WO2025093683A1

WO2025093683A1 - Wnt7 signaling agonists

Info

Publication number: WO2025093683A1
Application number: PCT/EP2024/080832
Authority: WO
Inventors: Benoit Vanhollebeke; Emilien NICOLAS; Marc VANHOVE
Original assignee: Neuvasq Biotechnologies Sa
Current assignee: Neuvasq Biotechnologies Sa
Priority date: 2023-11-03
Filing date: 2024-10-31
Publication date: 2025-05-08
Anticipated expiration: 2026-05-03

Abstract

The current invention relates to agonist molecules of the Wnt7 signaling pathway, wherein the agonist comprises one or more LRP binding domains such as one or more LRP5 and/or one or more LRP6 binding domains and one or more Gpr124 or Reck binding domains. The agonists are developed to be deployed in medical treatments, more particularly in the treatment of neurological and/ or ophthalmological diseases and/or disorders.

Description

WNT7 SIGNALING AGONISTS

FIELD OF THE INVENTION

The present invention relates to agonist molecules of the Wnt7 signaling pathway, more particularly to bispecific binding agents, even more particularly bispecific antibodies or bispecific antibody fragments that target two members of the Wnt7 receptor complex. The agonists are developed to be deployed in medical treatments, such as in the treatment of neurological and/or ophthalmological diseases and/or disorders.

BACKGROUND

Endothelial Wnt/ -catenin signaling acts as a master regulator of Blood-Brain Barrier (BBB) physiology in response to neural-derived Wnt7a/b ligands. Wnt/p-catenin signaling initiates the BBB differentiation cascade at the earliest steps of central nervous system (CNS) vascular invasion, and then maintains BBB function in adults. Recent evidence suggests that inhibition of Wnt signaling by conditional deletion of P-catenin signaling in cerebral endothelial cells (ECs) causes BBB breakdown and accelerates disease progression in stroke, glioblastoma, and multiple sclerosis murine models. Conversely, recombining a constitutively active form of p-catenin in the CNS endothelium is protective in models of brain cancer and stroke.

Wnt7a/b being the endogenous ligands controlling p-catenin-dependent BBB maturation, they are in principle, legitimate therapeutic agents to repair the dysfunctional BBB. However, activation of Wnt signaling via Wnt7 ligands has pleiotropic outcomes across a range of tissues and organs, in both health and disease. More so, the structural modalities of Wnt/Frizzled (FZD) interactions disqualify natural Wnt ligands as safe therapeutics. Interestingly, Wnt7 ligands activate Wnt signaling via two distinct types of membrane receptor complexes. The first receptor complex consisting of a member of the Frizzled (FZD) family receptors and LRP5 or LRP6 has broad tissue distribution, binds non-discriminately to Wnt7a/b and leads to systemic activation of Wnt signaling. The second one is the GPR124/RECK/FZD/LRP5 or LRP6 complex which is enriched at cerebral endothelial cells where GPR124 and/or RECK are expressed.

The observation that Wnt signalling at the BBB and CNS requires a different receptor complex compared to other cell types and tissues, opens the opportunity to develop CNS specific Wnt agonists. However, developing chemical or biologicals that bind to and stimulate a four-membered receptor complex is challenging. W02019/180204 for instance suggest many compounds but fails to disclose concrete data or experimental evidence demonstrating the availability of such molecules or that such molecules are effective in modulating Wnt signaling specifically in the CNS. The mere suggestion without enabling disclosure does not provide a viable pathway for developing such therapeutic agents.

The present invention provides a solution based on the unexpected observation that binding of a compound to only two specific members of the receptor complex is sufficient to activate the atypical GPR124/RECK/FZD/LRP5 or LRP6 mediated Wnt signalling.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides an agonist of the Wnt7 signalling pathway, preferably mediated by Frizzled (Fzd), Lipoprotein Receptor-related Protein (LRP) such as LRP5 or LRP6, G-protein coupled receptor (Gpr) 124 and Reversion-inducing cysteine-rich protein with Kazal motifs (Reck), wherein the agonist comprises one or more LRP binding domains such as LRP5 binding domains and/or LRP6 binding domains and one or more Gprl24 or Reck binding domains. In one embodiment, said agonist is a selective or specific agonist. In another or further embodiment, the one or more LRP domains bind an amino acid sequence having at least 75% sequence identity to an amino acid sequence selected from the list consisting SEQ ID N°: 3-5. In another or further embodiment, the one or more Gprl24 or Reck binding domains bind an amino acid sequence having at least 75% sequence identity to an amino acid sequence selected from the list consisting SEQ ID N°: 6-8. In another or further embodiment, the binding agent comprises or consists of an antibody or an antibody fragment, more particularly an immunoglobulin single variable domain (ISVD) or VHH. In another or further embodiment, the antibody or antibody fragment is bispecific. In another or further embodiment, the antibody or antibody fragment is tetravalent. In another or further embodiment, the antibody or antibody fragment is bivalent. In another or further embodiment, the antibody or antibody fragment is tetravalent or bivalent. In another or further embodiment, the antibody or antibody fragment is soluble. In another or further embodiment, the antibody or antibody fragment is a bispecific antibody, a bispecific antibody fragment, a bispecific ISVD, or a VHH. In another or further embodiment, the antibody or antibody fragment comprises one or more LRP binding domains that are LRP5 and/or LRP6 binding domains, and wherein said LRP6 binding domains comprise a CDR3 region as depicted in SEQ ID N°: 85, 86, 87, or 88 or consists of an amino acid sequence with maximally two amino acids different from SEQ ID N°: 85, 86, 87, or 88. In another or further embodiment, the antibody or antibody fragment comprises one or more LRP binding domains that are LR.P5 and/or LR.P6 binding domains, and wherein said LR.P6 binding domain comprises a CDR.2 region as depicted in SEQ ID N° : 81, 82, 83, or 84 and/or a CDR1 region as depicted in SEQ ID N°: 77, 78, 79, or 80. In another or further embodiment, the antibody or antibody fragment comprises an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62. In another or further embodiment, the antibody or antibody fragment comprises an IgG comprising a VH sequence according to SEQ ID N° 63, 65, or 67, and a VL sequence according to SEQ ID N° 64, 66, or 68, optionally separated by a linker sequence; preferably a VH sequence according to SEQ ID N° 63 and a VL sequence according to SEQ ID N° 64, VH sequence according to SEQ ID N°65 and a VL sequence according to SEQ ID N° 66, or a VH sequence according to SEQ ID N° 67 and a VL sequence according to SEQ ID N° 68. In another or further embodiment, the antibody or antibody fragment comprises an IgG comprising a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and a VL sequence according to SEQ ID N° 70, 72, 74, or 76, optionally separated by a linker sequence; preferably comprising a VH sequence according to SEQ ID N° 69 and a VL sequence according to SEQ ID N° 70, a VH sequence according to SEQ ID N° 71 and a VL sequence according to SEQ ID 72, a VH sequence according to SEQ ID N° 73 and a VL sequence according to SEQ ID N° 74, or a VH sequence according to SEQ ID N° 75 and a VL sequence according to SEQ ID N° 76.

In another or further embodiment, said agonist is not able to bind Fzd and/or does not comprise a Fzd binding domain. In another or further embodiment, said agonist does not comprise further binding domains over said one or more LRP binding domains and one or more Gprl24 or Reck binding domains. In another or further embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 or Reck binding domains. In another or further embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 binding domains. In another or further embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Reck binding domains. In another or further embodiment, said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Gprl24 binding domain. In another or further embodiment, wherein said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Reck binding domain. Also provided is a nucleic acid molecule or nucleic acid sequence encoding any of the agonists disclosed herein. Also a vector is provided comprising said nucleic acid.

In a second aspect, any of the herein disclosed agonists, nucleic acids or vectors are provided for use as a medicament. This is similar as saying that the invention provides methods of treatment comprising administering any of the herein disclosed agonists, nucleic acids or vectors to a subject in need thereof. In one embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use in gene therapy. In another or further embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use in blood-brain barrier endothelial cell-directed gene therapy. In another or further embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use to treat bloodbrain barrier integrity. In another or further embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use in the treatment of a neurological disorder. In a particular embodiment, the neurological disorder is selected from the list consisting of ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease intracranial atherosclerosis, and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorders, epilepsy, neurodegenerative disorders, dementia, vascular dementia, HIV-l-associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Charcot-Marie Tooth disease, dystonia, infectious brain diseases, traumatic brain injuries, migraine, neuroinflammation, COVID-19, and chronic traumatic encephalopathy and combinations thereof. In another or further embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use to treat blood-retina barrier (BRB) integrity. In another or further embodiment, any of the herein disclosed agonists, nucleic acids or vectors are provided for use in the treatment of an ophthalmological disease or disorder. Preferably said ophthalmological disease or disorder is selected from the group of: retinopathy, retinal vascular disorders such as Norrie disease, diabetic retinopathy, macular degeneration, familial exudative vitreoretinopathy, osteoporosis- pseudoglioma syndrome, retinal vein occlusion and retinopathy of prematurity.

DESCRIPTION OF FIGURES

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses. Throughout the drawings, corresponding reference numerals indicate like or corresponding parts and features.

Figure 1 is a schematic representation of the Gprl24/Reck/Fzd/LRP5 or LRP6 receptor complex (left) that mediates the atypical Wnt7 signaling at the BBB and the Fzd/LRP5 or LRP6 receptor complex (right) that mediates the canonical Wnt7 signaling.

Figure 2 is a schematic representation of the different bispecific antibodies AB0002, AB0005, AB0006 and AB0007.

Figure 3 shows the Wnt7 signaling induced by the different bispecific antibodies in STF cells expressing Fzdl, LRP6, Flag-Gprl24 and Reck (dark grey) or expressing Fzdl, LRP6, Gprl24 and Flag-Reek (light grey). The Wnt7 signaling is measured based on the luminescence signal typical for the STF cells and normalized in view of a Wnt7a control. Antibodies were tested in the presence of IWP-2, a small molecule inhibitor of Wnt processing and secretion.

Figure 4 shows the dose response curve for AB0007. The luminescence obtained upon adding AB0007 to STF cells transiently expressing Fzdl, LRP6, Reck and flag- Gprl24 was normalized in view of the activity of Wnt7a at saturation.

Figure 5 shows the luminescence signal in STF cells expressing Fzdl, LRP6 and flag- Gprl24 (light grey) and in STF cells expressing Fzdl, LRP6, flag-Gprl24 and Reck (dark grey) upon adding AB0002, AB0005, AB0006, AB0007, Wnt7a as positive control and an empty vector as negative control.

Figure 6 shows possible embodiments of compounds according to the current invention.

Figure 7 shows agonist activity of tested constructs in STF cells expressing Gprl24, Reck, Fzdl and LRP6 according to embodiments of the current invention.

Figure 8 illustrates the structure of the anti-Gprl24/LRP6 and anti-Reck/LRP6 bispecific antibodies used in Example 5, where the LRP6 VHH is fused to the N- terminus of the VL domain of the respective anti-Gprl24 or anti-Reck IgGs. Figure 9 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP6 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Figure 10 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 1.9 nM and 1.7 nM for 01E12-05A10 and 01B01-06C03, respectively.

Figure 11 shows a dose-response for bispecific antibodies 01B01-06A05 (A) and 01E12-06A05 (B) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 2.8 nM and 3.0 nM for 01B01-06A05and 01E12-06A05, respectively.

Figure 12 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP5 upon addition of the indicated bispecific antibodies (20 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Figure 13 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzd5 and LRP6. The EC50 values calculated from the curves were 4.2 nM and 1.5 nM for 01E12-05A10 and 01B01-06C03, respectively.

Figure 14 shows the luminescence signal in STF cells expressing Gprl24, Fzdl and LRP6 (no Reck) upon addition of the indicated bispecific antibodies (20 nM). In the Wnt7a control, Wnt7a was delivered via transient transfection, but the natural ligand showed no activity under these conditions, i.e. in the absence of Reck. An empty vector was used as a negative control.

Figure 15 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzd4 and LRP6 (A) or Gprl24, Reck, Fzd5 and LRP6 (B) upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Figure 16 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzd4 and LRP6. The EC50 values calculated from the curves were 3.7 nM and 1.3 nM for 01E12-05A10 and 01B01-06C03, respectively.

Fig ure 17 shows signaling activity of bispecific antibodies 01E12-05A10 and 01E12- 06C03 (10 nM) in hRMEC (Human Retinal Microvascular Endothelial Cells) (A) and hBMEC (Human Brain Microvascular Endothelial Cells) (B). Recombinant Wnt3a (13.4 nM) was used as a positive control. Wnt pathway activation was assessed by monitoring the level of Axin2 mRNA by qRT-PCR.

Fig ure 18 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hRMEC cells. Wnt pathway activation was assessed by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 66 pM and 32 pM for 01E12-05A10and 01B01-06C03, respectively.

Fig ure 19 shows that treatment of hRMEC cells with bispecific antibody 01B01- 06C03 (10 nM) for 6 h results in a detectable increase in the mRNA expression of the downstream mediators of the Wnt/3-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and NKD1 (Naked cuticle 1). The levels of Lefl and NKD1 mRNAs were monitored by qRT-PCR.

Fig ure 20 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hBMEC cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 17 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively.

Fig ure 21 relates to the response of bEnd.3 cells to (i) treatment with bispecific antibody 01E12-05A10 (1 nM), (ii) treatment with a control antibody obtained by combining an irrelevant VHH with the anti-Gprl24 IgG 05A10 (1 nM), (iii) treatment with a control antibody obtained by combining the anti-LRP6 VHH 01E12 with an irrelevant IgG (1 nM), or (iv) treatment with both control (1 nM each). Treatment was applied for 24 h, and Wnt pathway activation was assessed by monitoring the level of Axin2, PLVAP, Lefl and MFSD2A mRNAs by qRT-PCR. Figure 21 shows the levels of Axin2 (A), Lefl (B), PLVAP (C) and MFSD2A (D) mRNAs for treatment (i) with construct 01E12-05A10.

Figure 22 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in bEnd.3 (mouse brain endothelial) cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values

RECTIFIED SHEET (RULE 91) ISA/EP calculated from the curves were 25 pM and 61 pM for 01E12-05A10and 01B01- 06C03, respectively. As a positive control, cells were treated with different concentrations of recombinant Wnt3a (C). The EC50 value calculated from that curve was 6.6 nM.

Figure 23 shows a dose-response for bispecific antibodies 01E12-06C03 (A) and 01F11-06C03 (B) in bEnd.3 (mouse brain endothelial) cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 18 pM for both antibodies. As a positive control, cells were treated with different concentrations of recombinant Wnt3a (C). The EC50 value calculated from that curve was 6.6 nM.

Fig ure 24 shows that treatment of bEnd.3 cells with bispecific antibody 01E12- 05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 ("CHIR", 10 pM) for 24 h results in a detectable modulation in the level of the downstream mediators of the Wnt/P-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domaincontaining protein 2a) mRNAs, as well as in the level of the canonically Wnt- downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA, as monitored by qRT-PCR.

Figure 25 shows that treatment of bEnd.3 cells with bispecific antibody 01B01- 06C03 (10 nM) for 24 h results in a detectable modulation in the level of the downstream mediators of the Wnt/|3-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domaincontaining protein 2a) mRNAs, as well as in the level of the canonically Wnt- downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA, as monitored by qRT-PCR.

Figure 26 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP6 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Fig ure 27 shows a dose-response for bispecific antibodies 01B01-01F11 (A), 01E12- 01F11 (B), 01E12-03A04 (C) and 01B01-03A04 (D) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 4.2 nM, 1.3

RECTIFIED SHEET (RULE 91) ISA/EP nM, 4.3 nM, and 6.4 nM for 01B01-01F11, 01E12-01F11, 01E12-03A04 and 01B01- 03A04, respectively.

Figure 28 shows the luminescence signal in STF cells expressing human Gprl24, human Reck, human Fzdl and mouse LRP5 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Fig ure 29 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzd4 and LRP6 (A) or Gprl24, Reck, Fzd5 and LRP6 (B) upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

Fig ure 30 shows a dose-response for bispecific antibodies 01B01-01F11 (A), 01E12- 03A04 (B) and 01B01-03A04 (C) in STF cells expressing Gprl24, Reck, Fzd4 and LRP6. The EC50 values calculated from the curves were 5.5 nM, 5.9 nM, and 1.8 nM for 01B01-01F11, 01E12-03A04, and 01B01-03A04, respectively.

Figure 31 shows the modulation in bEnd.3 cells in the level of the downstream mediators of the Wnt/p-catenin signaling pathway Axin2, Lefl, and Mfsd2a mRNAs, as well as in the level of the canonically Wnt-downregulated protein PLVAP mRNA, as monitored by qRT-PCR, upon exposure to Reck-LRP6 bispecific antibodies (10 nM) for 24 h.

Fig ure 32 shows a dose-response for bispecific antibodies 01B01-01A08 (A), 01E12-03A04 (B) and 01E12-01F11 (C) in bEnd.3 cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 10 pM, 7 pM, and 7 pM, for 01B01-01A08, 01E12- 03A04 and 01E12-01F11, respectively.

