-like structure portion that has high affinity for an antigenic determinant or epitope by itself and contains one or more complementarity-determining regions (CDRs) responsible for such specificity. Non-limiting examples include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains, nanobodies, domain antibodies, and single-chain structures, such as a complete light chain or a complete heavy chain. An additional requirement for the "activity" of the fragment, in the context of the present invention, is that the fragment is capable of binding to LRRK2, is preferably an allosteric modulator of LRRK2, and more preferably is capable of increasing or decreasing LRRK2 activity in a subject. The term "immunoglobulin (Ig) domain" or more specifically "immunoglobulin variable domain" (abbreviated as "IVD") essentially means an immunoglobulin domain consisting of four "framework regions" referred to in the art and herein below as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," respectively; these framework regions are interrupted by three "complementarity-determining regions" or "CDRs," referred to in the art and herein below as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be designated as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.It is the immunoglobulin variable domain(s) (IVD) that confers specificity to an antibody by carrying the antigen-binding site. Typically, in a conventional immunoglobulin, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL will contribute to the antigen-binding site. That is, a total of six CDRs will be involved in forming the antigen-binding site. From the above definition, the antigen-binding domains of a conventional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule; known in the art) or of a Fab fragment, F(ab')2 fragment, Fv fragment, such as a disulfide-linked Fv or scFv fragment, or diabody (all known in the art) derived from such a conventional four-chain antibody will bind to their respective epitopes of the antigen in cooperation with a pair of (related) immunoglobulin domains such as light and heavy chain variable domains, i.e., a VH-VL pair of immunoglobulin domains. As used herein, an immunoglobulin single variable domain (ISVD) refers to a protein having an amino acid sequence comprising four framework regions (FR) and three complementarity-determining regions (CDRs) in the format FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The term "immunoglobulin domain" of the present invention also refers to an "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is equivalent to the term "single variable domain" and defines a molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "traditional" immunoglobulins or fragments thereof, in which two immunoglobulin domains interact at the specific two variable domains 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 at most three CDRs.Thus, a 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 consisting essentially of a single variable domain such that the single antigen-binding domain does not require interaction with another variable domain to form the functional antigen-binding unit).

[0028] In particular, the immunoglobulin single variable domain may be a Nanobody® (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies®, and Nanoclone® are registered trademarks of Ablynx NV (a Sanofi Company). For a general description of Nanobodies, reference is made to the further description below and to the prior art cited herein, e.g., as described in WO2008 / 020079. "VHH domains," also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, were originally described as antigen-binding immunoglobulin (Ig) (variable) domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (which are referred to herein as "VH domains") and the light chain variable domains present in conventional four-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and nanobodies, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001) and to the following patent applications:These are mentioned as general background art: WO94 / 04678, WO95 / 04079, and WO96 / 34103 from Vrije Universiteit Brussel; WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP1134231, and WO02 / 48193 from Unilever; WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016, and WO03 / 055527 from Vlaams Instituut voor Biotechnologie (VIB); Algonomics WO03 / 050531 by NV and Ablynx NV; WO01 / 90190 by the National Research Council of Canada; WO03 / 025020 (=EP1433793) by the Institute of Antibodies; and WO04 / 041867, WO04 / 041862, WO04 / 041865, WO04 / 041863, WO04 / 062551, WO05 / 044858, WO06 / 40153, WO06 / 079372, WO06 / 122786, WO06 / 122787, and WO06 / 122825 by Ablynx NV, and further published patent applications by Ablynx NV. As described in these references, Nanobodies (especially VHH sequences and partially humanized Nanobodies) can be characterized, inter alia, by the presence of one or more "hallmark residues" in one or more of the framework sequences. Further description of Nanobodies, including humanized and / or camelized Nanobodies, as well as other modifications, parts or fragments, derivatives or "nanobody fusions," multivalent or multispecific constructs (including some non-limiting examples of linker sequences), and different modifications to increase the half-life of Nanobodies, and their preparation, can be found, for example, in WO08 / 101985 and WO08 / 142164. Nanobodies form the smallest antigen-binding fragment that fully retains the binding affinity and specificity of a full-length antibody.Nbs possess exceptionally long complementarity-determining region 3 (CDR3) loops and convex paratopes, which allow them to penetrate into hidden cavities in target antigens.

[0029] As used herein, the terms "determining," "measuring," "assessing," "identifying," "screening," and "assaying" are used interchangeably and encompass both quantitative and qualitative determinations.

[0030] The terms "subject," "individual," or "patient," as used interchangeably herein, refer to any organism, such as a vertebrate, particularly any mammal, including both humans and other mammals, for which diagnosis, treatment, or prevention is desired, for example, a rodent, rabbit, cow, sheep, horse, dog, cat, llama, pig, or non-human primate (e.g., monkey). The rodent may be a mouse, rat, hamster, guinea pig, or chinchilla. In one embodiment, the subject is a human, rat, or non-human primate. Preferably, the subject is a human. In one embodiment, the subject is a subject who has or is suspected of having a disease or disorder, particularly a disease or disorder disclosed herein, and is also referred to herein as a "patient." However, it will be understood that the foregoing terms do not imply the presence of symptoms. The terms "treatment" or "treating" or "treat" may be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, hinders, controls, stops, reduces, or reverses the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the total elimination of all disease-related signs, symptoms, conditions, or disorders.

[0031] As used herein, the term "medicament" refers to a substance / composition used in therapy, i.e., in the prevention or treatment of a disease or disorder. According to the present invention, the term "disease" or "disorder" refers to any pathological condition, particularly a disease or disorder as defined herein.

[0032] Detailed Description The present invention relates to non-naturally occurring protein-based binding agents of the human LRRK2 protein, specifically allosteric modulators of LRRK2 activity. Mutations in the protein leucine-rich repeat kinase 2 (LRRK2), associated with Parkinson's disease (PD), usually lead to reduced GTPase activity and increased kinase activity. LRRK2 (dominant) mutations, of which G2019S is the most prevalent, cause late-onset Parkinson's disease mediated by increased phosphorylation of Rab proteins. Therefore, inhibition of kinase activity and / or increased GTPase activity may provide therapeutically beneficial outcomes. Indeed, although known ATP-competitive inhibitors of LRRK2 kinase activity have demonstrated potential therapeutic benefit in the treatment of PD, concerns remain about potential adverse side effects associated with the use of kinase inhibitors directed against the LRRK2 ATP-binding pocket [46, 81-82]. Although additional generations of compounds acting by orthosteric mechanisms are in clinical trials, the need for compounds with better safety profiles dictates alternative approaches through allosteric modulation of LRRK2 activity as a novel approach and within the scope of this disclosure.

[0033] The present invention relates to allosteric binding agents that bind predominantly to LRRK2 via a conformational epitope on a domain distinct from the ATP catalytic binding site on the kinase domain and / or without directly competing with ATP for its active site, and without competing with known binding agents of LRRK2, or specifically the LRRK2 kinase domain. The spectrum of binding agents described herein encompasses multiple layers of allosteric modulation of LRRK2, which will support the creation of novel, first-in-class LRRK2 therapeutics. Notably, the binding agents of the present invention all efficiently bind to LRRK2 from cell lysates both with LRRK2 overexpression and at endogenous LRRK2 expression levels, with K values ​​ranging from 10 to 200 nM. DThis reflects the high affinity binding confirmed by two independent biophysical methods. Further affinity maturation, generation of multiparatope constructs, and / or humanization may increase this human LRRK2 protein binding affinity even to subnanomolar affinities.

[0034] Furthermore, LRRK2-specific immunoglobulin single variable domains (ISVDs) provide the first protein-based LRRK2 binding factors that act by both an allosteric and a mixed (noncompetitive) inhibitory mechanism to inhibit LRRK2 activity. Similar to four of the five common PD mutations, treatment with type 1 ATP-competitive inhibitors has been shown to induce LRRK2 association with microtubules. This phenomenon of LRRK2 oligomerization on microtubules, with the concomitant blockade of microtubule-associated motor proteins, has been suggested as one of the causes behind LRRK2 pathology

[36] . Surprisingly, none of the ISVD-based LRRK2-specific binding factors described herein induce LRRK2 association with microtubules, and some even reverse LRRK2 translocation induced by the type 1 ATP-competitive kinase inhibitor MLI-2. These observations provide differential effects of the LRRK2 binding factors of the present invention and position them as candidates for further development into LRRK2 modulators with different modes of action and cellular profiles than currently available inhibitors.

[0035] Thus, the present invention provides allosteric LRRK2 protein-binding agents that bind to and maintain the cellular distribution of human LRRK2 in cells without affecting the protein's subcellular localization, thereby maintaining LRRK2 away from or not associated with microtubules. Thus, in one embodiment, the LRRK2-specific binding agents or modulators described herein do not induce LRRK2 accumulation on microtubules. Furthermore, the ISVD-based LRRK2 allosteric binding agents disclosed herein provide binding affinities that can be determined using methods known to those of skill in the art or exemplified herein, and that have a K as defined herein of 500 nM or less, preferably 200 nM or less, and more preferably between 150 nM and 10-fold lower, or 100-fold lower, or 1,000-fold lower, or 10,000-fold lower. D Regarding values.

[0036] More specifically, the binding agents most preferably allosterically modulate LRRK2 kinase activity in cells and / or in vitro. Although modulation of kinase activity can be linked to the LRRK2 monomer / dimerization cycle and to the GTPase activity of the protein, the LRRK2 modulators described herein focus on the inherent beneficial effects of applying ISVDs to target LRRK2, on the one hand, and on those effects obtained by high-affinity binding in a conformational manner that results in kinase activity modulation depending on the binding position, on the other hand.

[0037] Therefore, in specific aspects, LRRK2 allosteric binding agents include immunoglobulin single variable domains (ISVDs) or VHHs or nanobodies, which are used interchangeably herein, that bind to conformational epitopes and modulate LRRK2 protein conformation, thereby affecting its activity. Their effects were analyzed in cellulo for a broader panel of 18 different ISVD families. These ISVDs were categorized according to their regulatory profile (subcellular localization, kinase activity inhibition, or activation of LRRK2) and binding mode (different conformational epitopes and binding sites), which provide common features of in cellulo allosteric modulation of LRRK2. Moreover, their interactions with LRRK2 have been identified to predominantly reside outside the kinase domain, diversifying these highly specific protein-based binding agents from all known ATP-competitive kinase inhibitors, and even from the natural compound 5'-deoxyadenosylcobalamin (AdoCbl; the physiological form of vitamin B12;

[43] ; as shown in Example 10). In one aspect, the present invention relates to allosteric modulators of human LRRK2 that specifically bind to the LRRK2 protein with high affinity, wherein said modulators are distinct from or do not include naturally occurring LRRK2 modulators or binding factors, such as cobalamin, the physiological form of vitamin B12, or derivatives thereof.

[0038] Furthermore, in contrast to conventional LRRK2-specific antibodies, the LRRK2-modulating ISVD of the present invention is a small agent and therefore can also be applied to study the dynamic localization of endogenous LRRK2 in living cells (in vivo imaging).

[0039] Thus, the present invention identifies, for the first time, highly specific LRRK2-binding agents that allosterically regulate LRRK2 activity, a mechanism that can avoid further adverse effects, such as cellular translocation of the LRRK2 protein. Such allosteric binding factors can have significant activating or inhibitory effects on LRRK2. For this reason, kinase inhibitors are currently considered most therapeutically relevant. The only reported small compounds that inhibit in an allosteric manner are vitamin B12 derivatives

[43] , but their binding sites are predominantly located in the LRRK2 kinase domain, thereby providing at least a partially different binding site and mechanism of action compared to the allosteric ISVDs disclosed herein. Furthermore, ISVDs have been demonstrated to specifically bind to the interfaces of various LRRK2 domains, including the Roc domain, COR domain, kinase domain, WD40 domain, and / or the N-terminal (armadillo, ankyrin repeat, LRR) domain and the kinase or WD40 domain. Although a high-resolution structure of full-length LRRK2 is lacking and the precise epitope-binding site has yet to be confirmed, findings from ELISA and cross-link mass spectrometry data have provided a region that indicates where the allosteric ISVD binds, at distances of up to 35 Å, via these cross-links between proteins. From this information, it was concluded that the majority of allosteric modulators of LRRK2 described herein function by binding to conformational epitopes that are predominantly located outside the kinase domain and / or whose epitope-binding site residues are located at least outside the kinase active site.

[0040] The allosteric modulators described herein are "non-naturally occurring," "non-naturally occurring," or "unnatural" binding agents, which are used interchangeably herein. This refers to the fact that these binding agents or modulators do not naturally occur as such; i.e., technical steps or processes, such as immunization, are required to obtain such highly specific allosteric modulator-binding agents. In contrast, reported vitamin B12 derivative compounds that bind to the kinase domain of LRRK2 are known to naturally occur in four forms as cobalamins. Cobalamins actually refer to a group of complex, chemically closely related cofactors that require cobalt (Co) for function. Hydroxycobalamin is produced by bacteria, and cyanocobalamin (CNCbl) is a form derived during the purification of hydroxycobalamin (OHCbl) for therapeutic or nutritional supplement purposes. Both are further metabolized in the body to form the active forms adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).

[0041] As used herein, the terms "allosteric modulation," "allosteric activity," or "allosteric control" refer to the binding of an agent at an allosteric or regulatory site that is distinct from the enzymatic active or catalytic site of a protein and, therefore, distinct from the binding site of an orthosteric binding agent. Such catalytic sites of LRRK2 include the kinase domain active site and the GTPase domain active site. "Modulators" can be positive, negative, or neutral. A "positive allosteric modulator" increases the activity or response of LRRK2 by either increasing the probability that an agonist or ligand, such as ATP or GTP, will bind to LRRK2 (i.e., increasing affinity) or by increasing its ability to activate LRRK2 (i.e., increasing potency), or both. A "negative allosteric modulator" decreases agonist affinity and / or potency. A "neutral allosteric modulator" does not affect agonist activity but may prevent other modulators from binding to the allosteric site. Some modulators may also act as allosteric agonists. Allosteric binding agents may affect or modulate LRRK2 activity, for example, by inducing a conformational change in the LRRK2 protein upon binding. In one embodiment, the allosteric inhibitor of LRRK2 activity is a non-naturally occurring molecule and thus differs from naturally occurring compounds such as (metabolized) human cobalamin derivatives. Thus, in a specific embodiment, the allosteric modulator of LRRK2 is an LRRK2 inhibitor that is not cobalamin or a cobalamin derivative. As used herein, the term "derivative" includes, but is not limited to, the cobalamin derivatives AdoCbl, MeCbl, OHCbl, and CNCbl, and / or any naturally occurring compound containing the cobalamin backbone. Preferably, said allosteric modulator of LRRK2 that specifically binds to LRRK2 is a heterologous or exogenous compound when present in a cell, organism, or subject.

[0042] The present invention specifically relates to allosteric modulators of human LRRK2. It is clear to those skilled in the art that LRRK2 proteins of bacterial origin are highly diverse compared to the human LRRK2 protein (SEQ ID NO: 21), not only in terms of primary structure or amino acid sequence, but also in terms of secondary and tertiary structure, e.g., the fact that bacterial LRRK2 lacks a kinase domain. Human LRRK2 is a complex and larger protein, implying thorough design to generate specific binding factors that can strongly affect human LRRK2 activity. The binding agents or ISVDs and allosteric modulators described herein are obtained by well-defined immunization and selection strategies and provide novel binding agents with novel conformational epitopes of high therapeutic potential. ISVDs, or more specifically nanobodies, are known to act as stabilizers or chaperones in structural biology analyses and are also being developed as therapeutic agents. Targeting human LRRK2 with allosteric ISVDs or Nbs or active antibody fragments derived therefrom has not previously been demonstrated. Although their therapeutic potential may be complicated by the hurdles of intracellular targeting of LRRK2 and reaching the brain for PD treatment, their binding mode offers several unique approaches to producing and selecting novel compounds for improving LRRK2 drug delivery. Indeed, their conformational epitopes provide novel druggable pockets, and their highly specific and allosteric effects, combined with their sophisticated mode of action and preserved LRRK2 cellular localization, may potentially result in reduced toxicity risk. For drug delivery, as further described herein, their small size is advantageous for aiding in crossing the blood-brain barrier or for linking them to cargo or capturing them with a vehicle that crosses the BBB. Finally, nanobodies can also be applied therapeutically as intrabodies, potentially using gene therapy.

[0043] In one embodiment, the allosteric modulator of LRRK2 comprises a binding agent that specifically and predominantly binds to the Roc domain of LRRK2. In another embodiment, the allosteric modulator of LRRK2 comprises a binding agent that specifically and predominantly binds to the COR(-B) domain of LRRK2. In another embodiment, the allosteric modulator of LRRK2 comprises a binding agent that specifically and predominantly binds to the WD40 domain of LRRK2. In another embodiment, the allosteric modulator of LRRK2 comprises a binding agent that specifically and predominantly binds to the kinase domain of LRRK2. In another embodiment, the allosteric modulator of LRRK2 comprises a binding agent that specifically binds to the interface of various LRRK2 domains, meaning that it binds to residues on several domains, which may include the N-terminal (armadillo, ankyrin repeat, LRR) domain and the kinase or WD40 domain. In some embodiments, the allosteric modulator of LRRK2 specifically binds to a combination of residues present in any of the domains. In preferred embodiments, the allosteric modulators of LRRK2 described herein do not bind to the active site of the kinase or GTPase domain of LRRK2. In more preferred embodiments, the allosteric modulators of LRRK2 described herein do not bind to kinase domain amino acid residues, but rather specifically bind to an LRRK2 binding site that includes amino acid residues belonging to other protein domains of LRRK2.