Figure 33 shows BRB markers (Plvap and Claudin-5) expression and mouse immunoglobulin leakage (mlgG) in retina of Ndp WT and Ndp KO mice treated with bispecific antibody (5mg/kg) or vehicle as control. Images are representative of 2-3 animals per experimental group.

Figure 34 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP6 upon addition of bispecific antibodies 01E12-05A10 or 01B01-06C03 either in a tetravalent format (VHH-tethered IgG) or in a bivalent format (VHH-

RECTIFIED SHEET (RULE 91) ISA/EP tethered Fab) (20 nM). Wnt7a, delivered via transient transfection, was used as positive control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns novel means and method of agonizing or stimulating the Wnt7 signaling pathway, more particularly the atypical Wnt7 signaling pathway that is mediated or controlled by the four member receptor complex comprising Frizzled, LR.P5 or LRP6, Gprl24 and Reck. Even more particularly, the agonists herein provided are specific or selective agonist of the Wnt7 signaling pathway that is orchestrated by the GPR124/RECK/FZD/LRP5/6 complex. The binding agents herein disclosed do not significantly stimulate the canonical Wnt7 signaling mediated by Fzd/LRP5 or Fzd/LRP6 alone.

General definitions

In order that the present description can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description. The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims.

It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence", is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. "One or more" may for instance refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

RECTIFIED SHEET (RULE 91) ISA/EP It is understood that wherever aspects or embodiments are described herein with the language "comprising", otherwise analogous aspects or embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. Practitioners are particularly directed to Sambrook et al., Molecular Cloning : A Laboratory Manual, 4th ed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., current Protocols in Molecular Biology (Supplement 100), John Wiley & Sons, New York (2012), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The term "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance.

RECTIFIED SHEET (RULE 91) ISA/EP The present application relates to binding agents such as multispecific antibodies comprising at least two binding domains, one for binding LR.P5 or LRP6 and one for binding Gprl24 or Reck.

The term "antibody" as used herein, refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. "Antibodies" can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "immunoglobulin (Ig) domain" as used herein refers to a globular region of an antibody chain, or to a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold (Ig fold as named herein) characteristic of antibody molecules, which consists of a two-layer sandwich of about seven to nine antiparallel P-strands arranged in two 0-sheets, optionally stabilized by a conserved disulphide bond. The term "immunoglobulin (Ig) domain", includes "immunoglobulin constant domain", and "immunoglobulin variable domain" (abbreviated as "IVD"), wherein the latter means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or"CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site.

An "immunoglobulin domain" of this application also includes "immunoglobulin single variable domains" (abbreviated as "ISVD"), equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions

RECTIFIED SHEET (RULE 91) ISA/EP (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008/020079.

RECTIFIED SHEET (RULE 91) ISA/EP Immunoglobulin domains herein also include "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen-binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of 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 that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobody, including humanization and/or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08/101985 and WO 08/142164.

"Domain antibodies", also known as "Dabs", "Domain Antibodies", and "dAbs" (the terms "Domain Antibodies" and "dAbs" being used as trademarks by the GlaxoSmithKline group of companies) have been described in e.g., EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03/002609 as well as for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid

RECTIFIED SHEET (RULE 91) ISA/EP mammalians, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e., without being paired with a VL or VH domain, respectively, specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences. Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutical use in humans. It should also be noted that single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05/18629).

Immunoglobulin single variable domains such as Domain antibodies and Nanobody (including VHH domains and humanized VHH domains), represent in vivo matured macromolecules upon their production, but can be further subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molecule. Affinity-matured immunoglobulin single variable domain molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91 : 3809-3813, 1994), Shier et al. (Gene 169: 147- 155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). The process of designing/selecting and/or preparing a polypeptide, starting from an immunoglobulin single variable domain such as a Domain antibody or a Nanobody, is also referred to herein as "formatting" said immunoglobulin single variable domain; and an immunoglobulin single variable domain that is made part of a polypeptide is said to be "formatted" or to be "in the format of" said polypeptide. Examples of ways in which an immunoglobulin single variable domain can be formatted and examples of such formats for instance to avoid glycosylation will be clear to the skilled person based on the disclosure herein.

Immunoglobulin single variable domains such as Domain antibodies and Nanobody (including VHH domains) can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody (including VHH domains) may be immunoglobulin single variable domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and/or that

RECTIFIED SHEET (RULE 91) ISA/EP corresponds to a humanizing substitution (as 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, after which 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 sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody (including VHH domains) may be partially humanized or fully humanized. It should be noted that the immunoglobulin single variable domains, as well as the antigen-binding chimeric protein of the invention in their broadest sense are not limited to a specific biological source or to a specific method of preparation. For example but without the purpose of being limiting, the immunoglobulin single variable domains, in particular the antigenbinding chimeric proteins of the invention, can generally be obtained : (1) by isolating the VHH domain of a naturally occurring heavy chain antibody, and further engineering of the sequence to obtain the antigen-binding chimeric protein; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain, in a format fused to said scaffold protein of the antigen-binding chimeric protein; (3) by "humanization" of a naturally occurring VHH domain and/or scaffold protein or by expression of a nucleic acid encoding a such humanized VHH domain and/or scaffold protein, and/or antigen-binding chimeric protein; (4) by "mutation" of a naturally occurring VHH domain to reduce binding to pre-existing antibodies or by engineering of the scaffold protein fusion sites to obtain an antigen-binding chimeric protein of the invention with reduced binding to pre-existing antibodies as compared to the natural VHH; or (5) by using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se.

An "epitope", as used herein, refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule (e.g. a protein to which an immunoglobulin or part thereof, antibody, VHH or ISVD is binding). "Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact (e.g. physical or chemical) between two binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a

RECTIFIED SHEET (RULE 91) ISA/EP complex of more than two molecules. An interaction can be completely indirect (e.g. two molecules are part of the same complex with the help of one or more bridging molecules but don't bind in the absence of the bridging molecule(s)). An interaction may be partly direct or partly indirect: there is still a direct contact between two interaction partners, but such contact is e.g. not stable, and is stabilized by the interaction with one or more additional molecules. The term "binding pocket", "binding domain" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, associates with another chemical entity, compound, protein, peptide, antibody, single domain antibody or ISVD or VHH.

An epitope could comprise 1, 2 or 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance. A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3- dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3-dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. Similarly, conformational epitopes may here also consist of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation that is unique to the quaternary structure. The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, [3- sheet, among others), tertiary structure (e.g., the 3-dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a

RECTIFIED SHEET (RULE 91) ISA/EP polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labelling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to 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.

A "paratope" as used herein refers to the antigen-binding site and is the part of an antibody which recognizes and binds to an antigen. The paratope of the bispecific binding agent of current disclosure thus comprises the amino acid residues of the binding agent that bind the epitope of the LRP5 or LRP6 protein and the amino acid residues of the binding agent that bind the epitope of the Gprl24 or Reck protein.

Antibodies or binding agents in general can bind one or more epitopes of one or more targets.

"Bispecific" as used herein in the context of current disclosure means that the binding agent can bind two different epitopes or bind two different antigens simultaneously. "Multispecific" as used herein then refers to the ability of binding two or more different binding sites or antigens simultaneously. Multispecific binding agents thus include bispecific antibodies, but it can also refer to antibodies with the ability to bind to three or more targets.

Independent of the number of targets that can be bound by the binding agent, the binding agent can comprise multiple binding sites. The number of binding sites is referred to as the valency of the antibody. A "bivalent" antibody is a typical antibody structure with two binding sites. The common structure of natural antibodies comprises two identical binding sites that bind two identical antigenic determinants or epitopes. However, a bivalent antibody can also bind two different epitopes or two different targets. A "tetravalent" antibody is an antibody that has four binding sites. It can bind to four identical or four different antigenic determinants or epitopes, or it can bind two different epitopes or two different targets that are each bound by two binding sites.

RECTIFIED SHEET (RULE 91) ISA/EP The term "affinity" generally refers to the degree to which an antibody or other binding protein (as defined further herein) binds to a target protein so as to shift the equilibrium of target protein and binding protein toward the presence of a complex formed by their binding. Thus, for example, where an antibody and an antigen are combined in relatively equal concentration, an antibody of high affinity will bind to the antigen so as to shift the equilibrium toward high concentration of the resulting complex. The equilibrium dissociation constant KD (or KD) is commonly used to describe the affinity between a ligand and a target protein, or an antibody and its antigen. KD is the calculated ratio of koff/kon, between the antibody and its antigen and thus measures the propensity of a complex to fall apart into its component molecules. The association constant (k_on or kon) is used to characterize how quickly the antibody binds to its target. The dissociation constant (k_off or koff, also referred to as kdis, Kdis, Kd or kd) is used to measure how quickly an antibody dissociates from its target and is expressed as number of units that dissociated from a target per second. Hence, the lower koff is, the higher the affinity towards the target, koff and thus also KD is inversely related to affinity. A high affinity interaction is characterized by a low KD, a fast recognizing (high kon) and a strong stability of formed complexes (low koff). It will be appreciated that within the scope of the present application, the term "affinity" is used in the context of the antibody or antibody fragment that binds an epitope of the LRP5, LRP6, Gprl24 and/or Reck protein, more particularly the antibody or antibody fragment is "functional" in binding its target via the CDR regions of its immunoglobulin (Ig) domain.

"Amino acids" as used herein refer to the structural units (monomers) that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These chains are linear and unbranched, with each amino acid residue within the chain attached to two neighboring amino acids. Twenty amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids. Natural amino acids or naturally occurring amino acids are glycine (Gly or G), Alanine (Ala or A), Valine (Vai or V), Leucine (Leu or L), Isoleucine (He or I), Methionine (Met or M), Proline (Pro or P), Phenylalanine (Phe or F), Tryptophan (Trp or W), Serine (Ser or S), Threonine (Thr or T), Asparagine (Asn or N), Glutamine (Gin or Q), Tyrosine (Tyr or Y), Cysteine (Cys or C), Lysine (Lys or K), Arginine (Arg or R), Histidine (His or H), Aspartic Acid (Asp or D) and Glutamic Acid (Glu or E).

As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of

RECTIFIED SHEET (RULE 91) ISA/EP any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. The nucleic acid may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The nucleic acid may comprise single stranded or double stranded DNA or RNA. The nucleic acid may comprise modified bases or a modified backbone. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide. "Nucleotides" as used herein refer to the building blocks of oligonucleotides and polynucleotides, and for the purposes of present disclosure include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which are absent in nucleosides). A nucleotide without a phosphate group is called a "nucleoside" and is thus a compound comprising a nucleobase moiety and a sugar moiety. As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Naturally occurring nucleobases of RNA or DNA comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

"Nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, the (reverse) complement DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analogue. By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and

RECTIFIED SHEET (RULE 91) ISA/EP the like. "Coding sequence" is a nucleotide sequence, which 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'-terminus and a translation stop codon at the 3'- terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.

An "expression cassette" as used herein comprises any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription) : a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. Such cassettes can be constructed into a "vector". The term "vector" or alternatively "vector construct", "expression vector" or "gene transfer vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, and includes any vector known to the skilled person, including any suitable type, but not limited to, for instance, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. The construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold

RECTIFIED SHEET (RULE 91) ISA/EP Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

The terms "identical" or percent "identity" in the context of two or more nucleic acid or amino acid sequences refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues respectively that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of nucleotide or amino acid sequences.

The term "percent sequence identity" or "% sequence identity" or "percent identity" or"% identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.

One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et aL, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90: 5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389- 3402). In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain aspects, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative aspects, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453

RECTIFIED SHEET (RULE 91) ISA/EP (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain aspects, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain aspects, the default parameters of the alignment software are used.

One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequencesequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI (European Bioinformatics Institute).

In certain aspects, the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y/Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

According to the present disclosure, the degree of identity, between a given reference nucleotide sequence and a nucleotide sequence which is a homologue of said given nucleotide sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,

RECTIFIED SHEET (RULE 91) ISA/EP 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for a nucleic acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 nucleotides, preferably contiguous nucleotides. In a particular embodiment, the degree/percentage of similarity or identity is given for the entire length of the reference nucleic acid sequence.

The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. According to the present disclosure, the degree of identity, between a given reference amino acid sequence and an amino acid sequence which is a homologue of said given amino acid sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 amino acids, preferably contiguous amino acids. In a particular embodiment, the degree/percentage of similarity or identity is given for the entire length of the reference amino acid sequence.

"Homologue" or "homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar

RECTIFIED SHEET (RULE 91) ISA/EP biological and functional activity as the unmodified protein from which they are derived.

The term "defined by SEQ ID N°: X" or "as depicted in SEQ ID N°: X" as used herein refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID N°: X. For instance, a protein defined in/by SEQ ID N°: X consists of the amino acid sequence given in SEQ ID N°: X. A further example is an amino acid sequence comprising SEQ ID N°: X, which refers to an amino acid sequence longer than the amino acid sequence given in SEQ ID N°: X but entirely comprising the amino acid sequence given in SEQ ID N°: X (wherein the amino acid sequence given in SEQ ID N°: X can be located N-terminally or C-terminally in the longer amino acid sequence, or can be embedded in the longer amino acid sequence), or to an amino acid sequence consisting of the amino acid sequence given in SEQ ID N°: X.

An "agonist," as used herein, refers to a molecule (e.g., binding agent, ligand, or antibody) that, upon interaction with a receptor or another target in a signaling pathway, activates downstream signaling, thereby potentially initiating a biological response. In the context of the Wnt/beta-catenin signaling pathway, an agonist functions as an activator of this pathway, leading to the stabilization and accumulation of beta-catenin and subsequent transcriptional activation of Wnt target genes. The agonist may not necessarily exhibit strong binding affinity to the receptor but still effectively promotes pathway activation. In the present context, the term "agonist" may be used interchangeably with the term "activator", preferably of the Wnt/beta-catenin signaling pathway, more preferably of the Wnt7/beta-catenin signaling pathway.

Conversely, an "antagonist" is a molecule that interferes with or inhibits the activation of downstream signaling pathways, thereby preventing such biological response. In the context of the Wnt/beta-catenin signaling pathway, an antagonist functions by blocking or inhibiting the pathway, thereby preventing the accumulation of beta-catenin and the transcriptional activation of Wnt target genes. The antagonist does not activate the pathway and may act by preventing the agonist or natural ligand from initiating the signaling cascade. In the present context, the term "antagonist" may be used interchangeably with the term "inhibitor", preferably of the Wnt/beta-catenin signaling pathway, more preferably of the Wnt7/beta-catenin signaling pathway.

RECTIFIED SHEET (RULE 91) ISA/EP The terms "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, injury, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Those in need of treatment include those already diagnosed with the disorder as well as those prone or predisposed to contract the disorder or those in whom the disorder is to be prevented. For example, in tumor (e.g. cancer) treatment, a therapeutic agent can directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents or by the subject's own immune system.

As used herein, the terms "diagnosis", "prognosis" and/or "prediction" comprise diagnosing, prognosing and/or predicting a certain disease and/or disorder, thereby predicting the onset and/or presence of a certain disease and/or disorder, and/or predicting the progress and/or duration of a certain disease and/or disorder, and/or predicting the response of a patient suffering from a certain disease and/or disorder to therapy.

The term "statistically significantly" different is well known by the person skilled in the art. Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. The null hypothesis is the default assumption that nothing happened or changed. For the null hypothesis to be rejected, an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a. The p- value of a result, p, is the probability of obtaining a result at least as extreme, given that the null hypothesis were true. In one embodiment, a is 0.05. In a more particular embodiment, a is 0.01. In an even more particular embodiment, a is 0.001.

A peptide, polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A peptide, polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesized. Without limitation, a peptide, polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced

RECTIFIED SHEET (RULE 91) ISA/EP recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.

A conservative amino acid substitution is a substitution of one amino acid for another with similar characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

The term "biologically active" is interchangeable with terms such as "functionally active" or "functional", denoting that the proteins disclosed herein at least partly retain the biological activity or intended functionality of the respective or corresponding peptide, polypeptide or protein. Reference to the "activity" of a peptide, polypeptide or protein may generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.

Preferably, a functionally active protein may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding protein.

In certain embodiments, a functionally active protein may even display higher biological activity or functionality compared with the corresponding peptide, polypeptide or protein, for example may display at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about

RECTIFIED SHEET (RULE 91) ISA/EP 400%, or at least about 500% of the intended biological activity or functionality compared with the corresponding protein. By means of an example, where the activity of a given protein can be readily measured in an assay with a quantitative output, for example an enzymatic assay or a signaling assay or a binding assay producing a quantifiable signal, a functionally active fragment or variant of the peptide, polypeptide or protein may produce a signal which is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal produced by the corresponding protein.

In the present specification, references to any peptides, polypeptides, proteins, or nucleic acids denote the respective peptides, polypeptides, proteins, or nucleic acids as commonly known under the respective designations in the art. More particularly, the references to "Wnt" and in particular to "Wnt7", to "G-protein coupled receptor 124" (GPR124), "Reversion-inducing cysteine-rich protein with Kazal motifs" (RECK), "Frizzled" (FZD), or "lipoprotein receptor-related protein" (LRP) denote the respective peptides, polypeptides, proteins or nucleic acids, as apparent from the context, as commonly known under said designations in the art.

"Wnt7" in the present context refers to both "Wnt7a" and/or "Wnt7b", which are part of the Wnt-family of proteins. The terms "Wnt7" and "WNT7" are used interchangeably throughout this specification. "Wnt" or "Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Inti" refers to a family of genes and proteins comprising the homologous "Wingless" or "Wg" and "Integration site" or "Int". In a particular embodiment, Wnt is Wnt7a protein with amino acid sequence as depicted in SEQ ID N° : 1. In another particular embodiment, Wnt is Wnt7b protein with amino acid sequence as depicted in SEQ ID N° : 2.

A "Gprl24/RECK/FZD/LRP receptor complex", "Gprl24/RECK/FZD/LRP co-receptor complex", "Gprl24/RECK/FZD/LRP complex" or "Gprl24/RECK/FZD/LRP protein complex" broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one Gprl24 protein, at least one RECK protein, at least one FZD protein and at least one LRP protein. A GPR124/RECK/FZD/LRP receptor complex,

RECTIFIED SHEET (RULE 91) ISA/EP when located at the plasma membrane of a cell, is capable of activating Wnt/p- catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.

A "FZD/LRP receptor complex", "FZD/LRP co-receptor complex", "FZD/LRP complex" or "FZD/LRP protein complex" broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane associated protein complex comprising at least one FZD protein and at least one LRP protein. A FZD/LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt/p-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.

"Wnt signaling" and "Wnt/ 0-catenin signaling" are used interchangeably herein.

"Frizzled", "Fz", "FZ", "Fzd" or "FZD" as used herein denotes a family of at least ten atypical G protein-coupled receptors (GPCR) that serve as receptors in the Wnt signaling pathway and other signaling pathways. When activated, Frizzled leads to activation of Dishevelled in the cytosol. In certain particularly preferred embodiments, the terms denote Fzdl, Fzd4, Fzd5 and/or Fzd8.

In a particular embodiment, Fzd is Fzdl, wherein Fzdl is the protein with amino acid sequence as depicted in SEQ ID N° : 9.

"LRP" or "lipoprotein receptor-related protein" encompasses any and all lipoprotein receptor-related proteins, also known in the art as low-density lipoprotein receptor- related proteins or prolow-density lipoprotein receptor-related proteins. In certain particularly preferred embodiments, the terms denote LRP5, LRP6, or LRP5 and LRP6 (LRP5/6). In a particular embodiment, LRP is LRP5 with amino acid sequence as depicted in SEQ ID N°: 3. In another particular embodiment, LRP is LRP6 with amino acid sequence as depicted in SEQ ID N° : 4 or 5. As both LRP5 and LRP6 may exist as multiple isoforms, a person skilled in the art will understand that these isoforms are also understood as comprised herein.

The interaction between Wnt ligands, Frizzled receptors, and LRP is essential for the activation of the canonical Wnt pathway, which has wide-ranging effects on cell proliferation, differentiation, and tissue development. This binding event triggers a series of intracellular signaling events that ultimately lead to the stabilization and nuclear translocation of a protein called p-catenin. In the nucleus, p-catenin functions as a transcriptional co-activator, regulating the expression of target genes involved in various cellular processes.

RECTIFIED SHEET (RULE 91) ISA/EP "Reck", or "RECK" is a GPI-anchored membrane protein that stands for "Reversioninducing Cysteine-rich Protein with Kazal Motifs". In a particular embodiment, Reck is the protein with amino acid sequence as depicted in SEQ ID N°: 6.

"Gprl24" or "GPR124" is an orphan GPCR expressed in endothelial cells. Gprl24 is also named ADGRA2 or adhesion G protein-coupled receptor A2. In a particular embodiment, Gprl24 is the protein with amino acid sequence as depicted in SEQ ID N°: 7 or 8.

The terms "bind", "interact", "specifically bind" or "specifically interact" as used throughout this specification mean that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about lxl0⁴-fold or more greater, or at least about lxl0⁵-fold or more greater, than its affinity for a nontarget molecule.

The term "gene therapy" and/or "RNA therapy" as used herein refers to the introduction of an exogenous polynucleotide into a host cell for therapeutic or prophylactic purposes, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid- based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art. The RNA molecule may be any type of RNA molecule. For instance, the RNA molecule may be

RECTIFIED SHEET (RULE 91) ISA/EP cytoplasmic RI A, a nuclear RNA, an mRNA, antisense RNA or a noncoding RNA, preferably an mRNA.

The terms "host cell" and "host organism" may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E. coli, Salmonella typhimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tabacum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.

Except when noted, the terms "subject" or "patient" can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly a neurological disorder, neurovascular disorder or a central nervous system (CNS) disorder comprising neurovascular dysfunction; or to treat blood-retina barrier (BRB) integrity, or the treatment of an ophthalmological disease or disorder, such as a retinal vascular disorder or retinal degenerative disorder. Such subjects may include, without limitation, those that have been diagnosed with said condition, those prone to develop said condition and/or those in who said condition is to be prevented.

The term "therapeutically active/effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term "prophylactically active/effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. In

RECTIFIED SHEET (RULE 91) ISA/EP present context, the "therapeutically active amount" is used to refer to both the "therapeutically active amount" and "prophylactically active amount" as described above, unless the distinction is clear from the context. Methods are known in the art for determining therapeutically and/or prophylactically effective doses of a compound, a protein, a nucleic acid encoding the compound/protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein. The term "therapeutically effective dose" as used herein refers to an amount of a compound, a protein, a nucleic acid encoding the compound/protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein, that when administered brings about a positive therapeutic response with respect to treatment of a patient having a specific disease or disorder.

Detailed description

The inventors demonstrate herein the design of agonists capable of activating Wnt7 signaling selectively in cells expressing RECK and/or GPR124; for instance cells expressing GPR124, or cells expressing GP124 and RECK i.e. for instance in cerebral endothelial cells. It is further disclosed that such agonists are useful as therapeutics, such as particularly for the treatment of neurological disorders, neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction, or an ophthalmological disease or disorder in particular a retinal vascular disorder or retinal degenerative disorder. Hence, the invention allows providing inter alia agonists capable of stimulating Wnt/p-catenin signaling in cerebral endothelial cells (also referred to as "on-target activity") with only low cross-reactivity with other Frizzled pathways (i.e. "unwanted" or "off-target activity"), and useful as therapeutics, particularly for neurological disorders, neurovascular disorders or central nervous system (CNS) disorders comprising neurovascular dysfunction.

The inventors surprisingly discovered that the direct recruitment or binding of the agonist to LRP, such as LRP5 or LRP6, and at least Gprl24 or RECK is sufficient for stimulating Wnt7 signaling specifically through the Gprl24/RECK/FZD/LRP receptor complex.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be

RECTIFIED SHEET (RULE 91) ISA/EP combined with any other feature or features indicated as being preferred or advantageous.

Accordingly, in a first aspect, the invention provides an agonist of the Wnt7 signaling pathway, more particularly of the Wnt7 signaling pathway mediated by the Gprl24/RECK/Frizzled/LRP receptor complex, even more particularly of the Wnt7 signaling pathway mediated by the Gprl24/RECK/Frizzled/LRP (LRP5 or LRP6) receptor complex.

In one embodiment, said agonist is a selective or specific agonist of the Gprl24- and Reck-dependent Wnt/0-catenin pathway. The term "selective agonist of the Gprl24- and Reck-dependent Wnt/0-catenin pathway" or "specific agonist of the Gprl24- and Reck-dependent Wnt/0-catenin pathway" is to be understood as an agonist of said pathway that is able to activate said pathway with high selectivity and specificity (on-target activity), with no, no statistically significantly or only minimal activation of other Wnt signaling pathways (off-target activity). In embodiments, the agonist disclosed herein is an agonist of Gprl24- and Reck-dependent Wnt/0-catenin pathway that shows less than 20% off-target activity, more particularly less than 15%, 10%, 5% or 1% off-target activity, as measured in cell culture Super TOP- Flash assays (as known it the art and discussed in the example section below).

In embodiments, the agonist as disclosed herein is an agonist of the GPR124/RECK/Frizzled/LRP complex, wherein said agonist is capable of binding to LRP such as LRP5 or LRP6 and the GPR124 protein or to LRP such as LRP5 or LRP6 and the RECK protein. In embodiments, said agonist is not able to or has an impaired capacity to bind to or activate Frizzled.

In an embodiment, said agonist is not a Wnt7 protein or polypeptide, such as a Wnt7a or Wnt7b protein or polypeptide, or a mere variant thereof having one or more mutations in its sequence such as a mere variant having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mutations.

In certain embodiments, one or more and preferably all of Wnt7, GPR124, RECK, FZD and LRP as employed herein is or are of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.

RECTIFIED SHEET (RULE 91) ISA/EP A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be precursors of the respective peptides, polypeptides, proteins, or nucleic acids and may include parts that are processed away from mature molecules; or may represent sequences that are of mature molecules while also the precursor sequences are considered part of the present concept.

The terms encompass the peptides, polypeptides, proteins, or nucleic acids when forming a part of a living organism, organ, tissue, or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass the peptides, polypeptides, proteins, or nucleic acids when produced by recombinant or synthetic means.

Unless otherwise apparent from the context, reference herein to any peptide, polypeptide, protein, or nucleic acid also encompasses modified forms of said peptide, polypeptide, protein, or nucleic acid, such as forms bearing post-expression modifications including, for example, phosphorylation, glycosylation, palmitoylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, ubiquitination, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, and the like. A broader definition is given above.

The agonists as disclosed herein may have any of a variety of different structural formats or configurations. Said agonists may comprise polypeptides and/or nonpolypeptide binding moieties, e.g., small molecules. In particular embodiments, said agonists may comprise both a polypeptide region and a non-polypeptide binding moiety. In certain embodiments, said agonists may comprise a single polypeptide, or they may comprise two or more, three or more, or four or more polypeptides. In certain embodiments, said agonists may comprise one, two, three, or four polypeptides, e.g., linked or bound to each other or fused to each other.

When the agonists comprise a single polypeptide, they may be a fusion protein comprising one or more LRP binding regions or domains such as LRP5/6 binding regions or domains and one or more RECK or GPR124 binding domains. The binding regions may be directly fused or they may be connected via a linker, e.g., a polypeptide or chemical linker, including but not limited to any of those disclosed herein.

RECTIFIED SHEET (RULE 91) ISA/EP When the agonists comprise two or more polypeptides, the polypeptides may be linked via covalent bonds, such as, e.g., disulfide bonds, and/or noncovalent interactions. For example, heavy chains of human immunoglobulin IgG interact at the level of their CFI3 domains directly, whereas, at the level of their CFI2 domains, they interact via the carbohydrate attached to the asparagine (Asn) N84.4 in the DE turn.

In an embodiment, the agonists as described herein may be engineered.

In another or further embodiment, the agonists as described herein may be water soluble. By "water soluble", or short "soluble" it is meant a composition that is soluble in aqueous buffers in the absence of detergent, usually soluble at a concentration that provides a biologically effective dose of the polypeptide. The agonists disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of at least 25 pM and higher, e.g. at least 25 pM, 40 pM, or 50 pM, usually at least 60 pM, 70 pM, 80 pM, or 90 pM, sometimes as much as 100 pM, 120 pM, or 150 pM. In other words, the agonists disclosed herein typically form a substantially homogeneous aqueous solution at concentrations of about 2.5 mg/ml, about 5.0 mg/ml, about 10.0 mg/ml, about 15.0 mg/ml, about 20.0 mg/ml, about 25.0 mg/ml, about 30.0 mg/ml, or about 35.0 mg/ml or more.

The agonists as described herein may be engineered to facilitate binding between two polypeptides. For example, knobs-into-holes amino acid modifications may be introduced into two different polypeptides to facilitate their binding. Knobs- into- holes amino acid (AA) changes is a rational design strategy developed in antibody engineering, used for heterodimerization of the heavy chains, in the production of bispecific IgG antibodies. AA changes are engineered in order to create a knob on the CFI3 of the heavy chains from a first antibody and a hole on the CFI3 of the heavy chains of a second antibody. The knob may be represented by a tyrosine (Y) that belongs to the 'very large' IMGT volume class of AA, whereas the hole may be represented by a threonine (T) that belongs to the 'small' IMGT volume class. Other means of introducing modifications into polypeptides to facilitate their binding are known and available in the art. For example, specific amino acids may be introduced and used for cross-linking, such as Cysteine to form an intermolecular disulfide bond. In particular embodiments, the agonists comprise one or more binding regions derived from an antibody or antigen-binding fragment thereof, e.g., antibody heavy chains or antibody light chains or fragments thereof.

RECTIFIED SHEET (RULE 91) ISA/EP In an embodiment, the agonist as disclosed herein is capable of simultaneously binding to LRP proteins, more particularly LRP5 or LRP6, in addition to the GPR124 or the RECK proteins.

In an embodiment, the agonist of the Wnt7 signaling pathway comprises one or more LRP binding domains and one or more Gprl24 or Reck binding domains. In a further embodiment, said agonist comprises one, two, three, four, or five LRP binding domains, and one, two, three, four, or five Gprl24 or Reck binding domains. In a preferred embodiment, agonist comprises one or two LRP binding domains, and one or two Gprl24 or Reck binding domains, more preferably two LRP binding domains, and two Gprl24 or Reck binding domains.

In an embodiment, the LRP binding domains or binding regions are LRP5 and/or LRP6 binding domains or binding regions, and/or said LRP binding domains or binding regions are able to bind to LRP5 and/or LRP6.

In an embodiment, the agonist as described herein may be tetravalent or bivalent.

In an embodiment, the agonist as described herein is tetravalent.

In a particular embodiment, said agonist comprises: i) two regions that each specifically bind to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and/or LRP6 epitopes; and ii) two regions that each specifically bind to a set of one or more Reck or Gprl24 epitopes.

In an embodiment, each one or more LRP binding domain such as LRP5/6 binding domain is fused in tandem to either one or more Reck binding domains and/or one or more Gprl24 binding domains, and wherein said domains are optionally separated by a linker sequence.

Said linkers may be any linker known in the art, or described in this text. As a nonlimiting example, said linker is a 5-mer linker such as GSGGS (SEQ ID N° : 172).

In another embodiment, the agonist as described herein is bivalent.

In a particular embodiment, said agonist comprises: i) one region that specifically binds to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and/or LRP6 epitopes; and

RECTIFIED SHEET (RULE 91) ISA/EP ii) one region that specifically binds to a set of one or more Reck or Gprl24 epitopes.

In an embodiment, the LRP binding domain, such as LRP5/6 binding domain, is fused in tandem to a Reck or Gprl24 binding domain, and wherein said domains are optionally separated by a linker sequence.

Said linkers may be any linker known in the art, or described in this text. As a nonlimiting example, said linker is a 5-mer linker such as GSGGS (SEQ ID N°: 172).

In a particular embodiment, said agonist does not comprise a Fzd binding domain. According to particular embodiments, the agonist disclosed herein is not able to bind FZD.

In other or further embodiments, the agonist as disclosed herein cannot stimulate the Wnt7 signaling pathway in cells which are GRP124/RECK negative. In further embodiments, GRP124/RECK negative means having no or low expression of GPR124 and/or RECK proteins, no or low expression of GPR124 and/or RECK in a cell, no or low presence of GPR124 and/or RECK proteins in a cell or no or low presence of GPR124 and/or RECK proteins at a cell surface.

In other embodiments, GRP124/RECK positive then refers to the expression of GPR124 and/or RECK in a cell, presence of GPR124 and/or RECK proteins in a cell and/or presence of GPR124 and/or RECK proteins at a cell surface.

In other or further embodiments, FZD/LRP positive can mean the expression of FZD and/or LRP in a cell, presence of FZD and/or LRP protein in a cell, and/or presence of FZD and/or LRP proteins at a cell surface.

In particular embodiments, cells expressing GPR124, RECK, FZD and LRP proteins, more particularly LRP5 or LRP6, at its plasma membrane, are cells naturally expressing all GPR124, RECK, FZD and LRP proteins at the cell surface, such as a cerebral endothelial cell.

In some embodiments "the presence of RECK and GPR124" may refer to the presence of the RECK and GPR124 proteins at or close to the cell membrane, preferably in close proximity with the Frizzled and LRP proteins, more particularly LRP5 or LRP6. The close mutual proximity can facilitate the formation of the GPR124/RECK/Frizzled/LRP receptor complex under conditions conducive thereto,

RECTIFIED SHEET (RULE 91) ISA/EP such as when the agonist as taught herein is externally supplied to a cell. In other or further embodiments, when used in gene or RINA therapy, the agonist as taught herein could be expressed from the target cell population. On the other hand, the phrase "the absence of RECK and/or GPR124" may refer to the lack or nonoccurrence of the RECK and/or GPR124 proteins at the cell membrane. The absence of RECK and/or GPR124 at or close to the cell membrane may occur when a cell does not express, translate, or correctly translocate RECK and/or GPR124. The absence of RECK and/or GPR124 need not denote the complete absence of the RECK and/or GPR124 protein at the cell membrane, but may for example refer to an amount of RECK and/or GPR124 protein which is not detectable by, or falls below the sensitivity range of, conventional protein detection or quantification assays known by the person skilled in the art, such as for example immunoblotting, immunocytochemistry or immunofluorescence.