[0044] In another embodiment, the allosteric modulator that specifically binds to the LRRK2 protein can increase its kinase activity and / or decrease its GTPase activity. In other embodiments, the allosteric modulator that specifically binds to the LRRK2 protein can decrease, inhibit, or block its kinase activity and / or increase its GTPase activity. The terms "increase," "enhance," or "activate" are used interchangeably herein and refer to an increase in activity of at least 5% compared to a control without the allosteric modulator or a negative or unrelated control agent. The terms "increase," "enhance," or "activate" further refer to an increase in activity of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% compared to a control without the allosteric modulator or a negative or unrelated control agent. The terms "reduced," "reduced," "inhibition," or "preventing," as used interchangeably herein, refer to a reduction in activity of at least 5% compared to a control without an allosteric modulator or a negative or unrelated control. The terms "reduced," "reduced," "prevent," or "inhibit" further refer to a reduction in activity of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more than 50% compared to a control without an allosteric modulator or a negative control. The term "blocking" LRRK2 activity refers to a reduction in activity to undetectable levels compared to a control without an allosteric modulator or a negative control. As used herein, a "negative control" or an "irrelevant control" or a "control" or a "vehicle control" refers to a binding agent of similar properties (e.g., an irrelevant Nb) that is known not to bind to LRRK2 or that binds to LRRK2 but does not have any effect on its activity. A "control" can be a type or pool of molecules (such as an irrelevant Nb or compound) that are known to have no effect on LRRK2.

[0045] Inhibition of the kinase activity of LRRK2, as used herein, means that its ability to autophosphorylate and / or phosphorylate its substrate is reduced. Conversely, activation or enhancement of the kinase activity of LRRK2, as described herein, means that LRRK2 is affected by an allosteric regulator, resulting in increased autophosphorylation and / or substrate phosphorylation. Substrates referred to herein include, but are not limited to, the Rab proteins referenced in the examples, and also include peptides used in commercially available in vitro assays. Autophosphorylation has been reported at several LRRK2 amino acid positions, as known to those skilled in the art from the prior art.

[0046] In another embodiment, the allosteric modulator of LRRK2 protein activity comprises a compound, chemical substance, protein, peptide or peptidomimetic, antibody, antibody mimetic, single-domain antibody ISVD, or any active antibody fragment. As used herein, the term "compound" describes any molecule, either naturally occurring or synthetic, that can be designed, identified, screened, or generated and tested in an assay, such as a screening assay or drug discovery assay, or specifically in a method for identifying compounds capable of modulating LRRK2 activity. Thus, these compounds include organic and inorganic compounds. For high-throughput purposes, test compound libraries, such as combinatorial or randomized libraries that provide a sufficient range of diversity, can be used. Examples include, but are not limited to, natural compound libraries, allosteric compound libraries, peptide libraries, antibody fragment libraries, synthetic compound libraries, fragment-based libraries, phage display libraries, and the like. Such compounds may also be referred to as binding agents; as referred to herein, they may be "small molecules" or "small compounds," which refer to low molecular weight (e.g., <900 Da or <500 Da) organic compounds. Allosteric modulators also include chemicals and compounds characterized by low molecular weight, such as polynucleotides, lipids, or hormone analogs. Other biopolymer-based organic test compounds include small peptides or peptide-like molecules, or their derivatives, such as peptidomimetics containing synthetic amino acids (peptidomimetics) containing from about 2 to about 40 amino acids.

[0047] The compounds of the present invention include both those designed or identified using screening methods as specifically defined herein for the ISVDs of the present invention and those capable of conformationally binding to LRRK2. Such compounds can also be produced using screening methods based on the use of structural conformations obtained for LRRK2 in complex with the ISVDs of the present invention as presented herein. Candidate compounds and / or compounds identified or designed using the methods of the present invention can be any suitable compound, synthetic or naturally occurring, preferably synthetic. In one embodiment, synthetic compounds selected or designed by the methods of the present invention preferably have a molecular weight of less than or equal to about 5,000, 4,000, 3,000, 2,000, 1,000, or more preferably less than about 500 Da, or are preferably peptides. The compounds of the present invention are preferably soluble under physiological conditions. Such compounds may contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two chemical functional groups. The compounds may comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. The compounds may also include biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.Compounds can include, for example: (1) peptides, e.g., soluble peptides or peptidomimetics, including Ig-tail fusion peptides, and members of random peptide libraries and combinatorial chemistry-derived molecular libraries made from D- and / or L-configuration amino acids and / or conformationally constrained amino acid derivatives; (2) phosphopeptides (e.g., members of random and partially degenerate directed phosphopeptide libraries, (3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single-chain antibodies, nanobodies, and Fab, (Fab)2, Fab expression libraries, and epitope-binding fragments of antibodies); (4) non-immunoglobulin binding proteins, such as, but not limited to, avimers, DARPins, and lipocalins; (5) nucleic acid-based aptamers; and (6) organic and inorganic small molecules.

[0048] Synthetic compound libraries are commercially available from, for example, Maybridge Chemical Co. (Tintagel, Cornwall, UK), AMRI (Budapest, Hungary), and ChemDiv (San Diego, Calif.), Specs (Delft, The Netherlands), and ZINC15 (University of California). Additionally, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts can be readily produced. Additionally, natural or synthetic compound libraries and compounds can be readily modified by conventional chemical, physical, and biochemical means and used to produce combinatorial libraries. Additionally, numerous methods for producing combinatorial libraries are known in the art, including those involving biological libraries; spatially addressable parallel solid-phase or solution-phase libraries; synthetic library methods requiring deconvolution; "one bead, one compound" library methods; and synthetic library methods using affinity chromatography selection. While the biological library approach is limited to polypeptide or peptide libraries, the other four approaches are applicable to polypeptide, peptide, non-peptide oligomeric, or small molecule libraries of compounds. Compounds also include those that can be synthesized from leads generated by fragment-based drug design, and the binding of such chemical fragments is assessed by soaking or co-crystallizing such screening fragments with the crystals provided by the present invention, and then subjecting them to an X-ray beam and obtaining diffraction data.

[0049] Furthermore, compounds identified or designed using the methods of the present invention can be peptides or mimetics thereof. The isolated peptides or mimetics of the present invention can be conformationally constrained molecules, or alternatively, conformationally unconstrained molecules, such as unconstrained peptide sequences. The term "conformationally constrained molecules" refers to conformationally constrained peptides and conformationally constrained peptide analogs and derivatives. Additionally, amino acids can be replaced with various unencoded or modified amino acids, such as corresponding D-amino acids or N-methyl amino acids. Other modifications include the replacement of hydroxy, thiol, amino, and carboxyl functional groups with chemically similar groups. For peptides and their mimetics, other non-natural amino acids or chemical amino acid analogs / derivatives can be introduced as substitutions or additions. Peptidomimetics can also be used. Peptidomimetics are molecules that mimic the biological activity of peptides but are no longer peptidic in chemical nature. By strict definition, peptidomimetics are molecules that no longer contain any peptide bonds (i.e., amide bonds between amino acids). However, the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudopeptides, semi-peptides, and peptoids. Whether completely or partially non-peptidic, peptidomimetics for use in the present invention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of the active groups on the peptide on which the peptidomimetic is based. For example, a peptide or peptidomimetic can be designed to mimic the paratope or CDR of an ISVD described herein. Typically, as a result of this similar active site geometry, the peptidomimetic has an effect on a biological system that is similar to the biological activity of the peptide. In some cases, it is advantageous to use a mimetic of a given peptide rather than the peptide itself, because peptides usually exhibit two undesirable properties: (1) poor bioavailability; and (2) a short duration of action.Peptide mimetics offer an obvious bypass of these two major obstacles because the molecules involved are small enough to be both orally active and have a long duration of action. There are also significant cost savings and improved patient compliance associated with peptide mimetics because they can be administered orally compared to parenteral administration of peptides. Furthermore, peptide mimetics are generally cheaper to produce than peptides. Of course, those skilled in the art will recognize that the design of a peptide mimetic may require minor structural modifications or adjustments to chemical structures designed or identified using the methods of the present invention. Generally, chemical compounds or peptides identified or designed based on the ISVD of the present invention can be chemically synthesized and then tested for their ability to bind to and modulate LRRK2 activity using any of the methods described herein.

[0050] As previously described herein, allosteric modulators may also include larger polypeptides containing about 40 to about 500 amino acids, such as antibodies, antibody mimetics, antibody fragments, or antibody conjugates. LRRK2-binding agents preferably include protein-binding agents and / or binding agents that have allosteric or allosteric modulating activity upon binding to LRRK2. These allosteric compounds are defined as allosteric modulators that function as positive allosteric modulators (PAMs) of LRRK2 when they increase its (kinase) activity, or conversely, as negative allosteric modulators (NAMs) of LRRK2 activity, resulting in decreased LRRK2 (kinase) activity or inhibiting or blocking LRRK2 activity. In a specific embodiment, it is contemplated to provide an allosteric modulator of LRRK2 comprising an antibody or an active antibody fragment as defined herein. The allosteric modulator preferably specifically binds to LRRK2 and comprises an ISVD containing four framework regions and three CDR regions.

[0051] More specifically, said ISVD, nanobody or VHH, or active antibody fragment comprises the CDR1, CDR2, and CDR3 sequences provided for the CDRs of the ISVDs of SEQ ID NOs: 1 to 19, or any combination of said CDR sequences. The CDR region annotations for each Nb sequence described herein are shown in Table 3 for the specific CDR annotations used in the current analysis. Alternatively, slightly different CDR annotations known in the art can be applied herein to define CDR regions, such as AbM (AbM is an antibody modeling package from Oxford Molecular Ltd., described at http: / / www.bioinf.org.uk / abs / index.html), Chothia (Chothia and Lesk, 1987; J Mol Biol. 196:901-17), Kabat (Kabat et al., 1991; Sequences of Proteins of Immunological Interest. 5th edition, NIH publication 91-3242), or IMGT (LeFranc, 2014; Frontiers in Immunology. 5 (22): 1-22) annotations, all of which are applicable to identifying the CDR regions of the ISVDs disclosed herein in SEQ ID NOS: 1-19. To clarify the exact sequences covered by the alternative annotations, the CDRs are provided in FIG. 10 for the CA12610 Nb as an example relative to the currently applied annotations in Table 3.

[0052] It should be noted that, as is well known in the art for VH and 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 (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than permitted by the Kabat numbering). This generally means that 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 VH and VHH domains will usually range from 110 to 120, often between 112 and 115. However, it should be noted that smaller and larger sequences may also be suitable for the purposes described herein.

[0053] In specific embodiments, the ISVDs or Nbs that specifically bind to LRRK2 affect cellular kinase activity (see also the Examples and, in particular, Figure 2). The effect, in specific embodiments, can be envisioned as a kinase inhibitory effect by acting directly or indirectly on LRRK2 kinase enzymatic activity, observed as inhibition of LRRK2 autophosphorylation and / or inhibition of in cellulo substrate (Rab10) phosphorylation and / or inhibition of in vitro LRRK2 kinase activity toward a peptide substrate. Thus, in one embodiment, the allosteric modulators of LRRK2 described herein inhibit cellular kinase activity.

[0054] More specifically, the panel of Nbs exemplified herein (Nb1, Nb6, Nb23, and Nb42) that inhibit all such tested LRRK2 kinase activities, i.e., inhibit LRRK2 autophosphorylation and inhibit in cellulo substrate (Rab10) phosphorylation and / or inhibit in vitro LRRK2 kinase activity against peptide substrates, therefore block LRRK2 kinase activity itself. Nb42 was also classified into this group because it has a strong inhibitory effect on cellular LRRK2 autophosphorylation and Rab phosphorylation, but the effect on in vitro kinase activity could not be demonstrated in the presence of Nb42. Thus, in a specific embodiment contemplated by the Nbs exemplified herein, allosteric modulators of LRRK2 that are inhibitors of LRRK2 kinase activity per se are defined herein as comprising an ISVD in which CDR1, CDR2, and CDR3 consist of the CDRs of SEQ ID NOs: 1, 2, 3, or 6, with the CDR regions defined as in Tables 3-4 or FIG. 10 disclosed herein. These kinase-inhibitory Nbs are provided in two categories based on their binding epitopes. Nb1 (SEQ ID NO: 1) and Nb6 (SEQ ID NO: 2) bind exclusively to the C-terminal part of the COR domain (COR-B), whereas Nb23 (SEQ ID NO: 3) crosslinks with K2078 and K2091 on the C-terminal lobe of the kinase domain. The latter residues are located in close proximity to each other and to the S1292 autophosphorylation site, but are quite distant from the ATP-binding pocket. The observation that none of these Nbs binds to the ATP-binding pocket of the kinase, and that Nb1 and Nb6 even bind outside the kinase domain, indicates that these Nbs do not act by an ATP-competitive mechanism. Correspondingly, kinetic analysis indicates that all three Nbs act by a mixed inhibitory mechanism and have a preference for binding to the "ATP-free" LRRK2 conformation over the "ATP-bound" conformation. Notably, given that the COR-B domain is located at the center of the LRRK2 structure, this may indicate that these Nbs push the LRRK2 protein into a more "open" catalytically incompetent conformation.Nevertheless, in contrast to ATP-competitive type 1 inhibitors, none of these Nbs induced microtubule translocation of LRRK2.

[0055] The second group of Nbs contemplated herein (Nb17, Nb36, Nb38, Nb40, and Nb41) inhibit cellular Rab substrate phosphorylation while leaving autophosphorylation and peptide phosphorylation unaffected, suggesting that these Nbs either sterically interfere with the binding of larger Rab substrates or lock LRRK2 in a conformation that precludes Rab binding. Thus, in specific embodiments contemplated herein for the Nbs exemplified, allosteric modulators of LRRK2 that are inhibitors of LRRK2 kinase activity in that they prevent substrate phosphorylation are defined herein as comprising an ISVD in which CDR1, CDR2, and CDR3 consist of the CDRs of SEQ ID NOs: 4, 5, 7, 8, or 12, and the CDR regions are defined as in Tables 3-4 or Figure 10 disclosed herein.

[0056] Further embodiments relate to a third group of Nbs (Nb22, Nb28), which modulate LRRK2 by increasing both cellular (and, as shown for at least Nb22, in vitro) LRRK2 kinase activity. Thus, in specific embodiments contemplated for the Nbs exemplified herein, allosteric modulators of LRRK2 that seek to activate LRRK2 kinase activity are defined herein as comprising an ISVD in which CDR1, CDR2, and CDR3 consist of the CDRs of SEQ ID NO:9 or SEQ ID NO:18, and the CDR regions are defined as in Tables 3-4 or Figure 10 disclosed herein.

[0057] Finally, a fourth group of Nbs can be envisioned (Nb3, Nb9, Nb10, Nb13, Nb31, Nb37, and Nb39). While these do not appear to affect LRRK2 kinase activity, they also bind to LRRK2 outside the ATP-binding pocket with high affinity (nanomolar range) and still provide the previously mentioned effect of maintaining its cytoplasmic distribution. These kinase-neutral binding agents may be particularly suitable for use in detection or diagnostic assays requiring LRRK2 binding, or alternatively, for use in screening assays requiring an LRRK2 conformation accessible by binding by these Nbs. Thus, in specific embodiments contemplated herein for the Nbs exemplified herein, LRRK2 Nbs that are high-affinity kinase-neutral binders are defined herein as comprising ISVDs whose CDR1, CDR2, and CDR3 consist of the CDRs of SEQ ID NOs: 10, 11, and 13-17, and the CDR regions are defined as in Tables 3-4 or FIG. 10 disclosed herein.

[0058] Furthermore, provided herein is a panel of allosteric modulators of LRRK2 that prevent LRRK2 from associating with cellular microtubules, even when an ATP-competitive LRRK2 kinase inhibitor compound is present in the same cell. The ATP-competitive LRRK2 kinase inhibitors may also be defined herein as orthosteric binding factors of LRRK2. Thus, in a specific embodiment contemplated by the Nbs exemplified herein, an allosteric modulator of LRRK2 that binds with high affinity and blocks cellular LRRK2 translocation to microtubules when such translocation is normally (i.e., in the absence of a modulator) induced (e.g., by the presence of a type 1 ATP-competitive kinase inhibitor) is defined herein as comprising an ISVD in which CDR1, CDR2, and CDR3 consist of the CDRs of SEQ ID NOs: 3, 4, 5, and 9, and the CDR regions are defined as in Tables 3-4 or Figure 10 disclosed herein.

[0059] In another embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19. In another embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of sequences having at least 85% identity to any of the sequences SEQ ID NOs: 1-19, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1-19, with differences may reside in framework residues. In a specific embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of sequences having at least 85% identity to any of the sequences SEQ ID NOs: 1-19, or at least 90% identity thereto, or at least 95% identity thereto, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1-19, with differences may reside in framework residues, with the exception of llama germline hallmark residues present in said framework regions. More specifically, the latter FR residues that should not be altered correspond, for example, to residue 37 (Kabat N°; F or Y), which corresponds to residue 39 of SEQ ID NO:1; residues 44-45 (Kabat N°; QR, ER, or DR), which correspond to residues 46-47 of SEQ ID NO:1; residue 47 (Kabat N°; L, T, or F), which corresponds to residue 49 of SEQ ID NO:1; residue 78 (Kabat N°; G or V), which corresponds to residue 80 of SEQ ID NO:1; and residue 84 (Kabat N°; P), which corresponds, for example, to residue 89 of SEQ ID NO:1 (see also Figure 10, underlined / bold residues in the first sequence). In another aspect, the LRRK2 modulator comprises an ISVD comprising an amino acid sequence selected from the group consisting of a humanized variant of any of the sequences of SEQ ID NOs: 1-19, or a humanized variant of any of the sequences having at least 85% identity to SEQ ID NOs: 1-19, or at least 90% identity to SEQ ID NOs: 1-19, or at least 95% identity to SEQ ID NOs: 1-19, wherein the CDRs are identical to any one of the CDRs of SEQ ID NOs: 1-19 and the FR hallmark residues are identical to any one of SEQ ID NOs: 1-19, with humanized substitutions providing differences in residues elsewhere in the FR regions.