In other or further embodiments, said agonist herein disclosed is capable of activating the GPR124/RECK/Frizzled/LRP-mediated Wnt7 signaling, wherein said agonist does not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124.

In particular embodiments, the capability of the agonist herein disclosed to activate GPR124/RECK/Frizzled/LRP-mediated Wnt7 signaling, but not activate Frizzled/LRP- mediated Wnt signaling in the absence of RECK and/or GPR124, denotes the capability of the agonist to activate Wnt7 signaling in cells positive for GPR124, RECK, FZD and LRP, more particularly LRP5 or LRP6, but not in cells positive for FZD and LRP and negative for GPR124 and/or RECK, wherein the cells positive for GPR124, RECK, FZD and LRP, more particularly LRP5 or LRP6, and the cells positive for FZD and LRP and negative for GPR124 and/or RECK are otherwise substantially identical.

In particular embodiments, the capability of the agonist to activate GPR124/RECK/Frizzled/LRP-mediated Wnt7 signaling, but not activate the canonical Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124, denotes the capability of said agonist to activate the atypical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and/or GPR124.

The capability of activating Wnt7 signaling refers to the ability of the agonist as disclosed herein to mimic, reproduce or approximate the signal transduction effect and/or activity of a natural Wnt7 ligand binding to a Gprl24/RECK/FZD/LRP complex.

RECTIFIED SHEET (RULE 91) ISA/EP Activation of Wnt7 signaling may be suitably determined and/or quantitated by measuring the expression of one or more Wnt7 target genes, TCF reporter gene expression, beta-catenin stabilization, LRP phosphorylation, and/or translocation of Axin from cytoplasm to cell membrane as known in the art. For instance, activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of TCF gene (e.g., by RT-PCR or any other transcript detection method), a primary output of Wnt signaling. For example, a TCF reporter assay (also known as TOP/FOP or TOP flash) may be used to assess changes in the transcription of TCF/LEF controlled genes. The TCF reporter assay may be a luciferase reporter assay. Further for example, activation of Wnt signaling may be suitably determined and/or quantitated by measuring the expression of c-myc, n-myc, LEF1, or c-jun. Alternatively, activation of Wnt signaling may be determined by measuring the location, level and/or phosphorylation status of 0-catenin. A non-limiting example of such an assay is the "0-Catenin Redistribution Assay” (Thermo Scientific) which provides recombinant U20S cells stably expressing human 0-catenin fused to the C- terminus of enhanced green fluorescent protein (EGFP). The assay allows visualization and monitoring of the translocation of a GFP-p-catenin fusion protein from the membrane to the nucleus. Another way of determining activation of Wnt signaling is the visualization of Axin translocation, for example with a GFP-Axin fusion protein.

In particular embodiments, the agonist as disclosed herein may be considered capable of activating the central nervous system (CNS) Wnt7 signaling or the Gprl24/RECK/FZD/LRP5 or LRP6 dependent Wnt7 signaling, if the agonist enhances Wnt/p-catenin signaling at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 100-fold more, at least 250-fold more, at least 500-fold more, at least 750-fold more, at least 1000-fold more, at least lxl0⁴-fold more, or at least lxl0⁵-fold more compared to Wnt/p- catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

In particular embodiments, the agonist as disclosed herein may be considered to not activate (canonical) Wnt signaling if the compound enhances Wnt/0-catenin signaling less than 10-fold more, such as particularly at most 5-fold more or at most 2.5-fold more, or if the protein does not enhance or even reduces (e.g., 2-fold less or 5-fold less or 10-fold less) Wnt/0-catenin signaling compared to Wnt/ 0-catenin

RECTIFIED SHEET (RULE 91) ISA/EP signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

In particular embodiments, the agonist as disclosed herein may be considered to activate the GPR124/RECK/Frizzled/LRP5 or LRP6-mediated Wnt7 signaling (denoted "on-target activity"), but not activate Frizzled/LRP-mediated Wnt signaling in the absence of RECK and/or GPR124 (denoted "off-target activity"), if the on-target signaling activity is induced at least 30% and off-target activity is induced less than 20% by said agonist. In embodiments, said agonist can activate GPR124/RECK/Frizzled/LRP5 or LRP6-mediated Wnt7 signaling more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein, preferably more than 70% compared to a control situation, for example in the absence of said agonist.

In embodiments, said agonist can activate Frizzled/LRP-mediated Wnt in the absence of RECK and/or GPR124 less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein, preferable less than 10% compared to a control situation, for example in the presence of Wnt7. Before comparing the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity (denoted "on-target activity") and the Frizzled/LRP-mediated Wnt signaling activity in the absence of RECK and/or GPR124 (denoted "off-target activity" in this paragraph), on-target activity and off-target activity induced by the agonist may be normalized to on-target activity induced by wild-type Wnt7a or b, the latter for example set to represent 100% activity.

In one embodiment, the agonist of the present invention acts by Wnt7-specific RECK/GPR124/Frizzled/LRP5 or LRP6-mediated signaling, in which said agonist binds specifically to LRP5 or LRP6 and RECK in a FZD- and Gprl24-independent manner. The agonist thereby recruits Gprl24 via RECK to the FZD/LRP complex, thereby assembling the Wnt7-ligand specific RECK/GPR124/FZD/LRP signalosomes and activating the canonical Wnt7 signaling. According to further embodiments, the agonist of the disclosed invention thus induces the heteromerization of RECK and LRP5 or LRP6 proteins in the presence of Frizzled and GPR124.

In another embodiment, the agonist of the present invention acts by Wnt7-specific RECK/GPR124/Frizzled/LRP-mediated signaling, in which said agonist binds

RECTIFIED SHEET (RULE 91) ISA/EP specifically to LR.P5 or LR.P6 and Gprl24 in a RECK- and FZD-independent manner. The agonist thereby recruits RECK via Gprl24 to the FZD/LRP complex, thereby assembling the Wnt7-ligand specific RECK/GPR124/FZD/LRP signalosomes and activating the canonical Wnt7 signaling. According to further embodiments, the agonist of the disclosed invention thus induces the heteromerization of Gprl24 and LRP5 or LRP6 proteins in the presence of Frizzled and RECK.

Accordingly, in particular embodiments, the agonist as disclosed herein is able to bind to LRP5 and/or LRP6 proteins and a protein selected from Gprl24 and RECK, preferably at a cell membrane.

The skilled person shall further appreciate that a GPR124/RECK/FZD/LRP receptor complex, as envisaged herein, may include further component(s), which may or need not functionally modulate the complex. For example, the complex may include Dishevelled (Dvl), forming intracellular scaffolds capable of bridging GPR124 and Frizzled.

In particular embodiments, the agonist of the invention as disclosed herein induces or has a Wnt signaling activity of at least 35%, preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124/RECK/Frizzled/LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In preferred embodiments, the agonist as disclosed herein has at least 70% of the GPR124/RECK/Frizzled/LRP- mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In particular embodiments, the agonist of the invention as disclosed herein may activate GPR124/RECK/Frizzled/LRP-mediated Wnt signaling in the presence of all four co-receptors more than 30% of the activation by full-length wild-type Wnt7 protein. For example, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein.

In particular embodiments, the agonist of the invention as disclosed herein may activate Wnt signaling in the absence of one of the four co-receptors (i.e. RECK, Fzd, LRP or GPR124) less than 20% of the activation by full-length wild-type Wnt7 protein. For example, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein.

RECTIFIED SHEET (RULE 91) ISA/EP In one embodiment, the agonist of the invention is a binding agent comprising one or more LRP binding domains such as LRP5 or LRP6 binding domains and one or more Gprl24 or Reck binding domains. In a further embodiment, the binding agent is a fusion protein comprising a first polypeptide that can bind at least one LRP protein such as LRP5 or LRP6 protein and a second polypeptide that can bind at least one Gprl24 or Reck protein.

In an embodiment, the binding domains or polypeptides may be separated from each other via a linker or spacer sequence, such as the flexible GS or (Gly)n linker. Other suitable linker sequences will be known to the person skilled in the art.

In embodiments, the agonist disclosed herein can bind to the LRP protein, particularly to LRP5 or LRP6, for example to the extracellular domain of the LRP protein, to the DKK-binding site and/or the Wnt-binding site of the LRP protein. In more particular embodiments, the agonist disclosed herein is capable of binding to the DKKl-binding site of the LRP5 and/or LRP6 protein. In other particular embodiments the agonist as disclosed herein is capable of binding to ^-propeller— EGF-like domains 1 and 2 (P1E1P2E2) and/or p-propeller-EGF-like domains 3 and 4(P3E3P4E4) of the LRP protein, more particularly of the LRP5 and/or LRP6 protein.

In particular embodiments, the agonist as disclosed herein may be selective for the LRP5 or LRP6 proteins, for example having a specificity for LRP5 and/or LRP6 of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl0⁴-fold, or at least lxl0⁵-fold, compared to other nonpreferred LRP proteins.

In various embodiments, the binding agent of the application comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on LRP such as on LRP5 and/or LRP6. In an embodiment, the antigen-recognition domain recognizes one or more linear epitopes present on LRP such as LRP5 and/or LRP6. As used herein, a linear epitope refers to any continuous sequence of amino acids present on LRP such as LRP5 and/or LRP6. In another embodiment, the antigen-recognition domain recognizes one or more conformational epitopes present on LRP such as LRP5 and/or LRP6. As used herein, a conformation epitope refers to one or more sections of amino acids (which may be discontinuous) which form a three-dimensional surface with features and/or shapes and/or tertiary structures capable of being recognized by an antigen recognition domain.

RECTIFIED SHEET (RULE 91) ISA/EP In a particular further embodiment, the one or more LRP binding domains or targeting moieties, such as one or more LR.P5 and/or one or more LR.P6 binding domains or targeting moieties, bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the LRP5 or LRP6 proteins as depicted in SEQ ID N°: 3, 4 and 5. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids.

In a further particular embodiment, said fragment of the LRP5 protein is the amino acid sequence between position 32 and 614 or between position 644 and 1254 of SEQ ID N° : 3. In another further particular embodiment, said fragment of the LRP6 protein is the amino acid sequence between position 20 and 628 or between position 631 and 1244 of SEQ ID N°: 4.

In some embodiments, the one or more LRP5 and/or LRP6 binding domains comprise one or more antigen-binding fragments of an antibody. In some embodiments, the one or more antigen-binding fragments are selected from the group consisting of: IgG, scFv, Fab, and VHH or sdAbs. In some embodiments, the one or more antigenbinding fragments are humanized.

In other embodiments, the binding agent of the application further comprises a targeting moiety having an antigen recognition domain that recognizes an epitope present on Reck or Gprl24. In an embodiment, the antigen-recognition domain recognizes one or more linear epitopes or one or more conformational epitopes present on Reck or Gprl24.

In an embodiment, the agonist herein disclosed can bind to RECK proteins involved in Wnt signaling. RECK is composed of five N-terminal cysteine-knot (CK) motifs or regions (i.e. CK1, CK2, CK3, CK4 and CK5), a cysteine-rich domain (CRD) and three Kazal motifs preceding a Glycosylphosphatidylinositol (GPI)-anchor site. The CK motifs, the CRD and the Kazal motifs are located extracellularly. Accordingly, in embodiments, the agonist herein disclosed capable of binding to the RECK protein may bind the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and/or one or more of the Kazal motifs of the RECK protein, preferably said agonist binds to the CK4 and/or CK5 regions of the RECK protein.

Accordingly, in particular embodiments, the agent as disclosed herein is capable of binding to the CK4 and/or CK5 regions of RECK polypeptide. The CK4 motif spans

RECTIFIED SHEET (RULE 91) ISA/EP from amino acid C216 to C263, and the CK5 motif spans from amino acid C292 to C338 of the amino acid sequence of the human RECK protein annotated under NCBI Genbank accession number NP 066934.1 as disclosed elsewhere herein. Accordingly, the CK4 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence

CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID N°: 49) and the CK5 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence

CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC (SEQ ID N° : 50). In particular embodiments, the agent as disclosed herein is capable of binding to the amino acid sequence of SEQ ID N° : 49 and/or the amino acid sequence of SEQ ID N°: 50.

In another or further particular embodiment, the one or more Reck binding domains or targeting moieties bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the Reck protein as depicted in SEQ ID N° : 6. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In another further particular embodiment, said fragment of the Reck protein is the amino acid sequence between position 23 and 789 or between position 37 and 338 or between position 627 and 673 or between position 698 and 752 or between position 753 and 789 of SEQ ID N°: 6.

In an embodiment, said RECK-binding domain comprises a RECK specific antibody or comprises the RECK-binding region, preferably the variable region sequence or CDRs, of one or more RECK specific antibody or antibodies. Preferably, the RECK specific antibody is directed to the extracellular portion of the RECK polypeptide, such as directed to the CK4 and/or CK5 region(s) of the RECK polypeptide. Nonlimiting examples of RECK antibodies include antibodies which are known in the art and commercially available, such as from Abeam (e.g. ab88249 and ab899l5 specific for human full-length RECK), Cell Signaling Technology (e.g. #3433 specific for human, mouse, rat, monkey RECK (D8C7)), Santa Cruz (e.g. sc-373929 specific for C-terminus of human RECK).

In particular embodiments, the RECK-binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at

RECTIFIED SHEET (RULE 91) ISA/EP least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a RECK specific antibody.

In particular embodiments, the RECK-binding domain is RECK specific scFv, such as scFv comprising one or more (preferably all 6) CDR(s) of a RECK specific antibody.

In particular embodiments, the RECK-binding domain is RECK specific VHH , such as VHH comprising one or more (preferably all 3) CDR(s) of a RECK specific heavy chain antibody.

In particular embodiments, the RECK-binding domain of the agent as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different RECK specific antibodies. In particular embodiments, said RECK-binding domain of the agent as disclosed herein comprises, consists essentially of, consists of or is derived from (e.g., is a biologically active fragment and/or variant of) a RECK-binding polypeptide, such as a Wnt ligand or Wnt polypeptide. Preferably, the RECK-binding domain is derived from a Wnt7 polypeptide (e.g., Wnt7a or Wnt7b) as described elsewhere herein, more preferably from a human or murine Wnt7 polypeptide, even more preferably from a human Wnt7 polypeptide (e.g., human Wnt7a or human Wnt7b). For example, in certain embodiments, the RECK-binding domain may comprise, consist essentially of or consist of a RECK-binding fragment of Wnt7 (e.g., Wnt7a or Wnt7b), preferably of human or mouse Wnt7, more preferably of human Wnt7 (e.g., human Wnt7a or human Wnt7b) or variant thereof In a further example, in certain embodiments, the RECK-binding domain may comprise, consist essentially of or consist of an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to the amino acid sequence VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID N°: 46) or VEVVRASRLRQPTFLRLKQLRSYQKPMET (SEQ ID N°: 48).

In particular embodiments, said RECK-binding domain is derived from a Wnt7 polypeptide. In particular embodiments, said RECK-binding domain comprises, consists essentially of or consists of an amino acid sequence as set forth in HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID N° : 51),

VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID N°: 46) or

RECTIFIED SHEET (RULE 91) ISA/EP YEWRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID N°: 52), or an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the amino acid sequence SEQ ID N°: 51, SEQ ID N°: 46; or SEQ ID N° : 52. In particular embodiments, said RECK-binding domain derived from a Wnt7 polypeptide comprises, consists essentially of or consists of the amino acid sequence SEQ ID N°: 51, SEQ ID N° : 46; or SEQ ID N° : 52.

In particular embodiments, said RECK-binding domain comprises, consists essentially of, or consists of the amino acid sequence

XXXVXAXRXXXXXFLXIXXXXXYXKXXXX (SEQ ID N° : 54),

VXAXRXXXXXFLXIXXXXXYXK (SEQ ID N°: 55),

XXXVXAXRXXXXXFLXXXXXXXXXKXXXX (SEQ ID N° : 56) or

VXAXRXXXXXFLXXXXXXXXXK (SEQ ID N° : 57), wherein X is any amino acid, preferably wherein the amino acid sequence shows at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to any one of SEQ ID N° : 51, 52 or SEQ ID N° : 46.

In an embodiment, said Reck binding domain is chosen from the group of the RECK binding domain of the protein ADAMTS10, eg as depicted in SEQ ID N°: 45 (AA 823- 1103 of hADAMTSlO) or a part thereof; the Wnt7a linker region as depicted in SEQ ID N°: 46 or a multitude of said linker regions (eg 3 times the Wnt7a linker); or the extracellular domain of Gprl24 (or part thereof). In an embodiment, said RECK binding domain is derived from the N-terminal extracellular domain of Gprl24, having a sequence showing at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to SEQ ID N°: 58.

In an embodiment, said Reck binding domain may comprise, consist essentially of or consist of an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to the amino acid sequence as given in SEQ ID N°: 45 or SEQ ID N° : 58.