[0060] The term "humanized variant" of immunoglobulin single variable domains, such as domain antibodies and Nanobodies® (including VHH domains), refers to the amino acid sequence of said ISVD that represents the outcome of being subjected to humanization, i.e., to increase the degree of sequence identity with the closest human germline sequence. In particular, a humanized immunoglobulin single variable domain, such as a Nanobody® (including a VHH domain), can be an immunoglobulin single variable domain in which there is at least one amino acid residue (in particular at least one framework residue) that is and / or corresponds to a humanizing substitution (as further defined herein). Potentially useful humanizing substitutions can be ascertained 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, one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein), and the resulting humanized VHH sequence can be tested for affinity to the target, for stability, for ease and level of expression, and / or for other desired properties. In this manner, other or even more suitable humanizing substitutions (or suitable combinations thereof) can be determined by those skilled in the art by means of a limited degree of trial and error. Also, based on what has been described above, (the framework regions of) immunoglobulin single variable domains such as Nanobodies® (comprising VHH domains) can be partially or fully humanized. Humanized immunoglobulin single variable domains, and in particular Nanobodies, can have several advantages compared to corresponding naturally occurring VHH domains, such as reduced immunogenicity. In summary, humanizing substitutions should be chosen such that the resulting humanized amino acid sequences of the ISVD and / or VHH still retain favorable properties, such as antigen binding capacity and allosteric modulation capacity.Those skilled in the art will be able to select suitable humanization substitutions or combinations of humanization substitutions that optimize or achieve a desired or suitable balance between the favorable properties provided by the humanization substitutions on the one hand and the favorable properties of naturally occurring VHH domains on the other. Such methods are known to those skilled in the art. A human consensus sequence can be used as a target sequence for humanization, although other means are also known in the art. One alternative involves a method in which a person skilled in the art aligns several human germline alleles, such as, but not limited to, an alignment of IGHV3 alleles, and uses the alignment to identify suitable residues for humanization on the target sequence. Alternatively, a subset of the most homologous human germline alleles to the target sequence can be aligned as a starting point for identifying suitable humanization residues. Alternatively, a VHH can be analyzed to identify its closest homologs in human alleles, which are then used to design a humanized construct. Humanization techniques applied to camelid VHHs can also be performed by methods involving the replacement of specific amino acids, either alone or in combination. These replacements can be selected based on what is known from the literature, from known humanization efforts, and from human consensus sequences, by comparing them with natural VHH sequences or the human alleles most similar to the VHH sequence. As can be seen from the data on VHH entropy and VHH variability provided in Tables A-5 to A-8 of WO 08 / 020079, some amino acid residues in the framework regions (i.e., the bold / underlined hallmark residues in Figure 10) are more conserved between humans and camelids than others. Generally, any substitutions, deletions, or insertions are preferably made at less conserved positions, although the invention in its broadest sense is not limited thereto. Furthermore, amino acid substitutions are generally preferred over amino acid deletions or insertions. For example, the human-like class of camelid single domain antibodies contains hydrophobic FR2 residues typically found in conventional antibodies of human origin or from other species, but compensates for this loss of hydrophilicity by another substitution at position 103 that replaces the conserved tryptophan residue present in the VH of the two-chain antibody.Therefore, peptides belonging to these two classes exhibit high amino acid sequence homology to human VH framework regions, and the peptides can be directly administered to humans without the expectation of an unwanted immune response therefrom and without the additional effort of humanization. Indeed, some Camelidae VHH sequences exhibit high sequence homology to human VH framework regions, and the VHHs can be directly administered to patients without the expectation of an immune response therefrom and without the additional effort of humanization. Suitable mutations, particularly substitutions, can be introduced during humanization (see, for example, WO2012 / 175741 and WO2015 / 173325), for example, at at least one of positions 11, 13, 14, 15, 40, 41, 42, 82, 82a, 82b, 83, 84, 85, 87, 88, 89, 103, or 108, to generate polypeptides with reduced binding to existing antibodies. The amino acid sequences and / or VHHs of the invention may be suitably humanized at any framework residue(s), for example at one or more hallmark residues (as defined herein), or preferably at one or more other framework residues (i.e., non-hallmark residues), or any suitable combination thereof. As will be within the ability of one skilled in the art, depending on the host organism used to express the amino acid sequences, ISVDs, VHHs, or polypeptides of the invention, such deletions and / or substitutions may also be designed in such a way that one or more sites for post-translational modification are eliminated (e.g., one or more glycosylation sites at asparagine to be replaced by G, A, or S; and / or methionine oxidation sites). Alternatively, substitutions or insertions may be designed to introduce one or more sites for the 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 properties at positions 37, 44, 45, and / or 47 is replaced (Kabat No; see Table A-03 of WO2008 / 020079).Another example of humanization includes substitution of residues at FR1, e.g., positions 1, 5, 11, 14, 16, and / or 23, and / or 28; FR2, e.g., positions 40 and / or 43; FR3, e.g., positions 60-64, 73, 74, 75, 76, 78, 79, 81, 82b, 83, 84, 85, 93, and / or 94; and FR4, e.g., positions 103, 104, 105, 108, and / or 111 (see Tables A-05 to A08 of WO2008 / 020079; all numbering is according to Kabat). In one embodiment, said humanized variant comprises at least one substitution in any one of the ISVDs comprising SEQ ID NOs: 1-19 selected from the group of substitutions at the following positions (according to Kabat N°): residue 1 substitution to E or D; residue 14 to P; residue 23 to A; 40 to A; 43 to K; 60 to A; 61 to D; 62 to S; 63 to V; 64 to K; 73 to A; 76 to N; 81 to Q; 83 to R; 85 to E; 103 to W; 105 to Q, and / or 108 to L. More preferably, said humanized variant comprises at least one substitution in any one of the ISVDs comprising SEQ ID NOs: 1-19 selected from the group of substitutions at the following positions (according to Kabat N°): residue 1 substitution to E or D; residue 14 to P; 73 to A; 81 to Q; 83 to R; 85 to E; 105 to Q, and / or 108 to L. In another specific embodiment, the humanizing substitutions of at least SEQ ID NO: 1 and / or SEQ ID NO: 2 described herein result in the substitution of at least several residues in FR3, particularly substitutions at positions 60-62 and / or 60-64 (particularly for SEQ ID NO: 2).

[0061] In another embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10. In another embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of sequences having at least 85% identity to any of the sequences SEQ ID NOs: 1-10, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1-10, with differences being present in framework residues. In a specific embodiment, the allosteric modulator of LRRK2 comprises an ISVD comprising an amino acid sequence selected from the group consisting of sequences having at least 85% identity to any of the sequences SEQ ID NOs: 1-10, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1-10, with differences being present in framework residues, with the exception of llama germline hallmark residues present in said framework regions. In another aspect, the LRRK2 modulator comprises an ISVD comprising an amino acid sequence selected from the group consisting of a humanized variant of any of the sequences of SEQ ID NOs: 1-10 or a humanized variant of any of the sequences having 85% identity to SEQ ID NOs: 1-10, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1-10, with differences being present in the FR regions.

[0062] Another embodiment relates to allosteric modulators of LRRK2 as multispecific agents, including at least one allosteric LRRK2 modulator described herein, resulting in other forms of multispecific LRRK2 allosteric modulators, including multiparatopic LRRK2 modulators that bind to several different binding sites on LRRK2, or multivalent LRRK2 modulators that can increase the avidity for binding to LRRK2, or binding agents with different target specificities. For example, a "multispecific" form of an LRRK2 allosteric ISVD is formed by linking together two or more immunoglobulin single variable domains, at least one of which has a different specificity. Non-limiting examples of multispecific constructs include "bispecific" constructs, "trispecific" constructs, "tetraspecific" constructs, etc. To further illustrate this, any multivalent or multispecific (as defined herein) protein-binding agent of the present invention may suitably be directed to two or more different epitopes on the same antigen, for example, epitope 1 on one domain of LRRK2 and epitope 2 on another domain; or may be directed to two or more different antigens, for example, LRRK2 and serum albumin as half-life extensions. One of the most widely used techniques for increasing the half-life and / or reducing the immunogenicity of pharmaceutical proteins involves the attachment of a suitable pharmacologically acceptable polymer, such as polyethylene glycol (PEG) or a derivative thereof (e.g., methoxypolyethylene glycol (mPEG)). Other techniques for increasing the half-life of binding domains may involve engineering into bifunctional or bispecific domains (e.g., one or more ISVDs or active antibody fragments against LRRK2 are coupled to one ISVD or active antibody fragment against serum albumin, which helps to extend half-life), or into fusion of antibody fragments, particularly immunoglobulin single variable domains, with peptides (e.g., peptides against serum proteins such as albumin). Coupling to additional moieties will result in multispecific binding agents, as further disclosed herein.

[0063] The multivalent or multispecific binding agents of the present invention may also have (or be engineered and / or selected for) increased avidity and / or improved selectivity for a desired LRRK2 interaction and / or for any other desired property or combination of desired properties that can be obtained by use of such multivalent or multispecific binding agents. For example, a combination of one or more ISVDs that bind to epitope 1 and one or more ISVDs that bind to epitope 2 described herein results in a multispecific binding agent of the present invention with higher modulatory activity. The multispecific binding agent comprises at least the binding agents directed to epitope 1 and epitope 2, which may be coupled via a linker or spacer. By binding to LRRK2, the multispecific binding agent or multivalent ISVD may have an additive or synergistic impact on the allosteric modulatory activity of LRRK2. The multispecific LRRK2 allosteric modulators of the present invention may be coupled to a functional moiety, a targeting moiety, a half-life extending moiety, or a cell-penetrating carrier.

[0064] Given that modulation of LRRK2 activity is desirable for treating several neurological disorders, multispecific allosteric modulators of LRRK2 may, by way of non-limiting example, include a functional moiety capable of crossing the blood-brain barrier, or may be further fused or chemically coupled to a moiety capable of crossing the blood-brain barrier, for example, by receptor-mediated transcytosis. Indeed, the blood-brain interface severely limits the brain bioavailability of pharmaceutical drugs and compounds. Due to limited penetration of, for example, antibodies, active antibody fragments, or small molecules, large doses must be administered to achieve the desired efficacy. In addition to the risk of large doses inducing peripheral side effects in patients, such approaches are also undesirable due to economic and societal costs and the need for mass production capacity, especially in larger indications such as Parkinson's disease and Alzheimer's disease, which have millions of patients and where the production of biologicals can be a significant limiting factor. Although several means and methods for efficiently transporting compounds across the BBB have been reported (e.g., WO2015031673A2; WO2014033074A1; WO2015124540A1; WO2015191934A2), the type of BBB-crossing moiety will still be selected on a case-by-case and trial-and-error basis. Therefore, the present invention provides a multispecific LRRK2 allosteric modulator, which may include, for example, a (single domain) antibody targeting a blood-brain barrier (BBB) ​​receptor. This multispecific LRRK2 allosteric modulator can be intravenously injected, after which a BBB receptor-targeting antibody (or single variable domain antibody) will transport the complex across the BBB. However, further delivery methods and vehicles that may be suitable include nanoparticle delivery or lipid-based delivery systems, such as artificial exosomes, which may also be cell-specific and may be suitable for delivery of binding agents or multispecific binding agents as proteins or in the form of DNA (nucleic acids, vectors) to encode said binding agents or modulators [48-49].

[0065] Indeed, another aspect of the present invention relates to nucleic acid molecules comprising a nucleic acid sequence encoding an LRRK2-binding agent or allosteric modulator described herein. As used herein, "nucleotide sequence," "DNA sequence," or "nucleic acid molecule(s)" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term encompasses double- and single-stranded DNA, (reverse) complementary DNA, and RNA. It also encompasses known types of modifications, such as methylation, "capping," and substitution of one or more naturally occurring nucleotides with analogs. By "nucleic acid construct" is meant a nucleic acid sequence constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, and extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes containing non-native nucleic acid sequences, and the like. A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. A coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA; introns can also be present in certain circumstances.

[0066] One embodiment discloses an expression cassette comprising the nucleic acid molecule described above. More specific embodiments disclose expression cassettes in which elements for cell- or tissue-specific expression are present. An "expression cassette" includes any nucleic acid construct capable of directing expression of a gene / coding sequence of interest operably linked to the promoter of the expression cassette. An expression cassette is generally a DNA construct, preferably comprising (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence operably linked to a transcription initiation region, a homolog, variant, or fragment thereof, and a termination sequence including an RNA polymerase termination signal and a polyadenylation signal. It is understood that all of these regions should be operable in the biological cell, such as a prokaryotic or eukaryotic cell, to be transformed. The promoter region, including the transcription initiation region, preferably including an RNA polymerase binding site, and the polyadenylation signal, can be native to the biological cell to be transformed or can be derived from an alternative source, where the regions are functional in the biological cell. Such a cassette can be constructed as a "vector." A further aspect relates to a vector comprising said expression cassette or said nucleic acid molecule, the sequence of which encodes an allosteric regulator of LRRK2 as described herein.

[0067] As used herein, the terms "vector," "vector construct," "expression vector," or "gene transfer vector" are intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. More specifically, the vector may include any vector known to those skilled in the art. Any suitable type may be included, including, but not limited to, a plasmid vector, a cosmid vector, a phage vector, such as lambda phage, a viral vector, and more specifically, a lentivirus, adenovirus, AAV, or baculovirus vector, or an artificial chromosome vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a P1 artificial chromosome (PAC). Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in an in vitro expression system. Cloning vectors are commonly used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment. The construction of expression vectors for use in transfecting cells is also well known in the art and can therefore be accomplished by standard techniques (see, e.g., Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, NJ) and the Ambion 1998 catalog (Ambion, Austin, Tex.).

[0068] Furthermore, alternative embodiments relate to the use of the nucleic acid molecules, expression cassettes, or vectors described herein encoding the allosteric modulators of LRRK2 for production as intrabodies. Intrabodies, or "intrabodies," are antibodies or active fragments of antibodies that are heterologously expressed within a designated intracellular compartment, a process enabled by in-frame integration of intracellular trafficking signals. Intrabodies exert their function through exquisitely specific interactions with target antigens, resulting in the disruption or modification of the biological function of the target protein. Intrabodies can be expressed in any shape or form, such as intact IgG molecules or Fab fragments. More frequently, intrabodies are used in engineered antibody fragment formats and structures, such as scFv intrabodies, single-domain intrabodies, or bispecific tetravalent intradiabodies. For a review, see Zhu and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. _c Springer-Verlag Berlin Heidelberg). The allosteric modulators of LRRK2 described herein, optionally encoded by nucleic acid molecules or expression cassettes or present on vectors as described herein and resulting in intrabodies upon expression in a suitable host system, can also be used as tools to further investigate LRRK2 signaling, as diagnostic agents, for in vivo imaging, or as therapeutic agents when applicable forms of gene delivery are identified. Those skilled in the art are aware of currently applied methodologies of administration and delivery (see also Zhu and Marasco 2008).

[0069] The field of gene therapy for the nervous system has experienced explosive growth over the past five years, with ongoing human clinical trials for gene replacement and the recent approval of gene therapy for spinal muscular atrophy

[86] . Adeno-associated virus (AAV)-based systems are increasingly being used in clinical trials because they efficiently transduce both dividing and non-dividing cells, provide long-term transgene expression (after a single administration), and have low inherent toxicity

[87] . Various gene therapy strategies have been developed for PD

[88] . Where the (multispecific) allosteric modulators of LRRK2 are provided as nucleic acids or vectors, it is specifically contemplated that the modulators will be administered via gene therapy. As used herein, "gene therapy" refers to therapy performed by administering an expressed or expressible nucleic acid to a subject. In such applications, the nucleic acid molecules or vectors described herein allow for the production of allosteric modulators of LRRK2 in cells. Numerous methods for gene therapy are available in the art, including, for example, (adeno-associated) virus-mediated gene silencing or virus-mediated gene therapy (e.g., US20040023390; Mendell et al. 2017, N Eng J Med 377:1713-1722). Various delivery methods are well known to those skilled in the art, including, but not limited to, viral delivery systems, microinjection of DNA plasmids, biolistic methods of naked nucleic acid, and the use of liposomes. In vivo delivery by administration to individual patients typically occurs by systemic administration (e.g., intravenous, intraperitoneal infusion, or brain injection; e.g., Mendell et al. 2017, N Eng J Med 377:1713-1722). Where the (multispecific) allosteric modulators of LRRK2 are provided as nucleic acids or vectors, it is also contemplated that the modulators will be administered by delivery methods and vehicles including, more specifically, nanoparticles or lipid-based delivery systems, e.g., artificial exosomes.They may also be cell-specific and suitable for delivery of binding agents or multispecific binding agents as intrabodies or in the form of DNA to encode said binding agents or modulators [48-49].