RECTIFIED SHEET (RULE 91) ISA/EP In another or further particular embodiment, the one or more Gprl24 binding domains bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the Gprl24 protein as depicted in SEQ ID N°: 7 and 8. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In another further particular embodiment, said fragment of the Gprl24 protein is the amino acid sequence between position 34 and 344 or between position 82 and 106 or between position 107 and 130 or between position 131 and 154 or between position 156 and 178 or between position 190 and 240 or between position 247 and 344 of SEQ ID N°: 7.

In another or further embodiment, the binding agent of the application comprises a full-length multimeric protein that includes two heavy chains and two light chains. Each heavy chain includes one variable region (e.g. VH) and at least three constant regions (e.g. CHI, CH2 and CH3), and each light chain includes one variable region (VL) and one constant region (CL). As described above in the definitions section, the variable regions determine the specificity of the antibody and comprise three hypervariable regions also known as complementarity-determining regions (CDRs) that contribute to the antibody binding specificity.

In an embodiment, said Gprl24 binding domain is derived from RECK, preferably the human RECK sequence. The N-terminal part of RECK was reported to bind to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gprl24. In an embodiment, said Gprl24 binding domain comprises one or more Cysteine-Knot (CK) domains derived from RECK, preferably human RECK. In an embodiment, said binding agent comprises CK1 to CK5 of RECK, preferably human RECK (see SEQ ID N°: 47). In an embodiment, said GPR124 binding domain comprises a portion of CK1-5 of RECK, preferably hRECK. In an embodiment, said Gprl24 binding domain may comprise, consist essentially of or consist of an amino acid sequence having at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity to the amino acid sequence as given in SEQ ID N°: 47.

In some embodiments, the binding agent herein disclosed comprises a LRP targeting moiety such as a LRP5 or LRP6 targeting moiety and a Gprl24 or Reck targeting moiety, wherein at least one targeting moiety is an antibody fragment. The term

RECTIFIED SHEET (RULE 91) ISA/EP "antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has a high affinity for an antigenic determinant or epitope, and contains one or more CDRs accounting for such specificity. In some particular embodiments, at least one of the targeting moieties of the binding agent of the application is a single-domain antibody, an immunoglobulin single variable domain, a heavy-chain- only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody, an Affimer, a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a peptide aptamer, an alterases, a plastic antibodies, a phylomer, a stradobodies, a maxibodies, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, as described in US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446, and/or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also Storz 2011 MAbs 3: 310-317.

In particular embodiments, at least one of the targeting moieties of the binding agent of the application is a single-domain antibody, such as a VHH. The VHH may be derived from, for example, an organism that produces VHH antibodies such as a camelid, a shark, or the VHH may be a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies (see definition section above). In some embodiments, the single domain antibody as described herein is an immunoglobulin single variable domain or ISVD. In most particular embodiments, at least one of the targeting moieties of the binding agent is a VHH. In further most particular embodiments, said VHH comprises a single amino acid chain having four "framework regions" and three "complementary determining regions" or CDRs. As used herein, "framework region" refers to a region in the variable domain which is located between the CDRs. As used herein, "complementary determining region" or "CDR" refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets. In various embodiments, the binding agent of the application comprises a VHH or ISVD targeting LRP5 or LRP6 having a variable domain comprising at least one CDR1, CDR2, and/or CDR3 sequence. The CDR3 sequence represents an essential feature of a family of ISVDs, more

RECTIFIED SHEET (RULE 91) ISA/EP particularly VHHs. An ISVD family is defined herein as a group of ISVD amino acid sequences with high similarity, or even identical, in the CDR.3 sequence. By default, ISVDs belong to the same family when binding to the same target epitope. Variations in an ISVD family may be interesting if expression/stability/affinity/crystallization of a representative of that family is poor, as small deviations like single amino acid mutations occurring within one family may improve these properties.

In a specific embodiment, the ISVD or VHH targeting LR.P5 comprises a CDR3 region selected from the list consisting of SEQ ID N°: 38-41 or having an amino acid sequence with maximally two amino acids different to SEQ ID N°: 38-41 or with maximally one amino acid different to SEQ ID N°: 38-41.

In a further specific embodiment, said ISVD or VHH comprises a CDR.2 region as depicted in SEQ ID N°: 42 and/or a CDR1 region as depicted in SEQ ID N°: 43.

In a most specific embodiment, said anti-LRP5 ISVD or VHH comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID N° : 34, 35, 36, 37 or 53. In particular embodiments, said differences in amino acid sequence between said homologues and SEQ ID N°: 34, 35, 36, 37 or 53 are found in the framework regions.

In another specific embodiment, the ISVD or VHH targeting LRP6 and comprises a CDR3 as depicted in SEQ ID N°: 15, 24, 25, or 26 or having an amino acid sequence with maximally two amino acids different to SEQ ID N° : 15, 24, 25, or 26 or with maximally one amino acid different to SEQ ID N°: 15, 24, 25, or 26.

In a further specific embodiment, said ISVD or VHH comprises a CDR2 region as depicted in SEQ ID N°: 16, 27, 28 or 29 and/or a CDR1 region as depicted in SEQ ID N° : 17, 30, 31, 32 or 33.

In a most specific embodiment, said anti-LRP6 ISVD or VHH comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to any of SEQ ID N° : 14, 18-23.

In particular embodiments, said differences in amino acid sequence between said homologues and any of SEQ ID N° : 14, 18-23 are found in the framework regions.

RECTIFIED SHEET (RULE 91) ISA/EP In a further specific embodiment, the binding agent of the application targeting LR.P5 or LRP6 also comprises an ISVD or VHH targeting Reck.

In another further specific embodiment, the binding agent of the application targeting LRP such as LRP5 or LRP6 also comprises an ISVD or VHH targeting.

In one embodiment, the LRP binding domain, such as LRP5 or LRP6 binding domain, of the binding agent of the application or the first polypeptide that binds at least one LRP such as LRP5 or LRP6 of the fusion protein of the application comprises an antibody or any antibody fragment herein described such as an ISVD or a VHH, that binds LRP such as LRP5 or LRP6. In a particular embodiment, said antibody, antibody fragment, ISVD or VHH binding LRP6 comprises or consists of the amino acid sequence as depicted in SEQ ID N° : 14. In another particular embodiment, said antibody or antibody fragment binding LRP6 comprises or consists of the amino acid sequence as depicted in SEQ ID N° : 12 and/or 13.

In a further embodiment, the Gprl24 or Reck binding domain of the binding agent of the application or the second polypeptide that binds at least one Gprl24 or Reck of the fusion protein of the application comprises an antibody or any antibody fragment herein described such as an ISVD or a VHH, that binds Gprl24 or Reck.

In a further particular embodiment, the binding agent of the application is a bispecific antibody, antibody fragment or ISVD or VHH that preferably both targets LRP such as LRP5 or LRP6 and Gprl24 or Reck.

In another or further particular embodiment, said antibody, antibody fragment ISVD or VHH is tetravalent.

In a most particular embodiment, the binding agent of the application comprises a VHH targeting LRP6, wherein said VHH comprises or consists of the amino acid sequence as depicted in SEQ ID N° : 14.

As already described above, in an embodiment, agonist disclosed herein does not comprise a Fzd binding domain. In another or further embodiment, said agonist is not able to bind or not capable of binding to Fzd. This is particularly remarkable as it is not obvious based on what was previously known that such molecule, not capable of binding to Fzd and/or without Fzd binding domain and/or thus without

RECTIFIED SHEET (RULE 91) ISA/EP crosslinking said molecule with Fzd, can show agonist activity, i.e. can activate the Wnt/beta-catenin signaling pathway and thus can act as an activator of said signaling pathway. The inventors, however, were able to show that the agonist of the present invention, without Fzd binding domain and/or without the capability of binding Fzd, was able to activate said signaling pathway.

In an embodiment, the agonist does not comprise further binding domains over said one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

In an embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

In a preferred embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 binding domains. In another preferred embodiment, the binding domains of said agonist are selected from one or more LRP binding domains and one or more Reck binding domains.

In an embodiment, said agonist is a bispecific antibody with two binding domains. In a preferred embodiment, said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Gprl24 binding domain. In another preferred embodiment, said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Reck binding domain.

Similarly as described above in relation to said agonist not having a Fzd binding domain and/or not being able of binding Fzd, it could not be expected based on what was previously known that such molecule, having as binding domains only one or more LRP binding domains and one or more Gprl24 or Reck binding domains, or having only two binding domains chosen from an LRP binding domain and either a Reck binding domain or a Gprl24 binding domain, can show agonist activity, i.e. can activate the Wnt/beta-catenin signaling pathway and thus can act as an activator of said signaling pathway.

In another or further embodiment, the agonist described herein is capable of activating the Wnt/beta-catenin signaling pathway and thus can act as an activator of said signaling pathway in the absence of Reck.

In another or further embodiment, the agonist described herein comprises an anti- LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62.

RECTIFIED SHEET (RULE 91) ISA/EP

In another or further embodiment, the agonist described herein comprises an IgG comprising : - a VH sequence according to SEQ ID N° 63, 65, or 67, and

- a VL sequence according to SEQ ID N° 64, 66, or 68,

- the VH and VL optionally separated by a linker sequence.

Preferably, the agonist described herein comprises an IgG comprising :

- a VH sequence according to SEQ ID N° 63 and a VL sequence according to SEQ ID N° 64,

- a VH sequence according to SEQ ID N°65 and a VL sequence according to SEQ ID N° 66, or

- a VH sequence according to SEQ ID N° 67 and a VL sequence according to SEQ ID N° 68.

RECTIFIED SHEET (RULE 91) ISA/EP

In another or further embodiment, the agonist described herein comprises an IgG comprising:

- a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and - a VL sequence according to SEQ ID N° 70, 72, 74, or 76,

- the VH and VL optionally separated by a linker sequence.

Preferably, the agonist described herein comprises an IgG comprising:

RECTIFIED SHEET (RULE 91) ISA/EP - a VH sequence according to SEQ ID N° 69 and a VL sequence according to SEQ ID N° 70,

- a VH sequence according to SEQ ID N° 71 and a VL sequence according to SEQ ID 72, - a VH sequence according to SEQ ID N° 73 and a VL sequence according to

SEQ ID N° 74, or

- a VH sequence according to SEQ ID N° 75 and a VL sequence according to SEQ ID N° 76.

RECTIFIED SHEET (RULE 91) ISA/EP

In a preferred embodiment, the agonists described herein comprise: an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62, and either: an IgG comprising :

- a VH sequence according to SEQ ID N° 63, 65, or 67, and

- a VL sequence according to SEQ ID N° 64, 66, or 68,

- the VH and VL optionally separated by a linker sequence.

Or: an IgG comprising : - a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and

- a VL sequence according to SEQ ID N° 70, 72, 74, or 76,

- the VH and VL optionally separated by a linker sequence.

Based on the above, any combination of VHH and IgG listed above is deemed disclosed herein. Most preferred combinations are shown in the example section.

RECTIFIED SHEET (RULE 91) ISA/EP In a preferred embodiment, the VHH is fused to the N-terminus of the VL of the IgG, preferably using a linker such as a 5-mer linker, preferably having the sequence GSGGS (SEQ ID N° : 172). Preferably, there is no linker between the VL of the IgG and VH of the IgG.

Humanization

In one embodiment, the binding agent of current application comprises one or more immunoglobulin single variable domains or VHHs, for example targeting LRP such as LR.P5 or LRP6, that has been "humanized", i.e. one or more amino acid residues in the amino acid sequence of the VHH obtained by immunization is replaced by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being to increase the degree of sequence identity with the closest human germline sequence. 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 sequence(s), after which 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 sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a VHH domain may be partially humanized or fully humanized.

Therefore, in various embodiments, the binding agents of the application comprise a targeting moiety comprising an amino acid sequence having one or more amino acid mutations with respect to SEQ ID N° : 12, 13, 14, 18, 19, 20, 21, 22, 23, 34, 35, 36, 53 and/or 37. In various embodiments, the binding agent comprises a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID N° : 12, 13, 14, 18, 19, 20, 21, 22, 23, 34, 35, 36, 53 and/or 37. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and/or non-conservative substitutions.

RECTIFIED SHEET (RULE 91) ISA/EP In particular embodiments, the one or more amino acid mutations may be in the CDRs of the targeting moiety (e.g., the CDR1, CDR2 or CDR3 regions). In other particular embodiments, the one or more amino acid mutations may be in the framework regions of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 regions). In most particular embodiments, said one or more amino acid mutations are only present in the framework regions of said FRa binding agents.

"Conservative substitutions" may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices.

As used herein, "non-conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine 0-alanine, GABA and 5- Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, s-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, 0-alanine, fluoro-amino acids, designer amino acids such as 0 methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general).

Humanization can be performed using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions may be determined by methods known in the art, for example without the purpose of being limiting, by a comparison between the sequence of a VHH and the sequence of a naturally occurring human VH domain. In some embodiments, the humanizing substitutions are chosen such that the resulting

RECTIFIED SHEET (RULE 91) ISA/EP humanized VHHs still retain advantageous functional properties. Generally, as a result of humanization, the VHHs of the application may become more "human-like", while still retaining favourable properties such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. In various embodiments, the humanized VHHs of the application can be obtained in any suitable manner known in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material. Indeed, modification of the amino acid sequences may be achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

In various embodiments, the mutations that were introduced for example to humanize the binding agent do not substantially reduce the present binding agent's capability to specifically bind to the human LRP5 or LRP6 and Gprl24 or Reck and to agonize the Gprl24/Reck/Fzd/LRP5 or LRP6-mediated Wnt7 signaling.

Methods of production

The binding agents of the application, particularly the antibodies, more particularly the ISVDs or VHHs of the application are not limited to a specific biological source or to a specific method of preparation. Methods for producing the binding agents of the application are described herein. For example, DNA sequences encoding the binding agents of the application can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed.) (1993) and Ausubel et al. (1992). Alternatively, DNA sequences encoding the binding agents of the application can be chemically synthesized using methods known in the art. Synthetic DNA sequences can be ligated to other appropriate nucleotide sequences, including for example expression control sequences, to produce gene expression constructs encoding the desired FRo binding agents.

Accordingly, in various embodiments, the present application provides for isolated nucleic acids comprising a nucleotide sequence encoding the agonist of any of the

RECTIFIED SHEET (RULE 91) ISA/EP binding agents described in current application. One embodiment further discloses an expression cassette comprising said nucleic acid molecule. More specific embodiments disclose the expression cassette wherein elements for cell- or tissuespecific expression are present. Further embodiments relate to a vector comprising said expression cassette or said nucleic acid molecule. More particular, said vector may be a viral vector or lentiviral vector, preferably a viral vector specifically directed towards the central and/or peripheral nervous system (e.g., a brain-specific viral vector). In further particular embodiments, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant. In preferred embodiments, the viral vector is a blood brain barrier endothelial cellspecific viral vector. In further preferred embodiments, the viral vector is a blood brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.

Factors of importance in selecting a particular vector include inter alia: choice of recipient cell, ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to "shuttle" the vector between recipient cells of different species.

Expression vectors can be autonomous or integrative. A nucleic acid can be introduced into a cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle. The recombinant nucleic acid can be maintained extra-chromosomally or it can be integrated into the cell chromosomal DNA. Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and/or selection of those cells transformed with the desired nucleic acids. Expression vectors can also include an autonomous replication sequence (ARS). The ARS may comprise a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS18 or ARS68.

Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. The first and second insertable DNA fragments are each about 200 (e.g., about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous

RECTIFIED SHEET (RULE 91) ISA/EP to portions of the genomic DNA of the cell species to be transformed. A nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene. Integrative vectors can be linearized prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome. Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g., amplified) in bacterial cells such as Escherichia coli (E. coli). The vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu. The purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells.

In order to produce the binding agents of current application, expression vectors comprising a nucleic acid sequence encoding said binding agents can then be introduced into host cells through transfection, transformation, or transduction techniques. Hence, in various embodiments, the present application provides for a host cell comprising a nucleic acid encoding one of the binding agents of the present application. For example, nucleic acids encoding the binding agent of the application can be introduced into host cells by retroviral transduction. Illustrative host cells are E.coli cells, Chinese hamster ovary (CHO) cells, yeast cells such as Pichia spp., human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the binding agent of the application.

Following expression, the binding agents can be harvested and purified using techniques well known in the art, e.g. affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags or by chromatography. Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g. Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in eukaryotic host cells, e.g. CHO cells, it is first inserted into an expression vector containing for example, a suitable eukaryotic promoter, a secretion signal, enhancers, and various introns. In an embodiment, the binding agent of the application comprises a His tag, a FLAG-tag

RECTIFIED SHEET (RULE 91) ISA/EP and/or a Myc tag. In an embodiment, the binding agent of the application comprises a His tag and a proteolytic site to allow cleavage of the His tag.

Current application thus also provides a host cell comprising one of the binding agents described herein. Host cells comprising one of the nucleic acid molecules or the expression cassettes or the vectors of the application are provided herein as well. Host cells can be either prokaryotic or eukaryotic. Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis 2003 Virology 310: 1-7). Non-limiting examples of plant cells include tobacco cells, Arabidopsis cells, tomato cells, maize cells, algae cells, among others. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals.