[0070] In another embodiment, the kit may also include LRRK2-binding agents, ISVDs, and / or allosteric modulators as purified proteins, nucleic acids, or expression cassettes or vectors described herein, for example, for application in LRRK2 signaling research or as tools for structural biology and biochemical analysis of LRRK2.

[0071] A further aspect relates to the LRRK2-specific ISVD described herein, a nucleic acid molecule or vector encoding the LRRK2-specific binding agent, or a pharmaceutical composition comprising the same, for use as a diagnostic agent.

[0072] In certain embodiments, kits are provided that contain means for detecting LRRK2 protein, including an LRRK2 allosteric modulator or binding agent or ISVD described herein, allowing for detection or modulation of LRRK2 signaling in a system, which may be an in vitro or in vivo system. It is contemplated that these kits are provided for a specific purpose, such as for modulating LRRK2, for in vivo imaging, or for diagnosing the amount, response, or effect of altered LRRK2 in a subject. In another embodiment, the aforementioned kits are provided that contain means including a nucleic acid molecule, vector, or composition described herein. The means further provided by the kit will depend on the methodology used in the application and the purpose of the kit. For example, detection of a labeled LRRK2 allosteric modulator or binding agent, ISVD, or nucleic acid molecule described herein may be desirable for LRRK2 quantification at the nucleic acid or protein level. For protein-based detection, the kit will typically contain a labeled or coupled LRRK2 binding agent, e.g., an ISVD. Similarly, for detection at the nucleic acid level, the kit may contain a label for the nucleic acid, such as a primer or probe. Additionally, control agents, antibodies, or nucleic acids may also be provided with the kit. Reference or comparison standards, LRRK2 substrates or signaling components, reporter genes or proteins, or other means for using the kit may also be included. Of course, the kit may further include pharmaceutically acceptable excipients, buffers, carriers or delivery means, instruction manuals, etc.

[0073] Another aspect of the present invention provides a method for detecting the presence, absence, or level of LRRK2 protein in a sample, the method comprising: contacting the sample with an LRRK2-binding agent or ISVD described herein, and detecting the presence, absence, or level, i.e., quantifying the bound LRRK2 ISVD, which is optionally a labeled, conjugated, or multispecific LRRK2-binding agent. As used herein, a sample may be a sample isolated from the body, for example, a body fluid, including, inter alia, blood, serum, cerebrospinal fluid, or may be an extract, such as a protein extract, cell lysate, etc.

[0074] Furthermore, the LRRK2 allosteric modulators or binding agents, nucleic acid molecules, vectors, or pharmaceutical compositions containing the LRRK2-specific binding agents, particularly those containing the LRRK2-specific ISVDs described herein, can also be used for in vivo imaging.

[0075] For the purpose of in vitro or in vivo detection and / or imaging, the LRRK2-binding agent comprising the LRRK2-specific ISVD described herein may further comprise a detection agent, such as a tag or label, in some embodiments. For example, the ISVD, VHH, or Nb exemplified herein was also tagged with a 6-His-EPEA double tag (presented in SEQ ID NO: 20; for the EPEA tag, see also WO2011 / 147890A1). Such tags allow affinity purification and detection of the antibody or active antibody fragment of the present invention.

[0076] Some embodiments include an LRRK2-binding agent, ISVD, or allosteric modulator further comprising a label or tag, or more specifically, an LRRK2-binding agent labeled with a detectable marker. As used herein, the term detectable label or tag refers to a detectable label or tag that allows for detection and / or quantification of the LRRK2 modulator or binding agent described herein, and is meant to encompass any label / tag known in the art for these purposes. In particular, affinity tags, such as chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), poly(His) (e.g., 6xHis or His6), biotin or streptavidin, such as Strep-tag®, Strep-tag II®, and Twin-Strep-tag®; solubilization tags, such as thioredoxin (TRX), poly(NANP) and SUMO; chromatography tags, such as FLAG-tag; epitope tags, such as V5-tag, myc-tag, and FIA-tag; fluorescent labels or tags (i.e., fluorochromes / phores), such as fluorescent proteins. Preferred, but not limited to, labels include, but are not limited to, proteins (e.g., GFP, YFP, RFP, etc.) and fluorescent dyes (e.g., FITC, TRITC, coumarin, and cyanines); luminescent labels or tags, such as luciferase, bioluminescent, or chemiluminescent compounds (e.g., luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxetane, or GFP and its analogs); phosphorescent labels; metal chelators; and (other) enzymatic labels (e.g., peroxidase, alkaline phosphatase, beta-galactosidase, urease, or glucose oxidase); and radioisotopes. Also encompassed are combinations of any of the foregoing labels or tags. Technology for producing labeled polypeptides and proteins is well known in the art. LRRK2 allosteric modulators or binding agents comprising the LRRK2-specific ISVD of the present invention coupled to or further comprising a label or tag allow, for example, immunologically-based detection of the bound LRRK2-specific agent.Immuno-based detection is well known in the art and can be achieved by applying a variety of approaches. These methods are generally based on the detection of labels or markers, such as those described above. See, e.g., U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. In cases where multiple antibodies are reacted with a single array, each antibody can be labeled with a distinct label or tag for simultaneous detection. Further embodiments can include the introduction of one or more detectable labels or other signal-generating groups or moieties or tags, depending on the intended use of the labeled or tagged LRRK2 allosteric modulators or binding agents of the present invention. Other suitable labels will be apparent to those skilled in the art and include, for example, moieties that can be detected using NMR or ESR spectroscopy. Such labeled allosteric modulators of LRRK2, such as the LRRK2-specific ISVDs or nanobodies described herein, can be used, for example, in vitro, in vivo, or in situ assays (including immunoassays known per se, such as ELISAs, RIAs, EIAs, and other "sandwich assays"), and for in vivo imaging purposes, depending on the particular label choice.

[0077] Therefore, in another aspect, an in vitro method for detecting the localization and distribution of human LRRK2 protein in a biological sample is disclosed, comprising reacting the sample with an LRRK2-binding agent comprising an LRRK-specific ISVD described herein, and detecting the localization and distribution of said LRRK2 binding in said biological sample. As used herein, a biological sample may contemplate any sample derived from a biological system, and may include, for example, cells or extracts or in vitro samples of brain tissue, or body fluids such as cerebrospinal fluid or blood.

[0078] Another aspect of the present invention relates to pharmaceutical compositions comprising one or more allosteric modulators of LRRK2 described herein, or a nucleic acid molecule or vector described herein, and optionally a pharmaceutically acceptable carrier or diluent. These pharmaceutical compositions can be utilized to achieve a desired pharmacological effect upon administration to a patient in need thereof. A "pharmaceutically or therapeutically effective amount" of a compound or binding agent or composition is preferably an amount that produces a result or exerts an influence on the particular condition being treated. The allosteric modulators of LRRK2 or pharmaceutical compositions described herein can also function as "therapeutically active agents," which is used to refer to any molecule that has or can have a therapeutic effect (i.e., a curative or stabilizing effect) in the context of disease treatment (as further described herein). Preferably, a therapeutically active agent is a disease-modifying agent and / or an agent that has a curative effect on the disease. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable. That is, the material can be administered to an individual in conjunction with the compound without causing any undesired biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A pharmaceutically acceptable carrier is preferably one that is relatively non-toxic and harmless to the patient at concentrations consistent with the effective activity of the active ingredient, such that any side effects that may be attributable to the carrier do not impair the beneficial effects of the active ingredient. Suitable carriers or adjuvants typically include one or more of the compounds included in the following non-exhaustive list: large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.Such ingredients and procedures include those described in the following references, each of which is incorporated herein by reference: Powell, MF et al. ("Compendium of Excipients for Parenteral Formulations" PDA Journal of Pharmaceutical Science & Technology 1998, 52(5), 238-311), Strickley, RG ("Parenteral Formulations of Small Molecule Therapeutics Marketed in the United States (1999)-Part-1" PDA Journal of Pharmaceutical Science & Technology 1999, 53(6), 324-349), and Nema, S. et al. ("Excipients and Their Use in Injectable Products" PDA Journal of Pharmaceutical Science & Technology 1997, 51 (4), 166-171). As used herein, the term "excipient" is intended to encompass any substance that may be present in a pharmaceutical composition and is not an active ingredient, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, stabilizers, flavorings, or colorings. "Diluents," particularly "pharmaceutically acceptable carriers," include carriers such as water, saline, physiological saline solution, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and preservatives, may be included in such carriers. Such pharmaceutical compositions containing the aforementioned allosteric modulators of LRRK2 may also relate to nanoparticle-containing compositions or lipid-based exosome delivery vehicles, as discussed herein [48-49].

[0079] The allosteric modulators or binding agents or pharmaceutical compositions described herein can act as therapeutically active agents when useful for treating diseases related to LRRK2. The pharmaceutical compositions described herein can also include multispecific allosteric modulators of LRRK2 that may contain or be coupled to additional functional groups or moieties that are advantageous when administered to a subject.

[0080] Furthermore, the pharmaceutical compositions described herein containing an allosteric modulator of LRRK2 may further contain a compound known as an inhibitor of LRRK2 kinase activity, preferably a type I kinase inhibitor. The known LRRK2 kinase inhibitors of type I kinase inhibition are also known to induce LRRK2 translocation to microtubules, as described above

[36] .

[0081] In contrast to these prior art type I ATP-competitive kinase inhibitors, the LRRK2 allosteric modulators described herein, e.g., the ISVDs disclosed herein, do not induce microtubule redistribution of LRRK2 protein (see, e.g., Example 6). Furthermore, increased microtubule binding or translocation of LRRK2 protein is observed not only when ATP-competitive kinase inhibitors bind to cellular LRRK2, but also upon overexpression of various pathological LRRK2 mutants. Recent reports have linked such LRRK2 translocation events to potentially blocked transport on microtubules [36, 40]. LRRK2 allosteric modulators, including the LRRK2-specific ISVDs of the present invention, are different in the sense that they do not involve LRRK2 translocation. This further confirms that the LRRK2 allosteric modulators or ISVDs disclosed herein provide conformational LRRK2 binding in cells, which is different from "classical" ATP-competitive or known type I kinase inhibitors. Furthermore, the panel of LRRK2 allosteric modulators described herein functions to reduce or rescue microtubule localization when added to cells containing microtubule-translocated LRRK2 protein (e.g., due to prior treatment with small compounds that bind and inhibit LRRK2 kinase activity as type I inhibitors, or due to PD-mutant LRRK2). The surprising finding that this subset of ISVDs can even reverse LRRK2 microtubule redistribution upon inhibition with ATP-competitive kinase inhibitors provides further opportunities for applying these LRRK2 allosteric modulators as (combination) therapeutic agents, as this may be beneficial for avoiding any undesired effects of treatment with LRRK2 inhibitors.

[0082] Another aspect relates to the allosteric modulators of LRRK2 described herein, the nucleic acid molecules, vectors, or pharmaceutical compositions described herein for use as pharmaceuticals, more particularly for use in treating disorders or diseases related to or associated with LRRK2. As used herein, "LRRK2-related disorders" includes diseases currently known to be associated with, impacted by, or caused by altered LRRK2 activity. Most notably, this includes Parkinson's disease (Zimprich A, et al. (2004) Neuron 44:601-607; Paisan-Ruiz C, et al. (2004) Neuron 44:595-600) and Crohn's disease (Ridler C. (2018) Nat Rev Neurol. 14(3):126; Hui KY et al (2018) Sci Transl Med. 10(423); Rivas MA et al (2018) PLoS Genet. 14(5):el007329); and further, the immune response as a "host response to pathogens" (Gardet A et al (2010) J Immunol. 185(9):5577-85); an increased risk of cancer (Saunders-Pullman et al (2010) Mov Disord. 25(15): 2536-2541;Bjorg Johanne Waro & Jan O. Aasly (2018) Brain Behav. 8(1): e00858) and Alzheimer's disease (Zhao Y (2011) Neurobiol Aging. 2011 Nov;32(11):1990-3).

[0083] Specific embodiments relate to said allosteric modulators of LRRK2 described herein, nucleic acid molecules, vectors, or pharmaceutical compositions described herein for use in the treatment of Parkinson's disease.

[0084] While particular embodiments, specific configurations, and materials and / or molecules have been discussed herein for the methods, samples, and biomarker products according to the present disclosure, it should be understood that various changes or modifications in form and detail can be made without departing from the scope of the present invention. The following examples are provided to better illustrate particular embodiments, and should not be construed as limiting the present application. The present application is limited only by the claims. [Example]

[0085] Example 1. Generation of LRRK2-specific nanobodies. LRRK2 is a large and complex protein containing several domains: an armadillo domain, an ankyrin repeat domain, a leucine-rich repeat (LRR) domain, a RocCOR (the C-terminus of the Ras / Roc complex protein) supradomain, a kinase domain, and a WD40 domain (Figure 1B) [13-15]. As such, the protein possesses a highly unique combination of two catalytic activities: a GTPase activity mediated by the Roc domain and a Ser / Thr protein kinase activity

[16] . Although the mechanism of LRRK2 is still largely unknown, it is predicted to be regulated in a complex manner and to undergo large conformational changes during its functional cycle. One source of conformational changes in LRRK2 is mediated by nucleotide (GDP vs. GTP) binding to its RocCOR domain. Indeed, our previous studies on bacterial LRRK2 homologs have shown that GTP binding leads to the monomerization of LRRK2 dimers and is associated with the movement of secondary domains within each subunit [17,18]. Furthermore, recent in vitro and in vitro data have demonstrated that human LRRK2 can also cycle between monomeric and dimeric forms, although the precise nature of these conformational changes remains elusive [19-22]. Here, we aimed to identify Nb that could regulate LRRK2 activity by specifically binding to LRRK2 in one of its conformational states. In total, three immunizations using different llamas were performed with different protein constructs or conformational states of LRRK2 (Figure 7). In the first immunization strategy (Immunization 1), we immunized llamas with the LRRK2 RocCOR construct. After immunization, Nb were selected using a phage display panning approach with full-length LRRK2 as the bait protein. To subsequently increase the likelihood of obtaining Nbs that bind to LRRK2 in a specific nucleotide-induced conformation, we performed two additional immunizations with either LRRK2 bound to and in the presence of a large excess of GTPγS (a non-hydrolyzable GTP analog) (immunization 2) or LRRK2 bound to and in the presence of a large excess of GDP (immunization 3).Furthermore, to "trap" the protein in its nucleotide-specific conformation upon immunization, we performed mild cross-linking using the lysine-specific cross-linker DSS (Figure 8). After immunization, Nbs were selected using phage display panning with uncross-linked full-length LRRK2 in the presence of excess GTPγS (selection from the library originating from immunization 2) or GDP (selection from the library originating from immunization 3). In addition, to enrich for Nbs that bind to the Roc domain, the Nb library was subjected to two or three rounds of phage display using Roc protein bound to either GTPγS or GDP.

[0086] These different strategies ultimately resulted in a library of selected Nb ORFs cloned into the pMESy4 vector. These vectors were subjected to sequencing, and the Nbs were classified into different sequence families based on their CDR3 sequences (each Nb family exhibits a unique CDR3 sequence). This resulted in 49 Nb families originating from immunization 1, 70 Nb families originating from immunization 2 by selection against LRRK2-GTPγS, 4 Nb families originating from immunization 2 by selection against Roc-GTPγS, 44 Nb families originating from immunization 3 by selection against LRRK2-GDP, and 1 Nb family originating from immunization 3 by selection against Roc-GDP.

[0087] Further selection of Nbs was based on an ELISA screening step. A large subset of sequenced Nbs was expressed in E. coli at a small scale in a 96-deep-well plate format. After cell lysis, crude cell lysates were used to test binding in ELISA against full-length LRRK2 coated on the bottom side of an ELISA plate. Finally, 42 Nbs from different families and resulting from different immunization and selection strategies were selected based on good signals in ELISA (Table 1). These Nbs were expressed in E. coli at a larger scale and purified to homogeneity (Figure 9).

[0088] Table 1. List of purified LRRK2-specific Nbs. Selection of Nbs resulting from (1) immunization with RocCOR (RocCOR-GppNHp) and selection with full-length LRRK2, (2) immunization with cross-linked LRRK2-GTPγS (LRRK2-GTPγS(XL)) and selection with either LRRK2-GTPγS or Roc-GTPγS, and (3) immunization with cross-linked LRRK2-GDP (LRRK2-GDP(XL)) and selection with either LRRK2-GDP or Roc-GDP. Domain specificity (only for a subset of Nbs) determined from ELISA or cross-link MS is also indicated. [Table 1-1] [Table 1-2]

[0089] Example 2. Determining the domain specificity of LRRK2-directed Nbs. We recombinantly expressed and purified the RocCOR, Roc, COR-B, and kinase-WD40 (K-WD40) domain constructs of LRRK2 (Figure 7). We then performed ELISA, in which all these domain constructs were coated next to each other in parallel on the same ELISA plate, and then detected the binding of 42 purified Nbs. Figure 1 shows the results of this ELISA. As expected, most Nbs show binding to at least one of the LRRK2 and / or domain constructs used. However, two Nbs (CA13614 and CA13618) did not show any binding, and no further binding was observed (see also Table 1). All purified Nbs (7 Nbs) resulting from immunization with the RocCOR domain construct (Immunization 1) were found to specifically bind to the C-terminal subdomain (COR-B) of the COR domain. Of the Nbs obtained from immunization and selection with full-length LRRK2, the majority bind to the K-WD40 portion of the protein (18 Nbs). Another subset of Nbs derived from these immunizations (10 Nbs) showed robust binding to LRRK2 but were not observed to bind to any of the individual LRRK2 domain constructs. Therefore, we speculate that these Nbs either bind to the N-terminal region of LRRK2 (the armadillo-ankyrin-LRR domain) that was not covered by the individual domains in ELISA, or that these Nbs possess epitopes at the interface of two or more domains. Thus, we classify the Nbs as "full-length LRRK2 binders." To enrich for Nbs directed against the Roc domain, we specifically included a selection step with either Roc-GTPγS or Roc-GDP. This resulted in five Nbs directed against the Roc domain (Nb32 (CA16069), Nb33 (CA16070), Nb34 (CA16071), Nb35 (CA16072), and Nb42 (CA14259)). Four of these five gave strong signals at the Roc domain in ELISA as well.For the fifth Nb (Nb42, CA14259), binding to Roc was confirmed using analytical size-exclusion chromatography (data not shown). The epitope mapping results are summarized in Figure 1B.

[0090] Example 3. Cellular LRRK2 kinase activity is regulated by nanobodies targeting different LRRK2 domains. We then wanted to test whether any of the LRRK2-directed Nbs had the ability to bind to LRRK2 and regulate LRRK2 (kinase) activity in human HEK293T cells overexpressing LRRK2. Therefore, we selected a subset of 18 of the 41 purified Nbs, carefully covering Nbs originating from three different immunization strategies and targeting different LRRK2 domains (i.e., "full-length LRRK2," Roc, COR-B, and kinase-WD40). As a negative control, we also included an in-house-produced Nb against a completely unrelated bacterial protein ("irrelevant Nb"). To express these Nbs in human LRRK2 (wild-type) overexpressing HEK293 cells, we first recloned the Nb open reading frame into the pEGFP vector, resulting in the expression of the Nb fused at its C-terminus to an enhanced GFP molecule (the so-called "fluobody") [23, 24]. To test binding to cellular LRRK2, pull-down experiments were performed using magnetic GFP-nanotrap beads. This experiment showed that all tested Nbs were capable of pulling down LRRK2 under these conditions. Therefore, this indicates that these 18 Nbs are functional as intrabodies in the cytoplasmic context of human cells and have sufficiently high affinity to pull down their target proteins (Figure 12).

[0091] To assess the effects of Nbs on LRRK2 kinase activity, we monitored two physiologically and disease-relevant activities of LRRK2: phosphorylation of the endogenous substrate Rab10 at position T72 and LRRK2 autophosphorylation at position S1292 [25-30]. Both activities have previously been shown to be increased in most relevant LRRK2 PD mutants, including the common G2019S mutant. Therefore, in our cellular assays, we coexpressed LRRK2(G2019S), Rab29, and different Nb-GFP fusions in HEK293T cells (Figure 2a-c). Rab29 coexpression has previously been shown to boost LRRK2 autophosphorylation and Rab10 phosphorylation

[31] . Among the 18 selected Nbs, we detected a group that had no effect on LRRK2 kinase activity compared with the control (i.e., Nb3 (CA12614), Nb9 (CA13598), Nb10 (CA13599), Nb13 (CA13602), Nb31 (CA13620), Nb37 (CA14131), and Nb39 (CA14134) (Fig. 2d), while others strongly reduced LRRK2 autophosphorylation and / or Rab10 phosphorylation (Nb1 (CA12610), Nb6 (CA12618), Nb7 (CA12619), Nb8 (CA12620), Nb9 (CA13598), Nb10 (CA13599), Nb13 (CA13602), Nb31 (CA13620), Nb37 (CA14131), and Nb39 (CA14134) (Fig. 2d). Nb23 (CA13612), Nb42 (CA14259), Nb17 (CA13606), Nb36 (CA14130), Nb38 (CA14133), Nb40 (CA14135), and Nb41 (CA14136)) (Figure 2). In addition, several Nbs led to a significant increase in LRRK2 kinase activity, which is most pronounced for Nb28 (CA13617) and Nb22 (CA13611). Interestingly, Nbs appear to differentially affect LRRK2 autophosphorylation and Rab10 phosphorylation.Some of the binding factors inhibit both Rab10 phosphorylation and LRRK2 autophosphorylation at S1292 (e.g., COR-B binding factors Nb1 (CA12610) and Nb6 (CA12618)), Roc binding factor Nb42 (CA14259), or kinase-WD40 binding factor Nb23 (CA13612)), whereas others appear to inhibit Rab10 phosphorylation while inhibiting S1292 phosphorylation to a lesser extent (e.g., the "full-length LRRK2" binding factors Nb17 (CA13606), Nb36 (CA14130), and Nb38 (CA14133), or the "K-WD40" binding factors Nb40 (CA14135) and Nb41 (CA14136)). In contrast, other binding factors exhibit somewhat stronger inhibitory effects on LRRK2 autophosphorylation (eg, the "full-length LRRK2" binding factor Nb37 (CA14131) or the COR-B binding factor Nb3 (CA12614)).

[0092] Example 4. Epitope mapping by cross-linking mass spectrometry (CL-MS) reveals binding epitopes of activity-modulating Nbs. Following up on the above results, we decided to select 10 Nbs that regulate cellular LRRK2 (G2019S) activity for more thorough in vitro characterization. The following Nbs were selected for further characterization: Nb17 (CA13606), Nb36 (CA14130), Nb38 (CA14133) (directed to "full-length LRRK2" and inhibits cellular Rab10 phosphorylation), Nb39 (CA14134) (directed to "full-length LRRK2"), Nb42 (CA14259) (directed to the Roc domain and inhibits cellular Rab10 and autophosphorylation), Nb1 (CA12610), and Nb6 (CA12618) (directed to the COR-B domain and inhibits cellular Rab10 phosphorylation and autophosphorylation), Nb22 (CA13611) (directed to the kinase-WD40 domain and activates cellular Rab10 phosphorylation), Nb40 (CA14135), and Nb23 (CA13612) (directed to the kinase-WD40 domain and inhibit cellular Rab10 phosphorylation and Rab10 and autophosphorylation, respectively) (Figure 2d).

[0093] First, we used a cross-linking MS approach to gain more detailed insight into the precise binding epitopes of these 10 Nbs. Cross-linking mass spectrometry (CL-MS) has emerged as a powerful tool for structural investigation, and we previously studied LRRK2 by CL-MS and optimized the conditions for this protein [2]. Moreover, chemical cross-linking of LRRK2-Nb complexes followed by sequencing of the target peptides by mass spectrometry is also a versatile method to allow sensitive mapping of Nb-binding epitopes with high confidence [11,32]. CL-MS data revealed that different Nbs exhibit protein-protein cross-links to LRRK2 predominantly via one conserved lysine residue on the framework 3 region of the Nb, located in the loop connecting β-strands C" and D

[33] . This lysine residue is present in all selected Nbs, with the exception of Nb6 (CA12618), for which no corresponding cross-linking data could be obtained. In other Nbs, depending on the binding epitope, this lysine residue showed robust links to several lysine residues on LRRK2 (Fig. 3).

[0094] Overall, the CL-MS data are in excellent agreement with the domain mapping results using ELISA. Based on the ELISA experiments, Nbs Nb17 (CA13606), Nb36 (CA14130), Nb38 (CA14133), and Nb39 (CA14134) were flagged as "full-length LRRK2 binders" because they only showed binding to full-length LRRK2 and no significant binding was observed to any of the individual domain constructs tested. Correspondingly, CL-MS revealed that all these Nbs make multiple contacts with the N-terminal LRR domain of LRRK2, including the C-terminal end of the COR (CA14134), the kinase domain (CA13606 and CA14133), and the C-terminal part encompassing both the kinase and WD40 domains (CA14130). This finding clearly demonstrates that a significant portion of validated binding factors specifically recognize conformational epitopes instead of binding short linear peptide stretches. Furthermore, this indicates that the LRR domain "folds back" onto and is closely proximal to the C-terminal domain of LRRK2. For Nb42 (CA14259), only one cross-link with lysine K1502 on the Roc domain was identified, which is in good agreement with domain mapping in ELISA. Nb22 (CA13611), Nb23 (CA13612), and Nb40 (CA14135) were all identified as kinase-WD40 binding factors in ELISA. Correspondingly, the cross-linking data revealed interactions with the WD40 domain, the kinase domain, and both the WD40 domain and the C-terminal end of COR-B for these three Nbs, respectively. Interestingly, Nb1 (CA12610), identified as a COR-B binding factor in ELISA, cross-links with various lysines in different parts of the LRRK2 protein. Consistent with the ELISA data, cross-linking is found with K1833 on COR-B, but also with residues on other LRRK2 domains, including the adjacent kinase domain and the leucine-rich repeat.This finding is consistent with the very central location of the COR domain in a low-resolution structural model of the compact LRRK2 dimer. [2] Therefore, we hypothesize that CA12610 binding to the COR-B domain places it in the central cavity of the LRRK2 structure, in relatively close proximity to most other LRRK2 domains.

[0095] Example 5. Nb binding affinity to LRRK2 determined by microscale thermophoresis (MST) and biolayer interferometry (BLI). The binding affinities (dissociation constants K) of 10 Nbs to LRRK2 D To determine the KD of LRRK2, we used two methods in parallel: microscale thermophoresis (MST) and biolayer interferometry (BLI). In the MST experiments, we site-specifically labeled 10 Nbs at their C-termini with the m-TAMRA fluorophore using sortase-mediated coupling

[51] , and then titrated increasing amounts of full-length LRRK2 against these Nbs (Figure 13). With the exception of Nb23, which did not produce a change in thermophoretic behavior upon binding to LRRK2, MST signals were observed for all 10 Nbs. For the other Nbs, KD values ​​ranged from 25 nM to 150 nM (Table 2). In the BLI experiments, LRRK2 was first trapped on a streptavidin-coated biosensor using biotinylated Nb40 as a trapping agent (with the exception of Nb40, where Nb42 was used as a trapping agent to assess the binding of Nb40), after which the binding of all Nbs to LRRK2 was determined. Clear binding signals were obtained for all Nbs (including Nb23), with KD values ​​ranging from 10 nM to 200 nM (Figure 14). Overall, both methods show similar trends and affinity ranges. However, in general, BLI yields slightly higher affinities compared to MST. These differences are likely due to the experimental setup of MST, which uses LRRK2 in solution, and BLI, which uses LRRK2 trapped on the surface by means of a second Nb.

[0096] Table 2. Equilibrium dissociation constants (K ) for the binding of a set of 10 Nbs to LRRK2 assessed by two methods in parallel: microscale thermophoresis (MST) and biolayer interferometry (BLI). D ). [Table 2-1] [Table 2-2]

[0097] Example 6. Several LRRK2 activity-modulating Nbs inhibit LRRK2 kinase activity in vitro using different (allosteric) mechanisms. Next, we screened the effects of 10 selected Nbs on in vitro LRRK2 (wild-type) kinase activity. For this, we used the fluorescence-based PhosphoSens® Protein Kinase Assay (AssayQuant Technologies Inc.) with the LRRK2-optimized AQT0615 peptide as a substrate at a fixed concentration of 10 μM. The increase in fluorescence (λ) due to peptide phosphorylation catalyzed by LRRK2 was measured. exc :360nm;λ emm The ATP concentration (at 485 nm) was measured continuously over time under initial velocity conditions at either 0.1 or 1 mM ATP concentrations. Subsequently, 10 Nbs were added at a final concentration of 25 μM, and their effects on the initial velocity were determined in triplicate (Fig. 4A). Two negative controls, either no Nb or an irrelevant Nb, were also included. Furthermore, the LRRK2-specific ATP-competitive inhibitor MLi-2

[34] was added at 25 μM as a positive control. Additionally, these experiments were performed with LRRK2 in the presence of a large excess (500 μM) of either GDP or GTPγS (Fig. 4B), but no significant effect of nucleotides on the inhibition profile was observed.

[0098] Consistent with the in cellulo data, the WD40 domain-binding Nb22 (CA13611) activates the kinase activity of LRRK2 in vitro, with the addition of 25 μM Nb resulting in an approximately 30–50% increase in kinase activity compared to the control. At this point, we cannot exclude that Nb22 also interacts with the kinase domain, separate from the observed interaction with the WD40 domain. However, this observation supports a regulatory reciprocal influence between the kinase and WD40 domains. This is also in good agreement with a previous report showing that deletion of seven C-terminal amino acids leads to blockage of LRRK2 kinase activity

[35] . Furthermore, the very recently determined cryo-EM structure of the catalytic half of LRRK2 (Roc-COR-kinase-WD40) shows that the α-helix formed by the last 28 amino acids of LRRK2 following the WD40 domain folds back onto and interacts closely with the kinase domain

[36] .

[0099] In contrast, the addition of Nbs Nb17 (CA13606), Nb36 (CA14130), Nb38 (CA14133), and Nb40 (CA14135) resulted in only a very mild inhibition of AQT0615 phosphorylation (initial rates ranging between 70% and 95% of the control). This indicates that the observed inhibition of LRRK2-mediated Rab10 phosphorylation by the latter Nbs is not due to a direct effect on kinase activity per se. This is also suggested by the observation that these Nbs severely affected cellular Rab10 phosphorylation while having a less pronounced effect on autophosphorylation at position S1292, thus affecting Rab phosphorylation quite specifically. One exception appears to be Nb42, which inhibited both cellular LRRK2 autophosphorylation and Rab phosphorylation, but the inhibitory effect could not be observed in vitro.

[0100] Finally, three other Nbs, Nb1 (CA12610), Nb6 (CA12618), and (to a lesser extent) Nb23 (CA13612), have very clear and significant inhibitory effects on in vitro LRRK2 kinase activity. This is most striking for CA12610 and CA12618, which reduce kinase activity in this assay to levels that are only 20 to 30% of those of the "no Nb" and "irrelevant Nb" controls and only marginally above the activity of the MLi-2 control. This is consistent with the observation that all three Nbs severely inhibit cellular Rab10 and autophosphorylation, acting as true inhibitors of total LRRK2 kinase activity. Interestingly, CA13612 was found to bind directly to the kinase domain, whereas CA12610 and CA12618 achieve this effect by binding to the COR-B domain.

[0101] Because we found that Nb1 (CA12610), Nb6 (CA12618), and Nb23 (CA13612) significantly inhibited LRRK2 kinase activity in vitro, we performed dose-response analyses with these three Nbs sequentially. While keeping peptide and ATP substrate concentrations constant at 10 μM and 1 mM, respectively, the Nb concentrations were varied in two-fold serial dilutions from 200 or 150 μM to 0.006 μM (Figure 11A-C). Fits of these dose-response curves revealed IC values ​​of 8 ± 2 μM and 14 ± 3 μM for Nb1 and Nb6, respectively. 50 value, as well as an estimated IC of 65 μM for Nb23 50 gave the value.

[0102] Example 7. LRRK2 inhibitor Nb acts by an allosteric mechanism. For three Nbs (Nb1 (CA12610), Nb6 (CA12618), and Nb23 (CA13612)) identified as robustly inhibiting cellular LRRK2 autophosphorylation and Rab phosphorylation activity, as well as LRRK2 kinase activity toward peptide and Rab substrates in vitro, the results suggest that these Nbs target LRRK2 kinase activity itself. Interestingly, ELISA and CL-MS experiments suggested that Nb23 (CA13612) binds to the kinase domain, whereas Nb1 (CA12610) and Nb6 (CA12618) bind to the COR domain. This strongly suggests that at least the latter two Nbs act as allosteric kinase inhibitors. To further confirm this, we decided to determine the inhibitory mechanisms (competitive vs. uncompetitive vs. mixed / noncompetitive) of these three Nbs for ATP as a substrate using our peptide phosphorylation assay as the output. For this, full Michaelis-Menten curves were obtained at a fixed (presumably subsaturating) concentration of peptide substrate (10 μM) and various concentrations of ATP (Figure 11). For CA12610 and CA12618, linearization of the curves using a Lineweaver-Burk plot clearly shows intersecting lines to the left of the Y-axis, indicating mixed-type inhibition. This confirms that these Nbs are not competing with ATP for binding and that they inhibit the reaction by binding to an allosteric site. This is consistent with ELISA and CL-MS epitope mapping data. The observation that the linearized curves cross above the X-axis indicates that these Nbs inhibit the ATP-bound form (higher apparent K iU app ) than the ATP-unbound state of LRRK2 (lower apparent K iC app For CA12610, therefore, a global fit of the kinetic data using a mixed inhibition model yielded a K of 16 ± 4 μM with an α value of 1.8 ± 0.5. i app giving a K of 16 μM iC app(= affinity for apoLRRK2) and K of 30 μM iU app (= affinity for LRRK2 bound to ATP). For CA12618, the same model fit yielded a K of 5 ± 1 μM with an α value of 1.6 ± 0.4. i app and 5 μM K iC app and a K of 8 μM iU app For kinase domain-bound CA13612, the linearized curves intersect closer to the Y-axis, indicating a more ATP-competitive mechanism. However, the lines do not intersect exactly on the Y-axis, and V max app A systematic decrease in the K value is observed, again indicating a mixed inhibition mechanism. A fit of the mixed inhibition model for CA13612 yielded a K of 9 ± 1 μM with an α value of 7.5 ± 1.9. i app giving a K of 9 μM iC app and a K of 66 μM iU app The strong preference of CA13612 for the LRRK2 apo form over the ATP-bound form indicates that the Nb does not completely compete for ATP binding, but that ATP and CA13612 binding mutually repel each other. This suggests that the Nb binds near the ATP-binding pocket or that Nb binding forces LRRK2 into a conformation incompatible with ATP binding.