Animal or mammalian host cells suitable for harboring, expressing, and producing one of the FRa binding agents of the application include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al 1986 Som Cell Mol Genet 12: 555-556; Kolkekar et al 1997 Biochemistry 36: 10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO- Kl/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub & Chasin 1980 PNAS 77:4216), and dpl2.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al 1977 J Gen Virol 36:59, or GnTI KO HEK293S cells, Reeves et al 2002 PNAS 99: 13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney

RECTIFIED SHEET (RULE 91) ISA/EP cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather 1980 Biol Reprod 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a particular embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

The host cells described above can be transiently or stably transfected. Such transfection of DNA, such as nucleic acid molecules, expression cassettes or expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Molecular Cloning : A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2* Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.). The host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction.

Furthermore, in alternative embodiments, the use of the herein described nucleic acid molecules, expression cassettes, or vectors encoding the binding agents of the application, particularly the antibodies, antibody fragments, ISVDs or VHHs are provided for the production of said binding agent, antibodies, antibody fragments, ISVDs or VHHs. In a particular embodiment, said use is provided for production of an intrabody. An intracellular antibody or "intrabody" is an antibody or a fragment of an antibody that is heterologously expressed within a designated intracellular compartment, a process which is made possible through the in-frame incorporation of intracellular trafficking signals. An intrabody can be expressed in any shape or form such as an intact IgG molecule or a Fab fragment, more particularly as genetically engineered antibody fragment for example as single domain intrabodies or VHHs. For a review see Zhu, and Marasco, 2008 (Therapeutic Antibodies.

RECTIFIED SHEET (RULE 91) ISA/EP Handbook of Experimental Pharmacology 181. Ed. Springer-Verlag Berlin Heidelberg).

Medical applications

As described earlier, the atypical Wnt7 signaling mediated by the Gprl24/Reck/Fzd/LRP5 or LRP6 complex is of crucial importance to initiate bloodbrain-barrier differentiation and maintenance of BBB function, and blood-retina- barrier differentiation and maintenance of BRB function. Moreover, stimulating Wnt7 signaling in cerebral/retinal endothelial cells with impaired BBB/BRB can restore the BBB/BRB and ameliorate associated disease effects.

Therefore, in another aspect of the invention, any agonist, binding agent or fusion protein herein disclosed, but also any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector herein described is provided for use as a medicament. This is equivalent to providing a method of treatment, comprising the step of administering any agonist, binding agent or fusion protein herein disclosed, or any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector herein described to a subject or patient in need thereof.

In particular embodiments, the method of treatment is a method of treatment or prevention of a disorder presenting an abnormal Wnt signaling in RECK and/or GPR124 expressing cells, tissues and/or organs.

In a particular embodiment, said agonist, binding agent, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector or vector is provided for use in gene therapy, more particularly for use to treat blood-brain barrier integrity.

In another particular embodiment, said agonist, binding agent, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector or vector is provided for use in gene therapy, more particularly for use in blood-brain barrier endothelial cell-directed gene therapy.

Accordingly, also provided herein is a method for gene therapy, in particular central and/or peripheral nervous system-directed gene therapy, in a subject in need of said gene therapy comprising: introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid expression cassette or a vector as described herein; and expressing a therapeutically effective amount of the Wnt7 agonist encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject.

RECTIFIED SHEET (RULE 91) ISA/EP In particular embodiments, the agonists or the nucleic acids encoding said antibodies as taught herein are used in mRNA therapy, in particular in blood brain barrier endothelial cell-directed mRNA therapy.

Accordingly, also provided herein is a method for RNA therapy, preferably mRNA therapy, in particular central and/or peripheral nervous system-directed mRNA therapy, in a subject in need of said mRNA therapy comprising: introducing in the subject, in particular in the central and/or peripheral nervous system of the subject, a nucleic acid encoding any of the herein disclosed agonist; and expressing a therapeutically effective amount of the agonist encoded by the nucleic acid as taught herein in the subject, in particular the central and/or peripheral nervous system of the subject.

An advantage of the use of RNAs therapy, it is generally believed that RNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis.

Any other well-known methods of introducing nucleic acids into animal cells may be used herein. At the simplest, the nucleic acid can be directly injected into the target cell I target tissue. Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment ("gene gun" method), infection with viral vectors (i.e. derived from lentivirus, adeno-associated virus (AAV), adenovirus, retrovirus or antiviruses), electroporation, and the like. Other techniques or methods which are suitable for delivering nucleic acid (NA) molecules to target cells include the continuous delivery of an NA molecule from poly (lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) NA molecule(s) into micropumps delivering the product. Another possibility is the use of implantable drug-releasing biodegradable microspheres. Also envisaged is encapsulation of NA or providing NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticles or dendrimers, poly (lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc.; and co-injection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used. In particular embodiments, the Wnt7 agonist is provided in carriers, such as liposomes, lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, polymer nanoparticles, polymer micelle or dendrimers. In a preferred embodiment, said carriers are liposomes or lipid nanoparticles (LNPs).

In an embodiment, said carriers are lipid-based carriers. Said lipid-based carriers comprise one or more lipids. The one or more lipids can be in solid and/or liquid form. Said lipid-based carriers may be LNPs, lipoplexes, liposomes, phospholipid micelles, solid lipid nanoparticles, nanostructured lipid carriers or nano-emulsions. Lipid-based carriers useful according to the invention include, for example, cationic lipids, liposomes, in particular cationic liposomes, and micelles, and nanoparticles. Cationic lipids may form complexes with negatively charged nucleic acids. Any cationic lipid may be used according to the invention. Liposomes are phospholipid and cholesterol self-assembled bilayer membranes that enclose an aqueous core, where hydrophilic molecules can be incorporated. Hydrophobic compounds can also be incorporated in the lipid bilayer. Liposomes can be classified in (i) small unilamellar vesicles (SUVs); (ii) large unilamellar vesicles (LUVs) and (iii) multilamellar vesicles (MLVs), according to their size and lamellarity. Solid lipid nanoparticles (SLNs) have a spherical shape with an average diameter of 10-1000 nm. They are used as a colloidal NP drug delivery system in which lipid drug carrier solidifies at room temperature as well as at body temperature. Different solid lipids can be exploited to produce SLNs, such as, tripalmitin, cetyl alcohol, cetyl palmitate, glyceryl monostearate, trimyristin, tristearin, stearic acid, etc. SLNs comprise of solid lipid, such as triglycerides, fatty acids, waxes, partial glycerides, and polyethylene glycosylated lipid; emulsifiers, such as polysorbates, poloxamer and lecithin; and water. Nanostructured lipid carriers (NLC), comprise a blend of solid and liquid lipids which results in a partially crystallized lipid system and many have advantages such as enhanced drug loading capacity, drug release modulation flexibility and improved stability.

In an embodiment, said lipid-based carrier is a lipid nanoparticle. Solid lipid nanoparticles (SLNs, sLNPs), or lipid nanoparticles (LNPs), are nanoparticles composed of lipids that are suited to be used as a drug delivery vehicle for drug compounds, especially polynucleotides such as RNA or DNA.

In another embodiment, any agonist, binding agent or fusion protein herein disclosed, but also any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector herein described is provided for use in the treatment of a neurological disorder, and/or in the treatment of an ophthalmological disorder.

Also provided is a method of treating blood-brain-barrier dysfunction or treating a neurological disorder, or treating blood-retina-barrier dysfunction or treating an ophthalmological disorder such as a retinal vascular disorder or retinal degenerative disorder comprising the step of administering any agonist, binding agent or fusion protein herein disclosed, or any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector herein described to a subject or patient in need thereof. In a particular embodiment, said administration is performed by gene therapy, more particularly by blood-brain barrier endothelial cell-directed gene therapy or blood-retina barrier endothelial cell-directed gene therapy.

Also provided is a method of stimulating, activating or agonizing the Gprl24/Reck/Fzd/LRP5 or LR.P6 mediated Wnt7 signaling in a cell, comprising the step of administering a therapeutically effective amount of any agonist, binding agent, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector herein described to said cell, wherein said cell expresses at least Gprl24, Reck, Fzd, more particular Fzdl and a LRP protein selected from LRP5 and LRP6. In a further embodiment, said agonist, binding agent, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector cannot activate the canonical Wnt signaling or Wnt7 signaling in the absence of RECK and/or GPR124.

In a particular embodiment, a neurological disorder as used herein is a disorder selected from the list consisting of ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease intracranial atherosclerosis, and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorders, epilepsy, neurodegenerative disorders, dementia, vascular dementia, HIV-l-associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Charcot-Marie Tooth disease, dystonia, infectious brain diseases, traumatic brain injuries, migraine, neuroinflammation, COVID-19, and chronic traumatic encephalopathy and combinations thereof. "Neuroinflammation" can refer to a neurovascular inflammation. Further, neuroinflammation can be triggered by different factors. For example, in non-limiting embodiments neuroinflammation is caused by an injury such as traumatic brain injury and/or spinal cord injury, an infection such as viral or bacterial or fungal infection, exposure to a toxin or due to toxic metabolites, neurodegenerative disease, autoimmune disease, smoking or passive smoking, aging or any combination thereof.

Neuroinflammation can be caused by an infectious disease. Said infection may be of bacterial, fungal, parasitic or viral origin. In some instances, the infectious disease is a viral infection. In certain instances, the viral infection comprises infection by one or more of Herpesviridae (Herpes Zoster Virus (VZV) infection, Herpes Simplex Virus (HSV-1 or HSV-2), cytomegalovirus (CMV)), Paramoxyviridae, coronaviruses (SARS- CoV-1 or MERS-CoV or SARS-CoV-2, HCoVNL63, HCoV-229E, HCoV-OC43, or HKU1 or a variant thereof), influenza viruses (influenza virus A, group 1 (H1N 1), group 2 (H2N2), group 3 (H3N2), group 5 (H5N1, H5N2, H5N8) or group 7 (H7N7, H7N9), influenza B, influenza C), Zika virus, Japanese encephalitis virus, Epstein-Barr virus, Hepatitis B virus, Hepatitis C virus, Human immunodeficiency virus type 1 (HIV-1), Human papilloma virus (HPV), Human T-cell lymphotropic virus type I (HTLV-1) and Kaposi sarcoma herpesvirus (KSHV).

Neuroinflammation can be associated with lung condition such as, acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS), possibly associated with or arising from ventilator use, viral infection, sepsis, or systemic bacterial infections in a subject in need thereof have been established. Said lung condition can be caused by an infectious disease caused by bacterial, fungal, parasitic or viral infection. Preferably said infection is an infection causing respiratory problems or is a respiratory tract infection. Preferably said infection is a viral infection.

Neuroinflammation can be associated with a bacterial infection. Such inflammation is mostly caused by blood-borne bacteria that cross the blood-brain barrier (BBB) and finally invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main etiological causes of bacterial meningitis.

In the present context, prevention and/or treatment of neuroinflammation may include encephalitis. In certain instances, the encephalitis is viral encephalitis, such as COVID-19 associated encephalitis. Neuroinflammation may also be related to inflammation of the optic nerve. Neuroinflammation may also be related to acute disseminated encephalomyelitis, such as COVID-19 associated acute disseminated encephalomyelitis. Neuroinflammation may also be related to inflammation of the vitreous retinal ganglion cells.

In another or further embodiment, the ophthalmological disease or disorder is a retinal vascular disorder or retinal degenerative disorder, preferably said ophthalmological disease or disorder is selected from the group of: retinopathy, retinal vascular disorders such as Norrie disease, diabetic retinopathy, macular degeneration, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, retinal vein occlusion and retinopathy of prematurity.

In embodiments, the agonists here described and compositions comprising them are particularly suitable to be administered to a patient prone to develop or suffering from such disorder. In particular embodiments, the agonist or composition is administered orally or parenterally, e.g., intravenously, intraperitoneally, subcutaneously, or intraocularly.

In particular embodiments, the pharmaceutical composition as taught herein (see below) is administered to the subject by the injection (e.g., intravenously or intraocularly) or transplantation of allogeneic cells transformed with the vector comprising the nucleic acid or the nucleic acid expression cassette as taught herein. When administered, the injected or transplanted allogenic cells will transcribe and translate the nucleic acid encoding the Wnt7 agonist as taught herein in vivo.

The dosage or amount of the Wnt7 agonist as taught herein, optionally in combination with one or more other active compounds to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the disorder to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the Wnt7 agonist described in any of the embodiments above. In order to optimize therapeutic efficacy, the Wnt7 agonist as taught herein can be first administered at different dosing regimens. Typically, levels of the Wnt7 agonist in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject's age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.

Toxicity and therapeutic efficacy of the Wnt7 agonist as described herein or pharmaceutical composition of the invention as described in any of the embodiments, comprising the same can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., non-target cells) and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. In particular embodiments, the Wnt7 agonist as taught herein is administered using a sustained delivery system, such as a (partly) implanted sustained delivery system. Skilled person will understand that such a sustained delivery system may comprise a reservoir for holding the Wnt7 agonist as taught herein, a pump and infusion means (e.g., a tubing system). For example, the sustained delivery system may be a mini- osmotic pump system implanted in the brain.

In particular embodiment, the agonist as disclosed herein is the main or only active ingredient of the pharmaceutical composition.

In another embodiment, the pharmaceutical composition as described in any of the embodiments is combined with a second therapy, preferably chosen from surgery, chemotherapy, radiotherapy or immunotherapy.

Pharmaceutical Compositions and Formulations

Also disclosed herein are pharmaceutical compositions comprising any of agonist, binding agent, nucleic acid, nucleic acid expression cassette, or vector as described herein in any of the embodiments, with an optional pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline or phosphate buffered saline), solubilisers, colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disinteg rants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives, antioxidants, tonicity controlling agents, absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active substance, its use in the therapeutic compositions may be contemplated.

Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) or intraocularly use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.

In particular embodiments, any agonist, binding agent, bispecific antibody, nucleic acid, the nucleic acid expression cassette or the vector herein described that is part of the pharmaceutical composition is provided in a liposome or lipid nanoparticle.

Pharmaceutical compositions as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V.), intraocularly, intramuscular, intraperitoneal, intrathecal or intracisternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the methods and compositions can be, for example, systemic, local, tissue-specific, etc., depending on the specific needs of a given application.

For example, for oral administration, pharmaceutical compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.

For example, for oral or nasal aerosol or inhalation administration, pharmaceutical compositions may be formulated for administration in the form of aerosols or sprays, for example, as solutions, suspensions or emulsions of the compound as taught herein or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.

Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc. and are generally known in the art. For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. as generally known in the art. Injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents. The antibodies and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations.

Where aqueous formulations are preferred, such may comprise one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Various types of phospholipids are known in the art. Typically, a surfactant: active substance molar ratio in an aqueous formulation will be from about 10: 1 to about 1: 10, more typically from about 5: 1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.

In preferred embodiments, the pharmaceutical composition comprising the agonist of the invention or nucleic acid encoding the agonist, as taught herein is administered parenterally. More preferably, the pharmaceutical composition as taught herein is administered intravenously, for example by infusion, intratheca I ly, or intraocularly.

When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable nonirritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug.

One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

One skilled in the art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically-acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

EXAMPLES

The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

Example 1: A bispecific molecule agonizes Wnt7 signaling

The cerebral endothelial cells at the BBB express both Gprl24 and Reck that form a four-membered co-receptor complex with one of the ten Frizzled receptors and one LRP co-receptor selected from LRP5 and LRP6. In contrast to cells outside the CNS, Wnt7 signaling is not only controlled by the canonical pathway through Fzd/LRP5 or LRP6, but also by an atypical signaling pathway mediated by the Gprl24/Reck/Fzd/LRP5 or LRP6 receptor complex. The latter is of crucial importance to establish and maintain a functional, non-leaking blood-brain-barrier. To develop specific agonists of the atypical Wnt7 signaling pathway that can repair a dysfunctional BBB in patients with neurological disorders, several binding agents were developed that simultaneously target LRP5 or LRP6 on the one hand and on the other hand at least Gprl24 or Reck.

To demonstrate the proof-of-concept, we took advantage of Super Top Flash (STF) cells, i.e. HEK293 cells expressing Firefly Luciferase under the control of Wnt/B- catenin downstream signaling. The STF cells allow a luminescence-based read-out that is directly proportional to Wnt signaling. The STF cells were transiently transfected with vectors expressing Fzdl, LRP6, Reck with or without a Flag tag (Reck +/- flag) and Gprl24 with or without a Flag tag (Gprl24 +/- flag).

Upon expression of a Wnt7a construct, the luminescence signal was measured and set at 100% for normalization purposes.

Next, four different antibodies were constructed (Figure 2):

- AB0002: anti-LRP6 VHH fused to the N-terminus of each light chain (LC) of the anti-flag IgG (Figure 2A).

- AB0005: VH-CH1 of the anti-flag IgG fused to the N-terminus of the heavy chain (HC) of the anti-LRP6 IgG; LC (VL-CL) of anti-flag and of anti-LRP6 IgG's expressed separately, proper pairing being achieved by introducing complementary mutations in CL/CH1 pairs (Figure 2B). AB0006: VL of anti-LRP6 IgG fused to the N-terminus of the LC of the antiflag IgG; VH of anti-LRP6 IgG fused to the N-terminus of the HC of the antiflag IgG (Figure 20).