[0103] Example 8. Nb engages and colocalizes with LRRK2 at endogenous levels. Given the high-affinity binding of the 10 selected Nbs, we next tested whether they also had the ability to pull down LRRK2 at endogenous / physiological expression levels. Therefore, we turned to lysates of mouse RAW264.7 cells, which express LRRK2 at relatively high levels

[52] . Interestingly, we found that all 10 Nbs efficiently pulled down LRRK2 when added to these lysates (Figure S15), indicating that (1) the Nbs are cross-reactive with mouse LRRK2 and (2) their affinity is high enough to pull down endogenous levels of LRRK2 from cell lysates.

[0104] To test whether Nbs could trace and visualize endogenous LRRK2 in fixed cells, we generated green fluorescent protein (GFP)-Nb fusions (fluobodies) as previously described [23,53,54]. Recently, it has been shown that LRRK2 is recruited to phagosomes upon induction of phagocytosis in immune cells [55,56]. Therefore, RAW264.7 cells were transfected with these fluobodies and treated with zymosan, a dead yeast bioparticle, to induce phagocytosis. Confocal imaging of fixed cells showed that LRRK2 and Nb36 and Nb42 did not colocalize with endogenous LRRK2, and LRRK2 recruitment to phagosomes was barely detectable in cells transfected with Nb1, Nb6, Nb17, and Nb39 (Figure 16b). Importantly, four of the tested fluobodies (Nb22, Nb23, Nb38, and Nb40) clearly colocalized with LRRK2 on zymosan-containing phagosomes (Fig. S16a), demonstrating the ability of these Nbs to trace endogenous LRRK2 within cells.

[0105] Example 9. Expression of LRRK2-targeting nanobodies does not result in LRRK2 translocation to microtubules, and a subset of these nanobodies inhibits MLi-2-induced translocation. Pharmacological kinase inhibitors of LRRK2 from different structural classes induce cellular recruitment of LRRK2 to microtubules, similar to four of the five major PD-causing mutations [37-40]. Binding of LRRK2 to microtubules subsequently reduces kinesin- and dynein-mediated transport on microtubules

[36] . To examine whether our 10 identified LRRK2 kinase-modulating Nbs result in similar phenotypes, HEK293 cells were cotransfected with constructs encoding mScarlet-LRRK2 and GFP-Nb. For all 10 analyzed Nbs, confocal microscopy analysis showed that LRRK2 maintained its cytoplasmic distribution 48 hr after cotransfection with Nb; no translocation to microtubules was observed, indicating that Nb trap LRRK2 in a different conformation compared to classical inhibitors (Figure 5).

[0106] Next, we wanted to examine whether LRRK2-targeting Nbs have the ability to alter LRRK2 recruitment to microtubules induced by inhibition of LRRK2 kinase activity with the specific ATP-competitive inhibitor MLi-2

[41] . To do so, HEK293 cells cotransfected with mScarlet-LRRK2 and GFP-Nb constructs were treated with 1 μM MLi-2 (Figure 6). Interestingly, cells cotransfected with a subset of the tested Nbs did not exhibit MLi-2-induced recruitment to microtubules, and cytoplasmic blockade of LRRK2 was maintained. This rescue effect was most pronounced for Nbs CA13606, CA13611, CA13612, and CA14135. Recent structural information links LRRK2 microtubule translocation to the conformation of the kinase domain, with a closed conformation favoring LRRK2 binding to microtubules

[36] . Consequently, commonly used ATP-competitive type I LRRK2 inhibitors induce this effect. Additionally, it has previously been shown that the WD40 domain is essential for LRRK2-microtubule association

[40] . Furthermore, it has recently been reported that LRRK2 decoration on microtubules involves the WD40:WD40 dimerization interface, and blocking this protein-protein interaction abolishes the effect of MLi-2 on LRRK2 translocation to microtubules

[42] . Interestingly, our ELISA and CL-MS experiments demonstrated that all Nbs that inhibit MLi-2-induced microtubule localization interact with either the kinase or the WD40 domain. Therefore, it is conceivable that these Nbs trap LRRK2 in either a "kinase-open" conformation or a conformation that masks the WD40:WD40 interface.

[0107] Example 10. Nb inhibits LRRK2 through a binding site that is different from the previously described LRRK2 kinase inhibitors. Most currently described LRRK2 inhibitors bind directly to the ATP-binding pocket of the kinase domain. However, physiological forms of vitamin B12 have been reported to inhibit LRRK2 kinase activity by binding to a region of the kinase domain that does not overlap with the ATP-binding pocket

[43] . Two of the in vitro kinase activity-inhibiting Nb described herein (Nb1 and Nb6) bind to LRRK2 predominantly through interactions with the COR-B domain, thus proposing a novel mechanism of action, thereby excluding similar inhibitory mechanisms as those of previously described LRRK2 kinase inhibitors, all of which directly interact with the kinase domain. On the other hand, ELISA and cross-linking MS experiments showed that a third in vitro kinase activity-inhibiting Nb (Nb23) binds to LRRK2 via the kinase domain, but kinetic analysis also demonstrated that Nb23 is a mixed-type (non-ATP-competitive) inhibitor. To verify and confirm that Nb1, Nb6, and Nb23 bind to LRRK2 in a mode distinct from any known small-molecule kinase inhibitors and therefore from reported forms of vitamin B12, we performed a competitive ELISA titration experiment (Figure 17). In this experimental setup, ELISA experiments were performed using a fixed concentration of LRRK2 coated on the bottom side of an ELISA plate and a dilution series of one of the three Nbs ranging from 450 nM to 0.2 nM. Detection of Nb binding in ELISA was performed using their C-terminal EPEA tag, resulting in a dose-response titration curve that reflects the apparent affinity of the Nb. Subsequently, repeats of the setup were performed in the presence of a large excess of either the ATP-competitive inhibitor MLi-2 (1 μM)

[34] or the non-ATP-competitive Vitamin B12 derivative 5'-deoxyadenosylcobalamin (AdoCbl, 250 μM)

[43] . Additionally, as a positive control, repeats of the setup were performed in the presence of an excess (9 μM) of the corresponding untagged Nb.As expected, a very significant right-shift of the titration curves is observed when the corresponding untagged Nb is added as a direct orthosteric competitor, but no right-shift is observed for any of the three Nbs in the presence of either MLi-2 or AdoCbl. Therefore, this further demonstrates that (1) none of the three Group 1 Nbs described herein binds in the same pocket as the type I ATP-competitive inhibitor MLi-2, thus confirming that these Nbs act via a non-ATP-competitive allosteric inhibitory mechanism, and (2) none of the Nbs binds to the same epitope as AdoCbl, indicating that they act via an entirely novel allosteric mechanism.

[0108] Example 11. Creating multivalent and multiparatope nanobodies for increased affinity and potency. A panel of allosteric Nbs that bind to LRRK2 and inhibit LRRK2 kinase activity by targeting different, non-overlapping epitopes on different domains has been identified. Therefore, it is possible to genetically fuse combinations of such Nbs targeting different LRRK2 epitopes, resulting in multivalent / multiparatopic (i.e., binding to different epitopes on the same target) Nbs. Creating this multivalency is expected to result in significantly increased (apparent) affinity due to both additive and cooperative (avidity) effects. Furthermore, it is similarly expected that combinations of multiparatopic Nbs will synergistically increase kinase inhibitory potency compared to mixtures of individual Nbs [44, 45]. This is particularly true for Nbs that inhibit kinase activity by binding through different domains, such as, but not limited to, COR-B (e.g., CA12610 and C12618) and kinase domains (e.g., CA13612).

[0109] Flexible (G4S) for combining different nanobody building blocks xLinkers of various lengths can be used by changing the number of repeats x. Any combination of two or more different Nbs can be created on the pMESY4 vector. Furthermore, apart from changing the linker length, the order of Nbs on the fusion can also be changed. The resulting multiparatope Nbs can be expressed and purified in a similar manner to individual Nbs. First, the IC of the multiparatope construct 50 The (apparent) K values ​​of multiparatopic Nbs are determined using the PhosphoSens® protein kinase assay as described above and compared with corresponding cocktails of individual Nbs to detect synergistic effects of genetic linkages. D values ​​were also determined and these were compared to the K values ​​of individual Nbs using the MST or BLI methods as described herein. D The most promising Nbs will then be tested for their effect on cellular LRRK2 kinase activity.

[0110] material and method Protein expression and purification Full-length human LRRK2 was expressed and purified using previously developed protocols [1,2] with minor modifications to obtain LRRK2 bound to either specific nucleotides: guanosine-5'-(γ-thio)-triphosphate (GTPγS) or guanosine-5'-diphosphate (GDP). Briefly, full-length LRRK2 was cloned into the N-Strep / Flag-TAP (N-SF-TAP) pcDNA3.0 vector, which encodes a protein with an N-terminal Twin-Strep tag and a FLAG tag (NSF) [3]. HEK293T cells (CRL-11268; American Type Culture Collection) were transfected at between 50% and 70% confluence with 8 μg of plasmid DNA per 14 cm dish using polyethyleneimine (PEI) 25 kDa (Polysciences). After transfection, cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% (vol / vol) FBS (Sigma-Aldrich) and the appropriate antibiotics for 48 h in 14 cm dishes. After removal of medium, cells were resuspended in lysis buffer (1 mL per 14 cm dish) containing 50 mM HEPES (pH 8.0), 150 mM NaCl, 5 mM MgCl2, 2 mM DTT, 5% glycerol, and supplemented with 0.55% (v / v) Nonidet P-40, Complete protease inhibitor (Roche), and either 1 mM GTPγS or 1 mM GDP. Cell lysis was allowed to proceed for 1 h on an orbital shaker (10 rpm) at 4°C, and cell debris and nuclei were removed by centrifugation at 10,000 × g for 10 min. The lysate was incubated with Strep-Tactin beads (IBA, 500 μl bed volume / 15 mL cell lysate) for 2 h on an orbital shaker at 4°C. The beads were transferred to a microspin column (GE Healthcare) and thoroughly washed five times with wash buffer (50 mM HEPES (pH 8.0), 100 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.5 mM EDTA, 10% [vol / vol] glycerol) containing either 1 mM GTPγS or GDP.Elution was performed with 700-800 μL of the same wash buffer containing 2.5 mM D-desthiobiotin (IBA) and 1 mM of either GTPγS or GDP.

[0111] Initially, a domain construct of LRRK2 spanning the Roc and COR domains (RocCOR) was engineered with an N-terminal Twin-Strep tag and a C-terminal His 10The RocCOR coding region was expressed from the pBADcLIC vector, encoding a protein spanning aa residues 1334–1840 fused to a tag. In a later stage, a construct spanning residues 1293–1840, cloned into the pDEST-566 vector and encoding a protein with an N-terminal His6-MBP (maltose-binding protein) tag, was used. Protein expressed from the pBADcLIC vector was used for immunization and most screening experiments, but protein expressed from the pDEST-566 vector was also used in certain screening experiments. Expression and purification of the His6-MBP-tagged RocCOR was performed similarly to the purification of the COR-B construct (see further). The pBADcLIC vector containing the RocCOR coding region was transformed into an E. coli strain (E. coli RCEv9), which was custom-designed in-house for optimal expression of the RocCOR protein starting from the MC1061ΔacrB strain, using a previously described protocol [4,5]. The overnight culture was used to inoculate 4 L of TB medium (37 °C). When it reached an OD of approximately 0.7, protein expression was induced with 0.01% arabinose and allowed to proceed overnight at 20 °C. Cells were harvested and resuspended in a buffer containing 50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM MgCl, 2 mM β-mercaptoethanol, and 20 mM imidazole, supplemented with 1 mM PMSF, 1 μg / mL leupeptin, 0.1 μg / mL AEBSF, and 50 μg / mL DNase I. Finally, either 0.5 mM GDP or 0.5 mM guanosine-5'-[(β,γ)-imido]triphosphate (GppNHp) was added to the buffer prior to cell lysis. Cells were lysed using a Cell Disrupter (Constant Systems Ltd.), and after centrifugation, the cell lysate was loaded onto a 5 mL Ni-NTA column. The column matrix was first washed with 10 column volumes (CV) of resuspension buffer supplemented with 300 mM KCl and 5 mM ATP to reduce chaperone contamination.The column was then washed with 10 CV of buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl, 20 mM imidazole, 5% glycerol, 2 mM β-mercaptoethanol, and either 0.5 mM GDP or GppNHp, and the protein was eluted with the same buffer supplemented with 300 mM imidazole. After a concentration step, the final purification step consisted of gel filtration on a Superdex S20010 / 300 column using 30 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl, 5% glycerol, 1 mM DTT as buffer and supplemented with either 0.5 mM GDP or GppNHp.

[0112] The Roc domain construct spanning residues 1329-1520 was cloned into the pET-28a vector, which provides an N-terminal His6 tag, and the vector was transformed into the E. coli BL21(DE3) strain. An overnight culture was used to inoculate 4 L of TB medium (37°C). When it reached an OD of approximately 0.7, protein expression was induced with 0.1 mM isopropyl β-Dl-thiogalactopyranoside (IPTG) and allowed to proceed overnight at 20°C. Cells were harvested and resuspended in a buffer containing 30 mM HEPES pH 7.5, 250 mM NaCl, 10 mM MgCl2, 10 mM glycine, and 20 mM imidazole, supplemented with 1 mM PMSF, 1 μg / mL leupeptin, 0.1 μg / mL AEBSF, and 50 μg / mL DNase I. Cells were lysed using a Cell Disrupter (Constant Systems Ltd.), and after centrifugation, the cell lysate was loaded onto a 5 mL Ni-NTA column. After extensive washing with 10 CV of resuspension buffer, the protein was eluted with the same buffer containing 300 mM imidazole. The final purification step consisted of gel filtration on a Superdex S75 10 / 300 column using 30 mM HEPES pH 7.5, 150 mM NaCl, 5 mM MgCl2, 5% glycerol, and 1 mM DTT as the buffer.

[0113] A construct of the C-terminal part of the COR domain (COR-B), spanning residues 1672-1840, was cloned into the pDEST-566 vector, which provides an N-terminal His6-MBP tag. The vector was then transformed into the E. coli BL21(DE3) strain. An overnight culture was used to inoculate 4 L of TB medium (37 °C). Upon reaching an OD of approximately 0.7, protein expression was induced with 0.5 mM IPTG and allowed to proceed for 2 h at 20 °C. Cells were harvested and resuspended in a buffer containing 30 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA, and 1 mM DTT supplemented with 1 mM PMSF, 1 μg / mL leupeptin, 0.1 μg / mL AEBSF, and 50 μg / mL DNase I. Cells were lysed using a Cell Disrupter (Constant Systems Ltd.), and after centrifugation, the cell lysate was loaded onto a 5 mL MBPTrap column (GE Healthcare). After washing with 10 CV of resuspension buffer, the protein was eluted with the same buffer containing 10 mM maltose. The final purification step consisted of gel filtration on a Superdex S200 10 / 300 column using 30 mM HEPES pH 7.5, 150 mM NaCl as the buffer.

[0114] A domain construct of LRRK2 containing the kinase and WD40 domains (K-WD40) spanning residues 1876–2527 was cloned into the pFastBac vector (Invitrogen), encoding a protein with an N-terminal His tag. The protein was expressed in Sf9 cells (Invitrogen) and purified by affinity chromatography using a Ni-NTA matrix as previously described [6]. The final purification step consisted of gel filtration on a Superdex S75 10 / 300 column, buffered with 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 2 mM β-mercaptoethanol, and 0.1 mM GDP.

[0115] Nanobody (Nb) expression and purification was performed as previously described [7]. The open reading frames encoding Nb cloned into the pMESy4 vector (see further) were transfected into the non-suppressor E. coli WK6 (Su - The nucleotide sequence was transformed into 1 mM IPTG cells. The cells were grown in Terrific Broth (TB) medium at 37°C, and protein expression was induced with 1 mM IPTG. After overnight expression at 28°C, the cells were harvested by centrifugation and subjected to osmotic shock to obtain periplasmic extracts. This was followed by a dialysis step against a buffer consisting of 20 mM Tris-HCl pH 7.5, 150 mM NaCl. 2+ Nbs were purified using an affinity purification step with -NTA Sepharose.

[0116] immunization In total, three llama immunizations were performed: (1) with a RocCOR domain construct of human LRRK2; (2) with full-length LRRK2 in the presence of GTPγS; and (3) with full-length LRRK2 in the presence of GDP. To obtain LRRK2 in a specific nucleotide-bound state (GDP vs. GTPγS), all purification steps were performed in the presence of an excess of the respective nucleotide (see above). Furthermore, to ensure that the protein remained in a specific nucleotide-bound state during and after immunization, mild cross-linking was performed on the protein prior to immunization. Therefore, LRRK2 protein loaded with either 1 mM GTPγS or 1 mM GDP was incubated with the primary amine-specific cross-linker disuccinimidyl suberate (DSS) at a 1:20 molar ratio for 30 min, after which the reaction was quenched by adding excess Tris.

[0117] All three independent immunization strategies followed a 6-week protocol with weekly immunizations in the presence of GERBU adjuvant. All animal vaccinations were performed in strict accordance with good practice and EU animal welfare regulations. In immunization strategy (1), the RocCOR domain construct was used for immunization in the presence of 10 mM GppNHp. 200 μg of protein was injected in the first 2 weeks and 100 μg in the last 2 weeks. Immunization with full-length (partially cross-linked) LRRK2 in the presence of 10 mM GTPγS (immunization (2)) or 10 mM GDP (immunization (3)) was performed according to the following scheme: 300 μg protein was injected in week 1, 200 μg protein in week 2, and 100 μg protein in weeks 3–6. Blood was collected 4 days after the last injection.

[0118] Nanobody generation Construction of immune libraries and Nb selection by phage display were performed. A previously described protocol [7] was used with modifications to maximize the chances of selecting Nbs that specifically bind to different nucleotide forms of LRRK2. Briefly, starting from blood collected from llamas after immunization with the RocCOR domain (immunization 1), LRRK2 in the presence of GTPγS (immunization 2), and LRRK2 in the presence of GDP (immunization 3), respectively, variable domains of the heavy chain antibody repertoire were cloned into the pMESy4 phage display vector, which adds a C-terminal His6 tag and an EPEA tag (=CaptureSelect™ C tag) upon protein expression. This resulted in 8.3 × 10 8 , 1.8×10 9 , and 1.3 × 10 9Three independent immunization libraries of transformants were obtained. This Nb repertoire was expressed on the tip of filamentous phage after rescue with VCSM13 helper phage. In immunization 1, two consecutive rounds of phage display selection were performed against either solid-phase coated full-length LRRK2 or full-length LRRK2 trapped on beads with anti-flag M2 Ab (Merck). LRRK2 coating was performed in a coating buffer containing 50 mM HEPES pH 8.0, 150 mM NaCl, 5 mM MgCl2, and 5% glycerol supplemented with 100 μM GDP, and blocking was performed with 2% BSA. All binding and washing steps were performed in a wash buffer containing 50 mM HEPES pH 8.0, 150 mM NaCl, 5 mM MgCl2, 5% glycerol, and 0.05% Tween 20 supplemented with 100 μM GDP. In immunization 2, two consecutive rounds of phage display selection were performed using solid-phase coated full-length LRRK2. In addition, three consecutive rounds of phage display selection were performed using solid-phase coated LRRK2 Roc domain. In the coating step, the coating buffer was supplemented with 1 mM GTPγS, and in the binding and washing steps, the washing buffer was supplemented with 100 μM GTPγS. In immunization 3, one round of phage display selection was performed using solid-phase coated full-length LRRK2. In addition, two consecutive rounds of phage display selection were performed using solid-phase coated LRRK2 Roc domain. In the coating step, the coating buffer was supplemented with 1 mM GDP, and in the binding and washing steps, the washing buffer was supplemented with 100 μM GDP. Several single colonies were picked after each round of phage display selection, and the resulting Nb clones were classified into sequence families based on their CDR3 sequences using sequence analysis. To allow expression of Nb as a fluorescently (eGFP)-tagged intrabody (Fluobody) in HEK293T cells, the Nb ORF was recloned into the pEGFP-N1 vector using HindIII and BamHI restriction sites.This would result in the expression of Nbs fused to eGFP at their C-terminus (Nb-GFP). To allow expression of Nbs in HEK293T cells without GFP, a stop codon was introduced between the Nb and GFP coding sequences.

[0119] ELISA experiments Prior to Nb purification, binding of Nbs to LRRK2 was confirmed using ELISA screening with crude extracts of E. coli cells expressing each Nb. Full-length LRRK2 was solid-phase coated onto the bottom side of ELISA wells. The coating, blocking, binding, and washing buffers were kept the same as those used in phage display experiments and supplemented with the appropriate nucleotide (GTPγS or GDP). Binding of Nbs to LRRK2 was detected via their EPEA tag using 1:4000 CaptureSelect™ biotin anti-C tag conjugate (Thermo Fischer Scientific) in combination with 1:1000 streptavidin alkaline phosphatase (Promega). Color was developed by adding 100 μl of 4 mg / mL 4-nitrophenyl phosphate disodium solution (DNPP, Sigma-Aldrich) and measured at 405 nm.

[0120] After purification of the selected Nbs as described above, ELISA experiments were performed to determine their domain specificity. Full-length LRRK2 and Roc, COR-B, RocCOR, and K-WD40 domain constructs were solid-phase coated onto 96-well ELISA plates. The coating, binding, and washing buffers were kept the same as those used in the phage display experiments and supplemented with 100 μM GDP. ELISAs were developed in the same manner as described above.

[0121] In cell phospho-Rab assay HEK293T cells were cultured in DMEM (supplemented with 10% fetal bovine serum, 25 mM L-glutamine, and 0.5% Pen / Strep). For the assay, cells were seeded into 6-well plates and transfected at 50-70% confluency with individual Nb-GFP expression constructs, SF-tagged LRRK2 (G2019S), and FLAG-HA Rab29 using a homemade polyethylenimine (PEI)-based transfection reagent [8]. After 48 h, cells were lysed in lysis buffer [30 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonident-P40, Complete protease inhibitor cocktail, and phosphatase inhibitor cocktail II & III (all Sigma)]. Lysates were clarified by centrifugation at 10,000 × g and adjusted to a protein concentration of 1 μg / μl in 1× Laemmli buffer. Samples were then subjected to SDS-PAGE and Western blot analysis to determine LRRK2 pS1292 and Rab10 T72 phosphorylation levels as described below. Total LRRK2 and Rab10 levels were determined as a reference.

[0122] For Western blot analysis, protein samples were separated by SDS-PAGE using NuPAGE 10% Bis-Tris gels (Invitrogen) and transferred to PVDF membranes (Thermo Fisher). To allow simultaneous probing for LRRK2 on the one hand and Rab and Nb-GFP fusions on the other, the membranes were cut horizontally at the 140 kDa MW marker band. After blocking nonspecific binding sites with 5% nonfat dry milk in TBST (1 h, RT) (25 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20), the membranes were incubated overnight at 4°C with primary antibodies at the dilutions specified below. Phosphospecific antibodies were diluted in TBST / 5% BSA (Roth GmbH). Nonphosphospecific antibodies were diluted in TBST / 5% nonfat dry milk powder (BioRad). Phospho-Rab10 levels were determined with a site-specific rabbit monoclonal antibody anti-pRAB10(pT73) (Abcam, ab230261), and LRRK2 autophosphorylation was determined with a site-specific rabbit monoclonal antibody anti-pLRRK2(pS1292) (Abcam, ab203181), both at a dilution of 1:2,000. Total LRRK2 levels were determined with an in-house rat monoclonal antibody anti-pan-LRRK2 (clone 24D8; 1:10,000) [9]. Total Rab10 levels were determined with a rabbit monoclonal antibody anti-RAB10 / ERP13424 (Abcam, ab181367) at a dilution of 1:5,000. Nb-GFP fusion protein was detected with a rat monoclonal antibody anti-GFP (clone 3H9, ChromoTec) at a dilution of 1:2,000. For detection, goat anti-rat IgG or anti-rabbit IgG HRP-coupled secondary antibodies (Jackson ImmunoResearch) were used at a dilution of 1:15,000 in TBST / 5% nonfat dry milk powder. Antibody-antigen complexes were visualized on Hyperfilm (GE Healthcare) using the ECL plus chemiluminescence detection system (GE Healthcare).

[0123] Chemical Crosslinking / Mass Spectrometry (CL-MS)For chemical cross-linking, the LRRK2 protein solution was adjusted to a concentration of 3 μM (0.86 mg / mL). After extensive dialysis to remove the Tris buffer, each nanobody was added to the purified LRRK2 at a final molar ratio of 2:1 in the LRRK2 elution buffer (see Protein Purification). To allow complex formation, the protein mixture was incubated for 1 h at 4 °C under constant mixing. The cross-linking reaction was then carried out using the NHS-ester-based and CID-cleavable reagent disuccinimidyl sulfoxide (DSSO; Thermo Fisher Scientific)

[10] in a molar excess of 60:1 (reference nanobody). The cross-linking reaction was carried out for 30 min at room temperature under constant mixing. The reaction was then stopped by adding Tris-HCl (pH 7.5) solution to a final concentration of 10 mM and incubating for 15 min at room temperature. Proteins were finally precipitated with chloroform / methanol and then subjected to trypsin proteolysis as described [8]. The tryptic peptide solution was cleaned up using a StageTip and subjected to SEC separation to enrich cross-linked peptides as previously described [2]. Vacuum-dried fractions containing cross-linked peptides were individually analyzed using an Orbitrap Fusion mass spectrometer (Thermo Fisher) with MS2-MS3 fragmentation using default settings (ver. 3.0, build 2041). MSI scans were performed using the Orbitrap (FTMS, resolution = 60K) in the m / z range of 375–1500. MS2 scans were performed using CID (CE = 25%), and spectra were acquired using the Orbitap (FTMS) at 30K resolution. MS3 scans were performed using HCD (CE = 30%), and spectra were acquired using a linear ion trap. Thermo Scientific raw files were analyzed using the MS2-MS3 workflow provided by Proteome Discoverer 2.4. It uses XlinkX (ver. 2.4)

[11] for the detection of cross-linked peptides.Briefly, a global search of MS2 spectra was performed using Sequest HT against the human subset of the Swissprot database (v. 2019_02; 20,417 entries) supplemented with nanobody sequences. This was followed by FDR analysis (FDR = 0.01) using the Target Decoy PSM validator. The following settings were used in the Sequest analysis: trypsin was used as the enzyme; cysteine ​​carbamylation was used as the fixed modification; methionine oxidation, DSSO hydrolysis (K + 176.014 Da), DSSO Tris (K + 279.078 Da), and N-terminal acetylation were allowed as variable modifications.

[0124] For detection of crosslinked peptides using the XlinkX detection node, the acquisition strategy was set to MS2_MS3, and DSSO (158.004; K) was used as the crosslinker with a minimum S / N ratio of 1.5. For the XlinkX database search, the following parameters were used: trypsin was used as the enzyme. The precursor and fragment mass tolerances were set to 10 ppm (precursor), 20 ppm (FTMS), and 0.5 Da (ITMS), respectively. The search was performed using a database containing the LRRK2 sequence and the individual sequences of all nanobodies used. Carbamidomethyl was used as the fixed modification, and methionine oxidation was allowed as a variable modification. FDR-based analysis (XlinkX validator node) was performed using the Perculator setting with an FDR threshold of 0.01.

[0125] In the consensus step, identified cross-links were filtered by an identification score ≥ 20 (default value) to reduce the number of false positive hits. The filtered cross-link data was exported and visualized using xiNet

[12] .

[0126] In vitro peptide phosphorylation (kinase) assay The effect of purified Nb on LRRK2 kinase activity was determined using the PhosphoSens® Protein Kinase Assay (AssayQuant Technologies Inc.) with the optimized LRRK2 AQT0615 peptide as a substrate according to the manufacturer's instructions. Sequential kinase assays were performed in a total volume of 50 μL in black half-area 96-well plates. Each reaction mixture contained 10 μM AQT0615 peptide substrate / probe, either 0.1 mM or 1 mM ATP, and 500 μM GDP or GTPγS in a buffer consisting of 50 mM HEPES pH 7.5, 0.1% Brij-35, 50 mM NaCl, and 10 mM MgCl2. Reactions were initiated by the addition of LRRK2 to a final concentration of 80 nM in either the absence or presence of 25 μM Nb. Prior to addition, LRRK2 and Nb were preincubated on ice for 30 min. LRRK2-catalyzed phosphorylation of peptide substrates / probes was continuously followed at 30°C on a plate reader with excitation and emission wavelengths of 360 nm and 485 nm, respectively. Time traces were corrected by subtracting a "no LRRK2" control. Initial velocities were determined from the slope of the linear portion of the curve.

[0127] I C 50 To determine the r-value, Nb concentrations were varied using two-fold serial dilutions from 150 μM to 0.006 μM. A final LRRK2 concentration of 150 nM and an ATP concentration of 1 mM were used in these assays. Relative LRRK2 activity (compared to a "no nanobody" control) was plotted against the log Nb concentration and fitted to a three-parameter log(inhibitor) vs. response equation using GraphPad Prism software. All time traces were collected in triplicate.

[0128] Confocal microscopy and microtubule localization HEK293 cells were cultured in complete medium (high-glucose Dulbecco's modified Eagle's medium, 10% fetal bovine serum, and penicillin-streptomycin-glutamine (Gibco)). Cells were seeded onto 8-well μ-Slides (Ibidi) and transfected at 50–70% confluency with GFP-Nb and mScarlet-LRRK2 constructs using JetPEI reagent (Polyplus Transfection). After 24 h, cells were treated with either DMSO or 1 μM LRRK2 kinase inhibitor (MLi-2, cat. no. 5756, TOCRIS) for 90 min and then analyzed for localization. Data acquisition was performed with a Zeiss LSM800 confocal laser scanning microscope using a ×100 oil immersion objective. Image analysis of z-scans was performed using the Zeiss microscope software ZEN.

[0129] Determining the mechanism of kinase inhibition Inhibitory mechanisms and apparent K of CA12610, CA12618, and CA13612 i (K i app To determine the ATP value, the PhosphoSens® protein kinase assay (AssayQuant Technologies Inc.) was used in combination with the AQT0615 peptide substrate. Full Michaelis-Menten curves (relative velocity vs. [ATP]) in the presence of different Nb concentrations were collected at 30°C using a fixed concentration of AQT0615 (10 μM) and various concentrations of ATP in a buffer consisting of 50 mM HEPES pH 7.5, 0.1% Brij-35, 50 mM NaCl, 10 mM MgCl2, and 500 mM GDP. The LRRK2 concentration was chosen to obtain an initial velocity (linear fluorescence vs. time curve), and LRRK2, ATP, and Nb were preincubated for 30 min at 4°C prior to initiating the reaction by adding the peptide substrate. Michaelis-Menten curves for different Nb concentrations were calculated using GraphPad Prism using the equation:

number

[0130] Additionally, as a diagnostic tool, Michaelis-Menten curves were linearized using Lineweaver-Burk (double reciprocal) plots.

[0131] Pull-down experiments Fresh lysates from HEK293 cells overexpressing SF-tagged LRRK2 and GFP-tagged Nb were prepared in 100 μL ice-cold lysis buffer (10 mM Tris / HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) containing Complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich Cat. No. 11836170001) and protease inhibitor cocktail (Sigma, cat. no. P-2714). GFP-Nb was immunoprecipitated using magnetic GFP nanotrap beads (ChromoTek). The immune complexes were washed twice with 10 mM Tris / HCl pH 7.5 and subjected to immunoblot analysis by boiling the samples in sample buffer with reducing agent. Samples were separated on a 4-15% Tris-glycine gel (Mini-PROTEAN® TGX™ Precast Gel, Bio-Rad), transferred to a nitrocellulose membrane (GE Lifesciences), and processed for Western analysis. The membrane was blocked with 5% dry milk in Tris-buffered saline plus Tween-20 for 1 hour. To allow separate detection of LRRK2 and Nb, the membrane was cut horizontally at 180 kDa, and the upper section was probed with rat monoclonal anti-LRRK2 (clone 24D8, 1:1000, Gloeckner Lab) and the lower section was probed with rabbit anti-GFP antibody 1:2500 (MA5-15256, Invitrogen) and incubated overnight at 4°C with gentle shaking. The membranes were then washed three times for 10 min at room temperature with PBS containing 0.1% or 0.05% Tween-20, and then incubated for 1 h with anti-rat IgG-HRP (sc-2750, Santa Cruz Biotechnology) for LRRK2 or anti-rabbit HRP-conjugated (#7074, Cell Signaling, 1:5000) for GFP-Nb. The membranes were again washed three times for 10 min at room temperature with PBS containing 0.1% or 0.05% Tween-20.The membrane was coated with enhanced chemiluminescence (ECL) reagent (WesternSure PREMIUM, Li-COR Biosciences), and proteins were detected using a C-Digit imaging system (Li-COR Biosciences).

[0132] To test for Nb binding to endogenous (murine) LRRK2, fresh lysates of RAW264.7 cells were prepared in 10 mM Tris / HCl pH 7.5; 150 mM NaCl; 0.5% NP-40 supplemented with Complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich Cat#11836170001) and protease inhibitor cocktail (Sigma, cat. no. P-2714). Purified His-tagged Nb was added to the lysate at a final concentration of 1.5 mM, and the mixture was allowed to rotate overnight at 4 °C. His-tagged Nb was pulled down using magnetic Dynabeads (Invitrogen). Immune complexes were washed twice with 10 mM Tris / HCl pH 7.5 and subjected to immunoblot analysis in a similar manner as described above. To allow separate detection of LRRK2 and Nb, the membrane was cut horizontally at 180 kDa, and the upper part was probed with rabbit monoclonal anti-LRRK2 1:1000 ([MJFF2(c41-2)], ab133474, Abcam) and the lower part was probed with mouse anti-histidine tag antibody 1:1000 (AD1.1.10, Bio-Rad) and incubated overnight at 4°C with gentle shaking. The membranes were then washed three times for 10 min at room temperature with PBS containing 0.1% or 0.05% Tween 20 and then incubated with a secondary antibody: anti-rabbit HRP-conjugated (#7074, Cell Signaling, 1:500) or mouse IgG kappa-binding protein (m-IgGk BP) conjugated to HRP (sc-516102, Santa Cruz Biotechnology, 1:5000) for at least 1 hour. The membranes were again washed three times for 10 min at room temperature with PBS containing 0.1% or 0.05% Tween-20. The membranes were coated with enhanced chemiluminescence (ECL) reagent (WesternSure PREMIUM, Li-COR Biosciences), and proteins were detected using a C-Digit Imaging System (Li-COR Biosciences).