- AB0007: VL of anti-flag IgG fused to the N-terminus of the LC of the anti- LRP6 IgG; VH of anti-flag IgG fused to the N-terminus of the HC of the anti- LRP6 IgG (Figure 2D).

The sequences of the different antibody fragments are:

VL of anti-Flag (SEQ ID N° : 10):

DVLMTQIPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGV PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIR

VH of anti-Flag (SEQ ID N° : 11) :

QVQLQQSAAELARPGASVKMSCKASGYSFTTYTIHWVKQRPGQGLEWIGYINPSSGYAAYN QNFKDETTLTADPSSSTAYMELNSLTSEDSAVYYCAREKFYGYDYWGQGATLTVSS

VL of anti-LRP6 (SEQ ID N°: 12) :

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKKPKLLIYAASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK

VH of anti-LRP6 (SEQ ID N°: 13) :

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRDAPGKGLEWVASISSTSGSKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAKTYYDFWSGYYTFDYWGDGTLVTVS S anti-LRP6 VHH (SEQ ID N° : 14) :

DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYAD SVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS

The antibody constructs were transiently expressed in the Fzd l/LRP6/Reck/Gprl24 expressing STF cells wherein Reck or Gprl24 are flag tagged or untagged.

For all antibody constructs, the luminescence signal was measured and normalized using the Wnt7a construct data. Data for antibodies were obtained in the presence of the Wnt secretion inhibitor IWP-2. Data for Wnt7a and the control without Wnt7a were obtained without IWP-2. In the control experiments, the luminescence signal was determined only in cells expressing Gprl24, Reck, Fzd l and LRP6.

Based on Figure 3 it is clear that all four antibody constructs binding LRP6 and Gprl24 can stimulate the Wnt7 signaling pathway between 50% and 100% of the natural Wnt7a ligand. The signals for antibody constructs expressed in STF cells comprising the Flag tagged Reck protein were lower but still significantly higher compared to that from the control antibody. This data demonstrates that the atypical Wnt7 signaling mediated by Gprl24/Reck/Fzd/LRP5 or LR.P6 can be stimulated by a compound that binds only two of the four co-receptor proteins, more particularly LR.P5 or LR.P6 on the one hand and Gprl24 or Reck at the other hand.

Example 2: The bispecific antibodies act as functional Wnt7 surrogates

Next, a dose-response curve for the bispecific antibodies targeting LRP6 and Gprl24 was set up. STF cells were transiently transfected with vectors encoding the following proteins: Fzdl, LRP6, Reck and flag-Gprl24. Purified antibodies as explained in Example 1 or a control antibody (not shown) were added to the cells in an increasing concentration from 0 to 400 nM. Figure 4 shows the results obtained for AB0007 for the concentrations from 0 to 17 nM after normalizing versus the activity of Wnt7a at saturation. This allows to characterize the antibodies based on their signaling potential. For AB0007 this is calculated at 50% of maximum Wnt7a signaling (Figure 4). AB0007 was able to induce Wnt7 signaling in a dose-dependent manner with a EC50 of 1.7 nM.

Example 3: Reck-independent Wnt7 signaling

In a next experiment, it was investigated whether the above described antibodies need all four members of the receptor complex to induce Wnt7 signaling. STF cells were transiently transfected with vectors encoding the following proteins: Flag- Gprl24, Fzdl and LRP6. The cells were additionally transfected with Reck or not. Purified Flag/LRP6 bispecific antibodies were added to the cells at a concentration of 4 nM. As a control, cells transfected with vectors encoding Flag-Gprl24, Fzdl and LRP6, or Flag-Gprl24, Fzdl, LRP6, and Reck were additionally transfected with a vector encoding Wnt7a.

As can be seen in Figure 5, the Gprl24/LRP6 bispecific antibodies exert their agonistic activity in a Reck-independent manner.

Although the above described data obtained using flag-tagged versions of Gprl24 or Reck together with bispecific anti-LRP6/anti-Flag antibodies are convincing and supportive for multispecific binding agents that act as agonists of the atypical Wnt7 signaling pathway, new constructs are made to confirm the herein disclosed results.

First, several bispecific compounds are constructed comprising LRP5 and Gprl24 binding domains or comprising LRP6 and Gprl24 binding domains. Second, several bispecific compounds are constructed comprising LR.P5 and Reck binding domains or comprising LRP6 and Reck binding domains.

Sequences of the CDR regions of the anti-LRP5 and anti-LRP6 VHHs can be found in Table 1 and 2.

Examples of such constructs are given in Figure 6. Protein domains binding to Gprl24 or Reck fused (with or without a (G4S)2 linker) to an anti-LRP6 VHH into bispecific, tetravalent Fc-fusion formats. In the examples shown, the protein domain binding to Gprl24 is the hReck CK1-5 (27 - 340). Protein domains binding to Reck are chosen from hADAMSlO (823-1103) (SEQ ID N° 45); 3 x Wnt7a linker (linker-(GSG)- linker-(GSG)-linker) (SEQ ID N° 46 - the Wnt7a linker) or zGprl24-ECD (25 -742) (SEQ ID N°: 44).

Vectors encoding for four constructs (VHH anti-LRP6 -3xWnt7alinker-Fc and VHH anti-LRP6 -hRECK: ECD-Fc; with or without G4S linker) were transiently expressed in STF cells expressing Gprl24, Reck, Fzdl and LRP6. Results are shown in Figure 7. The tested constructs showed agonist activity.

The sequences of the different binding domains were: zGprl24 (25-742) (SEQ ID N°: 44) : AGCPELFSSGCSCTEDRSKAHPTPGTRRKVSCGGKELTETPEVSLLPNRTVSLNLSNNRIRML KNGSFAGLSSLEKLDLRNNLISTIMPGAFLGLTALRKLDLSSNRIGCLTPEMFQGLTNLTKLNI SGNIFSSLDPNVFMELHSLKLVNFHSEFLSCDCGLRWVPSFFRSGSARLGDETLCAYPRRLQN KPLRLLRESDLSCEGPLELHTLSLLPSQRQVVFKGDRLPFHCTASLVDKITALHWRQNGQPVT SDPTKGIHLEESVQHDCTFITSELILSNVHVEASGEWECVVSTGRGNTSCSVEIVVLENSASF CPEQKVNNNRGEFRWPRTLAGITSYQHCLQLRYPSLTLGGGVEQKKASRNCDRSGRWEEAD YSQCLYTNDITRILHTFILMPVNASNAVTLAHQVRSYTLEAAGFTDTVDVLYVAQMMHKFMDY VTELRELSEVLVEMGSNLMQVDDQILARAQREERACSSIVYTLETLAWPQLHSHAQDLSRYS RNIVMEAHLIRPAHFTGISCTVYQRREGAAGSQVHDGADLSLEQQLRFRCTTGTHNTSLNAF HLKNAVALATVSLPATLFPPNAPPDCKLQFVAFRNGRFFPFTSNFTGHSDLARRRGISTPVIYA GLDGCSMWNQSDPIIVSLRHTSPGHDPVAAHWNSQALGHHGSWSLDGCQLIHSDVSISTL RCSVLSNYAVLQEIPDFPGSPSIPVEVLHP hADAMTSlO (823-1103) (SEQ ID N° : 45) :

SLPPYSWHYAPWTKCSAQCAGGSQVQAVECRNQLDSSAVAPHYCSAHSKLPKRQRACNTE

PCPPDWVVGNWSLCSRSCDAGVRSRSVVCQRRVSAAEEKALDDSACPQPRPPVLEACHGPT CPPEWAALDWSECTPSCGPGLRHRVVLCKSADHRATLPPAHCSPAAKPPATMRCNLRRCPPA

RWVAGEWGECSAQCGVGQRQRSVRCTSHTGQASHECTEALRPPTTQQCEAKCDSPTPGD

GPEECKDVNKVAYCPLVLKFQFCSRAYFRQMCCKTCHGH hWnt7a linker ( 171-199) (SEQ ID N° : 46) :

VEPVRASRNKRPTFLKIKKPLSYRKPMDT hReck CK1-5 (27-340) (SEQ ID N° : 47) :

GLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNS SLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGS VCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVENYCASISPQLIHCVNNYTQSYPMRNPT DSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHP GVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVS MLTCLA

A similar experiment was repeated with constructs having LRP5 binding domains, obtaining similar results.

Materials and Methods

Expression plasmid constructs

All genes were cloned using pCS2+ vector as template. The identity of all constructs was confirmed by Sanger sequencing.

Super TOP-Flash reporter gene assay

Dual luciferase assays were performed using Super TOP-Flash HEK293 luciferase reporter cell lines as known in the art. The cells were transiently transfected as explained in the Examples. The assays were performed 48h after transfection and the activity was measured. The data shown (mean ± standard deviation (SD)) are pooled from three technical replicates.

Example 5: Generation of antibody-based Wnt7a mimics

Material and methods

Preparation of recombinant Gprl24, Reck, and LRP6 proteins

- Human and mouse Gprl24 extracellular domains (residues 34-344) were produced in a mammalian expression system either as Fc-fusion proteins or with a C-terminal 6-His extension, and purified by conventional chromatography techniques. - Human and mouse Reck extracellular domains (residues 27-340) were produced in a mammalian expression system with a C-terminal 6-His extension, and purified by conventional chromatography techniques.

- Purified human LRP6 extracellular domain (residues 20-630) was purchased from Sino Biological (cat. 17052-H08H).

- Mouse LRP6 extracellular domain (residues 20-628) was produced in a mammalian expression system with a C-terminal 6-His extension, and purified by conventional chromatography techniques.

Generation of anti-Gprl24, anti-Reck and anti-LRP6 antibodies

- Anti-Gprl24 and anti-Reck antibodies (IgGs) we isolated from naive Fab-on- phage, fully human libraries by performing multiple (up to four) rounds of selection on the corresponding antigens. Human and mouse antigens were alternated during selection to favor the identification of human/mouse cross- reactive antibodies. HEK293 cells transiently expressing the corresponding, full-length human antigens were also use at specific stages of phage libraries selection, typically round 4.

- Anti-LRP6 nanobodies (VHHs) we isolated from naive VHH-on-phage libraries by performing multiple (up to four) rounds of selection on recombinant LRP6. Human and mouse antigens were alternated during selection to favor the identification of human/mouse cross-reactive VHHs. HEK293 cells transiently expressing the corresponding, full-length human LRP6 protein were also use at specific stages of phage libraries selection, typically round 4.

Generation of anti-Gprl24/LRP6 and anti-Reck/LRP6 bispecific antibodies

- Anti-LRP6 VHHs were fused to the N-terminus of the VL (variable light) domain of either anti-Gprl24 or anti-Reck IgGs (human IgGl with Fc- silencing L234A, L235A, P329G "LA - LA - PG" mutations - Lo et al., J. Biol. Chem. 292, 3900-3908, 2017) via a 5-mer linker (GSGGS - SEQ ID N°: 172). Constructs displaying agonist activity were identified by screening in STF (Super Top Flash) cells transiently expressing human Gprl24, Reck, Frizzled- 1 (Fzdl), and LRP6.

Constructs with agonist activity could be identified by combining the following anti-Grpl24 or anti-Reck IgGs with some or any of the below-listed LRP6 VHHs: o Gprl24 IgGs: 05A10 (SEQ ID N° 63 and 64), 06C03 (SEQ ID N° 65 and 66), 06A05 (SEQ ID N° 67 and 68) O Reck IgGs: 01A08 (SEQ ID N° 69 and 70), 01F11 (SEQ ID N° 71 and 72), 03A04 (SEQ ID N° 73 and 74), 07B05 (SEQ ID N° 75 and 76) o LRP6 VHH: 01F11 (SEQ ID N° 61), 01B01 (SEQ ID N° 59), 01E12 (SEQ

ID N° 60), 02G02 (SEQ ID N° 62)

Figure 8 illustrates the structure of the anti-Gprl24/LRP6 and anti-Reck/LRP6 bispecific antibodies, where the LRP6 VHH is fused to the N-terminus of the VL domain of the respective anti-Gprl24 or anti-Reck IgGs.

Each construct is tetravalent, meaning it has two binding sites for Gprl24 or Reck and two binding sites for LRP6, unless explicitly mentioned otherwise.

5.1: Gorl24/LRP6 or Gorl24/LRP5 bisoecific antibodies

Gorl24/LRP6

Identification of bispecific constructs with agonist activity

STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

- Cells transfected with empty vector were used as a negative control ("Control")

Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal.

Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

Luminescence measurements were used to assess activation of the Wnt/ - catenin pathway

Results: all tested constructs were capable of activating the Wnt/0-catenin pathway to a level that exceeded the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 9. STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the indicated concentrations

Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 1.9 nM and 1.7 nM for 01E12-05A10 and 01B01-06C03, respectively), as shown in Figure 10.

Two additional bispecific antibodies were tested subsequently:

Bispecific antibodies 01B01-06A05 (A) or 01E12-06A05 (B) were added to the cells at the indicated concentrations

Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 2.8 nM and 3.0 nM for 01B01-06A05and 01E12-06A05, respectively), as shown in Figure 11.

Gorl24/LRP5

STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP5

Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP5 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

Bispecific antibodies were added to the cells as indicated at a concentration of 20 nM Luminescence measurements were used to assess activation of the Wnt/ - catenin pathway

Results: bispecific antibodies 01E12-05A10, 01B01-06C038<. 01E12-06C03were capable of activating the Wnt/ -catenin pathway in STF cells expressing hLRP5 instead of hLRP6, as shown in Figure 12. This indicates (1) that VHHs 01B01 & 01E12 exhibit cross-reactivity with hLRP5 and (2) that agonist activity can be achieved by engaging either Gprl24 & LRP6 or Gprl24 & LRP5.

Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 4.2 nM and 1.5 nM for 01E12-05A10and 01B01-06C03, respectively), as shown in Figure 13.

Reck independency

STF cells were transfected with vectors encoding human Gprl24, Fzdl & LRP6

Cells transfected with vectors encoding human Gprl24, Fzdl, LRP6 & Wnt7a were used as an additional control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

Bispecific antibodies were added to the cells as indicated at a concentration of 20 nM

Results: all tested bispecific antibodies were capable of activating the Wnt/fJ-catenin pathway in a Reck-independent manner, i.e. in the absence of Reck, as shown in Figure 14. By contrast, and as predicted, the natural ligand Wnt7a showed no activity in the absence of Reck.

Fzd receptor dependency

STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6 (A), or Gprl24, Reck, Fzd5 & LRP6 (B) - Cells transfected with empty vector were used as a negative control ("Control")

Cells transfected with vectors encoding human Gprl24, Reck, Fzd4, LRP6 & Wnt7a (A), or Gprl24, Reck, Fzd5, LRP6 & Wnt7a (B) were used as additional controls ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

Results: all tested constructs were capable of activating the Wnt/ -catenin pathway in the presence of Gprl24, Reck, LRP6, and either Fzd4 or Fzd5 (also Fzdl, see above), to a level that exceeded the level of activation achieved with the natural ligand under these experimental conditions, as shown in Figure 15. Based on the data, the bispecific antibodies described here can use any of the 10 Fzd receptors present in human for activity.

STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6

Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 3.7 nM and 1.3 nM for 01E12-05A10and 01B01-06C03, respectively), as shown in Figure 16.

Bispecific antibodies display agonist activity in hRMEC and hBMEC hRMEC (Human Retinal Microvascular Endothelial Cells) (A) and hBMEC (Human Brain Microvascular Endothelial Cells) (B) were treated with either recombinant Wnt3a (13.4nM) or the indicated antibodies (10 nM)

Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

Results: both tested bispecific antibodies were capable of activating the Wnt pathway to an extent that was comparable to the level of pathway activation achieved with the natural ligand Wnt3a, as shown in Figure 17. Bispecific antibodies 01E12-05A10& 01B01-06C03 are highly potent agonists in hRMEC hRMEC (Human Retinal Microvascular Endothelial Cells) were treated with either 01E12-05A10 (A) or 01B01-06C03 (B) at the indicated concentrations Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

Results: both tested bispecific antibodies were capable of activating the Wnt pathway with low EC50 values (EC50 = 66 pM and 32 pM for 01E12-05A10and 01B01-06C03, respectively), as shown in Figure 18.

01B01-06C03regulates other mediators of the Wnt/fi-catenin pathway in hRMEC cells hRMEC cells were treated with 01B01-06C03 (10 nM) for 6 h

- The level of the downstream mediators of the Wnt/ -catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and NKD1 (Naked cuticle 1) mRNAs were monitored by qRT-PCR.

Results: treatment of hRMEC cells with 01B01-06C03resulted in a moderate but detectable increase in the expression of Lefl and NKD1, as shown in Figure 19.

Bispecific antibodies 01E12-05A10& 01B01-06C03are highly potent agonists in hBMEC hBMEC (Human Brain Microvascular Endothelial Cells) were treated with either 01E12-05A10 (A) or 01B01-06C03 (B) at the indicated concentrations Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

Results: both tested bispecific antibodies were capable of activating the Wnt pathway with low EC50 values (EC50 = 17 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively), as shown in Figure 20.