[0133] Microscale thermophoresis and biolayer interferometry measurements Equilibrium binding affinity (K D We performed microscale thermophoresis (MST) experiments to determine the affinity of Nb to LRRK2. Therefore, Nb were site-specifically labeled with m-TAMRA at their C-terminus using sortase-mediated peptide exchange

[57] . Nb were recloned into the pHEN29 vector, which was subsequently used to express and purify Nb bearing a C-terminal LPETGG-His6-EPEA tag. Exchange of the latter peptide with m-TAMRA-labeled GGGYK peptide (GenicBio, Shanghai, China) was performed using sortase, and unlabeled Nb and unincorporated peptide were removed using Ni-NTA and SEC purification steps, respectively. MST measurements were performed using a Monolith NT.115 instrument (Nanotemper technologies) by titrating a fixed concentration of m-TAMRA-labeled Nb (50–100 nM, depending on the affinity of the Nb) with various concentrations of LRRK2 (16-point, 3:1 dilution series). Experiments were performed in 50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl2, 5% glycerol, 0.1% BSA, 0.05% Tween, and 500 μM GDP (with the exception of Nb42, where 500 μM GTPγS was used). After a 30 min incubation at 4 °C, samples were loaded into the capillary, and measurements were performed at 25 °C using 50-70% LED power and 80% laser intensity (laser on time: 30 s, laser off time: 5 s). All experiments were performed in triplicate. Initially, data were processed using MO affinity analysis software, and the final K D Values ​​were obtained by fitting the MST signal (5–15 s on time) vs. [LRRK2] curve to a second-order binding isotherm by GraphPad Prism 7.

[0134] Biolayer interferometry (BLI) measurements were performed using an Octet Red 96 (Forte Bio, Inc.) system in a buffer containing 50 mM HEPES pH 8.0, 150 mM NaCl, 10 mM MgCl2, 5% glycerol, 0.1% BSA, and 0.05% Tween at 25 °C with a shaking speed of 1000 rpm. Binding of Nb to FL-LRRK2 was performed by first trapping LRRK2 on a streptavidin-coated (SA) biosensor via a high-affinity LRRK2-specific biotinylated Nb (either Nb40 or Nb42). The biotinylated Nb was first loaded onto a pre-equilibrated SA biosensor at a concentration of 5 μg / mL. The Nb-loaded sensor was then used to trap FL-LRRK2 from a 50 nM LRRK2 solution. Finally, the sensor was used to monitor the association (600s to 1000s) and dissociation (600s to 1000s) of the entire set of Nbs (to assess the binding of Nb40, a sensor with Nb42 as the trapping agent was used; for all other Nbs, Nb40 was used as the trapping agent). All experiments were performed in triplicate. The equilibrium dissociation constants (K D ) was obtained by fitting the dose-response curve, resulting from plotting the association signal from 100 s to 800 s (depending on the association rate of Nb) versus Nb concentration, to the Langmuir equation using GraphPad Prism 7.

[0135] immunostaining RAW264.7 cells (ATCC® SC-6003™) were transfected with the peGFP-Nb construct using JetOPTIMUS (Polyplus transfection) for 24 hours and then stimulated with zymosan particles (Sigma-Aldrich) (50 mg / mL) for 30 minutes. LRRK2 immunostaining was performed as previously described

[78] . Briefly, cells were fixed with 4% (w / v) paraformaldehyde for 30 minutes, followed by treatment with 100% EtOH at -20°C. Samples were permeabilized with PBS / 0.5% Triton X-100 and blocked with 3% BSA. Primary antibodies (rabbit anti-LRRK2, c41-2, Abcam, and mouse anti-GFP, G6539, Sigma-Aldrich) were diluted in PBS containing 3% BSA and 0.5% Triton X-100 and incubated overnight at 4°C. After three 5-min washes with PBS, fluorescently labeled secondary antibodies (anti-rabbit Alexa Fluor 568 and anti-mouse Alexa Fluor 488, Invitrogen) were added in a similar manner to the primary antibodies and incubated for 1 h at room temperature. Cells were washed twice with 1x PBS and mounted with Fluroshield antifade reagent containing DAPI (Sigma-Aldrich). Images were acquired using a Zeiss LSM800 confocal laser scanning microscope. Image analysis of z-scanning was performed using the Zeiss microscope software ZEN.

[0136] Competitive ELISA To assess any possible competition between the binding of nanobodies Nb1, Nb6, or Nb23 and either Mli-2 or 5'-deoxyadenosylcobalamin (AdoCbl), a competitive ELISA experiment was performed. The ELISA experiment was performed similarly as described above. A fixed concentration of LRRK2 was coated on the bottom side of the ELISA plate, and a 1:4 dilution series of Nb1, Nb6, or Nb23 ranging from 450 nM to 0.2 nM was used. Detection of Nb binding was performed using the C-terminal EPEA tag of the Nb as previously described, resulting in a dose-response titration curve reflecting the apparent affinity of the Nb. This setup was performed with each dilution series either in the absence or presence of a large excess of either the ATP-competitive inhibitor Mli-2 (1 μM), the non-ATP-competitive VitB12 derivative 5'-deoxyadenosylcobalamin (AdoCbl, 250 μM), or an excess of the corresponding untagged Nb (9 μM). A "no antigen control" in which LRRK2 was not coated on the bottom side of the well was also included. Absorbance signals (A405nm) were read at different time points, and A405nm at 0.5 h (Nb6 and Nb23) or 10 h (Nb1) was plotted as a function of Nb concentration. Measurements in the presence of Mli-2 and AdoCbl were performed in triplicate (with the exception of Nb1 in the presence of AdoCbl, which was performed in duplicate).

[0137] Sequence Listing SEQ ID NOs: 1-19: LRRK2 nanobody sequences (without 6xHis / EPEA C-terminal tag) (Table 3) >SEQ ID NO: 20: 6x histidine and EPEA C-terminal tag SEQ ID NO: 21: Human leucine-rich repeat kinase 2 amino acid sequence (T1647S mutant of Q17RV3_UniProt used herein; 2527 aa) >SEQ ID NO: 22: N-terminal SF tag (flag and twin-strep) used for expression of human LRRK2 of SEQ ID NO: 21 (without N-terminal Met). The cDNA used herein to encode human LRRK2 was originally described in

[50] , and the construct including the N-terminal SF tag (SEQ ID NO: 22) was originally described in [1]. >SEQ ID NOs: 23-79: CDR1, CDR2, and CDR3 sequences of Nbs of SEQ ID NOs: 1-19. As annotated in Table 3 and as provided in Table 4.

[0138] Table 3. LRRK2-specific nanobody sequences. The Nanobodies provided in the examples contain the corresponding sequences of SEQ ID NOs: 1-19 and a C-terminal 6xHis / EPEA tag. CDR annotations are labeled herein according to the current analysis (+Table 4) (see also Figure 10 for alternative annotations). [Table 3]

[0139] Table 4. Sequences of the CDR1, CDR2, and CDR3 regions annotated in Table 3 for SEQ ID NOs: 1-19. [Table 4]

[0140] Aspects of the Disclosure A non-natural allosteric modulator of leucine-rich repeat kinase 2 (LRRK2) that specifically binds to human LRRK2.

[0141] The allosteric modulator of LRRK2 described above does not include a cobalamin derivative.

[0142] The allosteric modulator of LRRK2 as described above, which inhibits or enhances LRRK2 activity.

[0143] The allosteric modulator of LRRK2 specifically binds to a binding site of human LRRK2 that is different from the ATP catalytic site of LRRK2.

[0144] The allosteric modulator of LRRK2 described above, which specifically binds to a binding site of human LRRK2 that does not exclusively include the kinase domain, or a binding site that is distinct from the kinase domain.

[0145] The allosteric modulators of LRRK2 include small molecules, chemical compounds, proteins, peptides, peptidomimetics, antibodies, antibody mimetics, single domain antibodies, immunoglobulin single variable domains (ISVDs), or active antibody fragments.

[0146] The allosteric modulator of LRRK2 comprises an ISVD, wherein the ISVD comprises four framework regions (FR) and three complementarity determining regions (CDRs) according to the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(1).

[0147] The allosteric modulator of LRRK2 as described above, comprising an ISVD, wherein in the ISVD: CDR1 consists of a sequence selected from the group of CDR1 sequences of SEQ ID NOs: 1 to 19; CDR2 consists of a sequence selected from the group of CDR2 sequences of SEQ ID NOs: 1 to 19; The CDR3 consists of a sequence selected from the group of CDR3 sequences of SEQ ID NOs: 1 to 19.

[0148] The allosteric modulator of LRRK2, comprising an ISVD, wherein the ISVD comprises any of the sequences of SEQ ID NOs: 1 to 19, or a sequence having at least 85% amino acid identity thereto, or a humanized variant thereof.

[0149] A multispecific allosteric modulator of LRRK2, comprising at least one of said modulators defined herein.

[0150] 1. An in vitro method for detecting the amount of LRRK2 protein in a sample, comprising: interacting the sample with the LRRK2-specific ISVD or with the multispecific agent comprising the LRRK2-specific ISVD; and Detecting the presence or absence or level of the interacted LRRK2-specific ISVD; The method comprising:

[0151] A pharmaceutical composition comprising an allosteric modulator of LRRK2, a multispecific allosteric modulator of LRRK2, a nucleic acid molecule encoding an allosteric modulator of LRRK2 or a multispecific allosteric modulator of LRRK2, or a vector comprising said nucleic acid molecule.

[0152] The pharmaceutical composition as described above, further comprising a compound that is an ATP-competitive LRRK2 kinase inhibitor.

[0153] The allosteric modulator of LRRK2, the multispecific allosteric modulator of LRRK2, the nucleic acid molecule encoding the allosteric modulator of LRRK2 or the multispecific allosteric modulator of LRRK2, a vector comprising the nucleic acid molecule, or a pharmaceutical composition for use as a pharmaceutical, or for use as a diagnostic agent or for in vivo imaging.

[0154] The allosteric modulator of LRRK2, a multispecific allosteric modulator of LRRK2, a nucleic acid molecule encoding an allosteric modulator of LRRK2 or a multispecific allosteric modulator of LRRK2, a vector comprising the nucleic acid molecule, or a pharmaceutical composition for use in the treatment of disorders related to LRRK2, particularly for use in the treatment of Parkinson's disease.

[0155] 1. An in vitro method for detecting the localization and distribution of human LRRK2 protein in a biological sample, comprising: reacting the biological sample with the LRRK2-specific ISVD or a multispecific agent comprising the LRRK2-specific ISVD; and detecting the localization and distribution of said LRRK2-specific ISVD in said biological sample; The method comprising:

[0156] A nucleic acid molecule encoding said allosteric modulator of LRRK2 or said multispecific allosteric modulator of LRRK2.

[0157] A vector, preferably a viral, lentiviral or adenoviral vector, comprising said nucleic acid molecule.

[0158] reference [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8]

Claims

1. 1. An allosteric modulator of leucine-rich repeat kinase 2 (LRRK2) that specifically binds to human LRRK2, wherein the binding leaves the LRRK2 protein non-associated with microtubules in cells, The allosteric regulator comprises a VHH, VHHs contain three complementarity determining regions: CDR1, CDR2 and CDR3 as set forth in SEQ ID NO: 1; CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:2; CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:3; CDR1, CDR2 and CDR3 as represented in SEQ ID NO:4; CDR1, CDR2 and CDR3 as represented in SEQ ID NO:5; CDR1, CDR2 and CDR3 as represented in SEQ ID NO:6; CDR1, CDR2 and CDR3 as represented in SEQ ID NO:7; CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:8; CDR1, CDR2 and CDR3 as set forth in SEQ ID NO:9; or CDR1, CDR2 and CDR3 as represented in SEQ ID NO: 10; and wherein the CDR regions are annotated according to Kabat, MacCallum, IMGT, AbM, or Chothia; or VHH is represented by the following formula (1): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (1) and wherein: CDR1 consists of the sequence of SEQ ID NO: 23, CDR2 consists of the sequence of SEQ ID NO: 42, and CDR3 consists of the sequence of SEQ ID NO: 61; CDR1 consists of the sequence of SEQ ID NO: 24, CDR2 consists of the sequence of SEQ ID NO: 43, and CDR3 consists of the sequence of SEQ ID NO: 62; CDR1 consists of the sequence of SEQ ID NO: 25, CDR2 consists of the sequence of SEQ ID NO: 44, and CDR3 consists of the sequence of SEQ ID NO: 63; CDR1 consists of the sequence of SEQ ID NO: 26, CDR2 consists of the sequence of SEQ ID NO: 45, and CDR3 consists of the sequence of SEQ ID NO: 64; CDR1 consists of the sequence of SEQ ID NO: 27, CDR2 consists of the sequence of SEQ ID NO: 46, and CDR3 consists of the sequence of SEQ ID NO: 65; CDR1 consists of the sequence of SEQ ID NO: 28, CDR2 consists of the sequence of SEQ ID NO: 47, and CDR3 consists of the sequence of SEQ ID NO: 66; CDR1 consists of the sequence of SEQ ID NO: 29, CDR2 consists of the sequence of SEQ ID NO: 48, and CDR3 consists of the sequence of SEQ ID NO: 67; CDR1 consists of the sequence of SEQ ID NO: 30, CDR2 consists of the sequence of SEQ ID NO: 49, and CDR3 consists of the sequence of SEQ ID NO: 68; CDR1 consists of the sequence of SEQ ID NO: 31, CDR2 consists of the sequence of SEQ ID NO: 50, and CDR3 consists of the sequence of SEQ ID NO: 69; or CDR1 consists of the sequence of SEQ ID NO: 32, CDR2 consists of the sequence of SEQ ID NO: 51, and CDR3 consists of the sequence of SEQ ID NO: 70; An allosteric regulator of the LRRK2.

2. K of binding to LRRK2 D 2. The allosteric modulator of claim 1, wherein the LRRK2 activity is in the range of 200 nM or less.

3. 3. The allosteric modulator of LRRK2 of claim 1 or 2, which affects LRRK2 kinase activity in cells and / or in vitro.

4. The VHH is any of the sequences set forth in SEQ ID NOs: 1 to 9; or A sequence having at least 85% amino acid identity with any of SEQ ID NOs: 1 to 9, wherein the CDRs are identical to the CDRs of SEQ ID NOs: 1 to 9 and differences may exist in framework residues; or A humanized variant of any of the sequences of SEQ ID NOs: 1 to 9, wherein the CDRs are identical to those of SEQ ID NOs: 1 to 9, the FR hallmark residues are identical to those of any one of SEQ ID NOs: 1 to 9, and with humanizing substitutions that provide differences in residues elsewhere in the FR regions. The allosteric modulator of LRRK2 of claim 3, comprising:

5. The allosteric modulator of LRRK2 according to any one of claims 1 to 4, which inhibits LRRK2 kinase activity in a cell.

6. The allosteric modulator of LRRK2 according to any one of claims 1 to 4, which inhibits LRRK2 substrate phosphorylation in a cell.

7. The allosteric modulator of LRRK2 according to any one of claims 1 to 4, which increases LRRK2 kinase activity in a cell.

8. 5. The allosteric modulator of LRRK2 of any one of claims 1 to 4, which prevents LRRK2 association with microtubules in a cell, optionally when in the presence of an ATP-competitive LRRK2 kinase inhibitor compound.

9. A multispecific allosteric modulator of LRRK2 comprising at least one of the allosteric modulators of LRRK2 according to any one of claims 1 to 8.

10. A nucleic acid molecule encoding the allosteric modulator of LRRK2 of any one of claims 1 to 8 or the multispecific allosteric modulator of LRRK2 of claim 9.

11. A vector comprising the nucleic acid molecule of claim 10.

12. The vector of claim 11, wherein the vector is a viral vector.

13. The vector of claim 12, wherein the viral vector is a lentiviral or adenoviral vector.

14. i) interacting the sample with an allosteric modulator of LRRK2 according to any one of claims 1 to 4; and ii) detecting the presence or absence or protein level of said allosteric regulator of LRRK2 bound to LRRK2; An in vitro method for detecting the amount of LRRK2 protein in a sample, comprising:

15. i) reacting a biological sample with an allosteric modulator of LRRK2 according to any one of claims 1 to 4 and / or optionally a labeled form of said allosteric modulator; ii) detecting the localization and distribution of said allosteric modulator in said biological sample; An in vitro method for detecting the localization and distribution of human LRRK2 protein in a biological sample, comprising:

16. 14. A pharmaceutical composition comprising an allosteric modulator of LRRK2 according to any one of claims 1 to 8, a multispecific allosteric modulator of LRRK2 according to claim 9, a nucleic acid molecule according to claim 10, or a vector according to any one of claims 11 to 13.

17. 17. The pharmaceutical composition of claim 16, further comprising an ATP-competitive LRRK2 kinase inhibitor compound.

18. 18. An allosteric modulator of LRRK2 according to any one of claims 1 to 8, a multispecific allosteric modulator of LRRK2 according to claim 9, a nucleic acid molecule according to claim 10, a vector according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 or 17, for use as a pharmaceutical.

19. 18. An allosteric modulator of LRRK2 according to any one of claims 1 to 8, a multispecific allosteric modulator of LRRK2 according to claim 9, a nucleic acid molecule according to claim 10, a vector according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 or 17, for use in the treatment of an LRRK2-related disorder.

20. 18. An allosteric modulator of LRRK2 according to any one of claims 1 to 8, a multispecific allosteric modulator of LRRK2 according to claim 9, a nucleic acid molecule according to claim 10, a vector according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 or 17, for use in the treatment of Parkinson's disease or Crohn's disease.

21. 18. An allosteric modulator of LRRK2 according to any one of claims 1 to 8, a multispecific allosteric modulator of LRRK2 according to claim 9, a nucleic acid molecule according to claim 10, a vector according to any one of claims 11 to 13, or a pharmaceutical composition according to claim 16 or 17, for use as a diagnostic agent or for in vivo imaging.

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