- Agonist activity requires engagement of both Gprl24 and LRP6 bEnd.3 cells (mouse brain endothelial cells) were treated with 01E12-05A10, with an irrelevant VHH linked to 05A10, or 01E12 linked to an irrelevant IgG (1 nM), or with both 'irrelevant VHH'-05A10and 01E12- 'irrelevant IgG' (1 nM each), for 24 h o 01E12-05A10 o 'irrelevant VHH'-05A10 o 01E12- 'irrelevant IgG' - The levels of the Axin2, Lefl, Mfsd2a, and PLVAP mRNAs were monitored by qRT-PCR

Results: As shown in Figure 21, activity was detected only with the construct capable of engaging both Gprl24 and LRP6 (i.e. 01E12-05A10).

Bispecific antibodies 01E12-05A10& 01B01-06C03are highly potent agonists in bEnd.3 cells bEnd.3 cells (mouse brain endothelial cells) were treated with either 01E12- 05A10 (A), 01B01-06C03 (B), or recombinant Wnt3a (C) at the indicated concentrations

Results: both tested bispecific antibodies were capable of activating the Wnt pathway in this particular cell line with EC50 values markedly lower than the one of the natural ligand Wnt3a (EC50 = 25 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively, vs. 6.56 nM for Wnt3a). Maximum signal amplitude was also larger for the bispecific antibodies compared to Wnt3a, as shown in Figure 22.

Bispecific antibodies 01E12-06C03& 01Fll-06C03are highly potent agonists in bEnd.3 cells bEnd.3 cells (mouse brain endothelial cells) were treated with either 01E12- 06C03 (A), 01F11-06C03 (B), or recombinant Wnt3a (C) at the indicated concentrations

Results: both tested bispecific antibodies were capable of activating the Wnt pathway in this particular cell line with EC50 values markedly lower than the one of the natural ligand Wnt3a (EC50 = 18 pM for both 01E12-06C03and 01F11-06C03, vs. 6.1 nM for Wnt3a). Maximum signal amplitude was also larger for the bispecific antibodies compared to Wnt3a, as shown in Figure 23.

01E12-05A10regulates other mediators of the Wnt//3-catenin pathway in bEnd.3 cells bEnd.3 cells were treated either with 01E12-05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 ("CHIR", 10 pM) for 24 h

- The level of the downstream mediators of the Wnt/ -catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domain-containing protein 2a) mRNAs, as well as the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA were monitored by qRT-PCR.

Results: treatment of bEnd.3 cells with both CHIR 99021 and 01E12-05A10resulted in an increased expression of Lefl and Mfsd2a, and a decreased expression of PLVAP, as shown in Figure 24.

bEnd.3 cells were treated with 01B01-06C03 (10 nM) for 24 h

Results: treatment of bEnd.3 cells with 01B01-06C03 resulted in an increased expression of Lefl and Mfsd2a, and a decreased expression of PLVAP, as shown in Figure 25.

Agonist activity can be achieved with bivalent molecules

- Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

- The following antibodies were added to the cells at a concentration of 20 nM (see also Figure 34B) o 01F11-06C03, 01B01-06C03: see elsewhere o 01E12-06C03 (VHH-Fab): VHH-Fab fragment derived from 01F11- 06C03 o 01E12-05A10 (VHH-Fab): VHH-Fab fragment derived from 01B01- 06C03

- Luminescence measurements were used to assess activation of the Wnt/ - catenin pathway

Results: All constructs, whether tetravalent (VHH-tethered IgG) or bivalent (VHH- Fab) showed a similar activity, as shown in Figure 34A.

5.2: Reck/LRP6 bisoecific antibodies

Identification of bispecific constructs with agonist activity

Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

Results: all tested constructs were capable of activating the Wnt/0-catenin pathway to a level that, for some constructs at least, was close to the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 26.

- Bispecific antibodies 01B01-01F11 (A), 01E12-01F11 (B), 01E12-03A04 (C) or 01B01-03A04 (D) were added to the cells at the indicated concentrations Luminescence measurements were used to assess activation of the Wnt/ - catenin pathway

Results: All tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 4.2 nM, 3.4 nM, 1.3 nM, and 6.4 nM for 01B01-01F11, 01E12-03A04, 01E12-01Flland 01B01-03A04, respectively), as shown in Figure 27. - STF cells were transfected with vectors encoding human Gprl24, human Reck, human Fzdl & mouse LRP5

- Cells transfected with vectors encoding human Gprl24, human Reck, human Fzdl, mouse LRP5 & human Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

- Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

Results: All tested antibodies were capable of activating the Wnt/ -catenin pathway in STF cells expressing mouse LRP5, as shown in Figure 28, indicating (1) that VHHs 01B01 & 01E12 exhibit at least some degree of cross-reactivity with mouse LRP5 and (2) that agonist activity can be achieved by engaging Reck and either LRP6 or LRP5.

Fzd receptor dependency (1)

- STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6 (Fig 27A), or Gprl24, Reck, Fzd5 & LRP6 (Fig 27B)

- Cells transfected with vectors encoding human Gprl24, Reck, Fzd4, LRP6 & Wnt7a (A), or Gprl24, Reck, Fzd5, LRP6 & Wnt7a (B) were used as additional controls ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

Results: All tested constructs were capable of activating the Wnt/ -catenin pathway in the presence of Gprl24, Reck, LRP6, and either Fzd4 or Fzd5 (also Fzdl, see above), to a level that equaled or exceeded the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 29.

Based on the data, the bispecific antibodies described here can use any of the 10 Fzd receptors present in human for activity. Fzd receptor dependency (2)

- STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6

- Bispecific antibodies 01B01-01F11 (A), 01E12-03A04 (B), or 01B01-03A04 (C) were added to the cells at the indicated concentrations

Results: As shown in Figure 30, all tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 5.5 nM, 5.9 nM, and 1.8 nM for 01B01-01F11, 01E12-03A04, and 01B01-03A04, respectively).

Reck/LRP6 Abs regulate several Wnt/fi-catenin target genes in bEnd.3 cells

- bEnd.3 cells were treated with the indicated Abs (10 nM) for 24 h

- The level of the downstream mediators of the Wnt/ -catenin signaling pathway Axin2 (axis inhibition protein 2), Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domain-containing protein 2a) mRNAs, as well as the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA were monitored by qRT-PCR

Results: as shown in Figure 31, treatment of bEnd.3 cells with Reck/LRP6 bispecific Abs resulted in an increased expression of Axin2 (A and B), Lefl (C) and Mfsd2a (D), and a decreased expression of PLVAP (E and F).

Bispecific antibodies 01B01-01A08, 01E12-03A04 and 01E12-01F11 are highly potent agonists in bEnd.3 cells

- bEnd.3 cells were treated with 01B01-01A08 (A), 01E12-03A04 (B) or 01E12- 01F11 (C) at the indicated concentrations for 24 h

- Wnt pathway activation was assessed by monitoring the mRNA level of Axin2 by qRT-PCR

Results: as shown in Figure 32, 01B01-01A08, 01E12-03A048<. 01E12- OlFllactivated the Wnt pathway in this particular cell line with EC50 value of 10 pM, 7 pM, and 7 pM, respectively.

Example 6: in vivo data

Antibodies are constructed as described above. Bispecific antibody 01E12-05A10 improves Blood-Retina-Barrier (BRB) properties in Ndp KO pups

The Ndp gene is responsible for producing norrin, a protein that plays a key role in the development and maintenance of the retina and its blood vessels. In the present knockout mice, a truncated version of Norrin is expressed which is not able to activate the pathway. This truncated Norin expression leads to developmental abnormalities, including uncomplete superficial vascularization of the retina and leaky BRB. Said model is generally used to study diseases like Norrie disease, which affects the eye and causes blindness, as well as other retinal conditions.

- 01E12-05A10 was administered intraperitoneally to Ndp-WT and Ndp-KO pups as indicated on the timeline (Figure 33A). NaCI was used as vehicle control. Representative pictures of n=2-3 animals/group.

Retinas were carefully dissected and stained for leaky vessel marker (Plvap), BRB junction protein (Claudin-5) and mouse immunoglobulin (mlgG)

Results show, as shown in Figure 33B:

Plvap expression is decreased in Ndp-KO animals treated with 01E12-05A10, Claudin-5 is increased in both Ndp-WT and Ndp-KO animals treated with 01E12-05A10. Arrows point capillaries for which Claudin-5 expression is detected (as compared to NaCI counterpart for which no expression of Claudin-5 is detected in capillaries) mlgG pathological leakage in Ndp-KO animals is decreased in animals receiving 01E12-05A10. No leakage is observed in WT animals

Thus: 01E12-05A10 improves BRB properties of Ndp-KO animals.

This example is repeated with all other antibodies as described above and similar results are obtained.

The present invention is in no way limited to the embodiments described in the examples and/or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention. Overview of sequences

SEQ ID N°:l (mature human Wnt7a)

LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFG KELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSA DIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWT TLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEE DPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTC SERTEMYTCK

SEQ ID N°:2 (mature human Wnt7b)

LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFG QELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSA DVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCW TTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCE EDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNT

SEQ ID N°:3 (human LRP5)

MEAAPPGPPWPLLLLLLLLLALCGCPAPAAASPLLLFANRRDVRLVDAGGVKLESTIVVSGLED AAAVDFQFSKGAVYWTDVSEEAIKQTYLNQTGAAVQNVVISGLVSPDGLACDWVGKKLYWT DSETNRIEVANLNGTSRKVLFWQDLDQPRAIALDPAHGYMYWTDWGETPRIERAGMDGSTR KIIVDSDIYWPNGLTIDLEEQKLYWADAKLSFIHRANLDGSFRQKVVEGSLTHPFALTLSGDT LYWTDWQTRSIHACNKRTGGKRKEILSALYSPMDIQVLSQERQPFFHTRCEEDNGGCSHLCL LSPSEPFYTCACPTGVQLQDNGRTCKAGAEEVLLLARRTDLRRISLDTPDFTDIVLQVDDIRHA IAIDYDPLEGYVYWTDDEVRAIRRAYLDGSGAQTLVNTEINDPDGIAVDWVARNLYWTDTGT DRIEVTRLNGTSRKILVSEDLDEPRAIALHPVMGLMYWTDWGENPKIECANLDGQERRVLVN ASLGWPNGLALDLQEGKLYWGDAKTDKIEVINVDGTKRRTLLEDKLPHIFGFTLLGDFIYWTD WQRRSIERVHKVKASRDVIIDQLPDLMGLKAVNVAKVVGTNPCADRNGGCSHLCFFTPHAT RCGCPIGLELLSDMKTCIVPEAFLVFTSRAAIHRISLETNNNDVAIPLTGVKEASALDFDVSNN HIYWTDVSLKTISRAFMNGSSVEHVVEFGLDYPEGMAVDWMGKNLYWADTGTNRIEVARLD GQFRQVLVWRDLDNPRSLALDPTKGYIYWTEWGGKPRIVRAFMDGTNCMTLVDKVGRAND LTIDYADQRLYWTDLDTNMIESSNMLGQERVVIADDLPHPFGLTQYSDYIYWTDWNLHSIER ADKTSGRNRTLIQGHLDFVMDILVFHSSRQDGLNDCMHNNGQCGQLCLAIPGGHRCGCAS HYTLDPSSRNCSPPTTFLLFSQKSAISRMIPDDQHSPDLILPLHGLRNVKAIDYDPLDKFIYWV DGRQNIKRAKDDGTQPFVLTSLSQGQNPDRQPHDLSIDIYSRTLFWTCEATNTINVHRLSGE AMGVVLRGDRDKPRAIVVNAERGYLYFTNMQDRAAKIERAALDGTEREVLFTTGLIRPVALVV DNTLGKLFWVDADLKRIESCDLSGANRLTLEDANIVQPLGLTILGKHLYWIDRQQQMIERVE KTTGDKRTRIQGRVAHLTGIHAVEEVSLEEFSAHPCARDNGGCSHICIAKGDGTPRCSCPVH LVLLQNLLTCGEPPTCSPDQFACATGEIDCIPGAWRCDGFPECDDQSDEEGCPVCSAAQFPC ARGQCVDLRLRCDGEADCQDRSDEADCDAICLPNQFRCASGQCVLIKQQCDSFPDCIDGSD

ELMCEITKPPSDDSPAHSSAIGPVIGIILSLFVMGGVYFVCQRVVCQRYAGANGPFPHEYVSG TPHVPLNFIAPGGSQHGPFTGIACGKSMMSSVSLMGGRGGVPLYDRNHVTGASSSSSSSTK ATLYPPILNPPPSPATDPSLYNMDMFYSSNIPATARPYRPYIIRGMAPPTTPCSTDVCDSDYSA SRWKASKYYLDLNSDSDPYPPPPTPHSQYLSAEDSCPPSPATERSYFHLFPPPPSPCTDSS

SEQ ID N°:4 (human LRP6) MGAVLRSLLACSFCVLLRAAPLLLYANRRDLRLVDATNGKENATIVVGGLEDAAAVDFVFSHG LIYWSDVSEEAIKRTEFNKTESVQNVVVSGLLSPDGLACDWLGEKLYWTDSETNRIEVSNLD GSLRKVLFWQELDQPRAIALDPSSGFMYWTDWGEVPKIERAGMDGSSRFIIINSEIYWPNGL TLDYEEQKLYWADAKLNFIHKSNLDGTNRQAVVKGSLPHPFALTLFEDILYWTDWSTHSILAC NKYTGEGLREIHSDIFSPMDIHAFSQQRQPNATNPCGIDNGGCSHLCLMSPVKPFYQCACPT GVKLLENGKTCKDGATELLLLARRTDLRRISLDTPDFTDIVLQLEDIRHAIAIDYDPVEGYIYWT

DDEVRAIRRSFIDGSGSQFVVTAQIAHPDGIAVDWVARNLYWTDTGTDRIEVTRLNGTMRKI LISEDLEEPRAIVLDPMVGYMYWTDWGEIPKIERAALDGSDRVVLVNTSLGWPNGLALDYDE GKIYWGDAKTDKIEVMNTDGTGRRVLVEDKIPHIFGFTLLGDYVYWTDWQRRSIERVHKRS AEREVIIDQLPDLMGLKATNVHRVIGSNPCAEENGGCSHLCLYRPQGLRCACPIGFELISDMK TCIVPEAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDFDVTDNRIYWTDISLKTISRAF

MNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNRIEVSKLDGQHRQVLVWKDLDSP RALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTLVPNVGRANGLTIDYAKRRLYWTDLD TNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIYWTDWSRRSIERANKTSGQNRTIIQGHL

DYVMDILVFHSSRQSGWNECASSNGHCSHLCLAVPVGGFVCGCPAHYSLNADNRTCSAPTT FLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVRAIDYDPLDKQLYWIDSRQNMIRKAQEDGS QGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYWTCEATNVINVTRLDGRSVGVVLKGEQDRPR AVVVNPEKGYMYFTNLQERSPKIERAALDGTEREVLFFSGLSKPIALALDSRLGKLFWADSDL RRIESSDLSGANRIVLEDSNILQPVGLTVFENWLYWIDKQQQMIEKIDMTGREGRTKVQARI AQLSDIHAVKELNLQEYRQHPCAQDNGGCSHICLVKGDGTTRCSCPMHLVLLQDELSCGES

QFQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGKHKKCDHNVDC SDKSDELDCYPTEEPAPQATNTVGSVIGVIVTIFVSGTVYFICQRMLCPRMKGDGETMTNDYV VHGPASVPLGYVPHPSSLSGSLPGMSRGKSMISSLSIMGGSSGPPYDRAHVTGASSSSSSST KGTYFPAILNPPPSPATERSHYTMEFGYSSNSPSTHRSYSYRPYSYRHFAPPTTPCSTDVCDSD YAPSRRMTSVATAKGYTSDLNYDSEPVPPPPTPRSQYLSAEENYESCPPSPYTERSYSHHLYPP PPSPCTDSS

SEQ ID N°:5 (human LRP6)

MGAVLRSLLACSFCVLLRAAPLLLYANRRDLRLVDATNGKENATIVVGGLEDAAAVDFVFSHG

LIYWSDVSEEAIKRTEFNKTESVQNVVVSGLLSPDGLACDWLGEKLYWTDSETNRIEVSNLD GSLRKVLFWQELDQPRAIALDPSSGFMYWTDWGEVPKIERAGMDGSSRFIIINSEIYWPNGL TLDYEEQKLYWADAKLNFIHKSNLDGTNRQAVVKGSLPHPFALTLFEDILYWTDWSTHSILAC NKYTGEGLREIHSDIFSPMDIHAFSQQRQPNATNPCGIDNGGCSHLCLMSPVKPFYQCACPT GVKLLENGKTCKDGATELLLLARRTDLRRISLDTPDFTDIVLQLEDIRHAIAIDYDPVEGYIYWT DDEVRAIRRSFIDGSGSQFVVTAQIAHPDGIAVDWVARNLYWTDTGTDRIEVTRLNGTMRKI

LISEDLEEPRAIVLDPMVGYMYWTDWGEIPKIERAALDGSDRVVLVNTSLGWPNGLALDYDE GKIYWGDAKTDKIEVMNTDGTGRRVLVEDKIPHIFGFTLLGDYVYWTDWQRRSIERVHKRS

AEREVIIDQLPDLMGLKATNVHRVIGSNPCAEENGGCSHLCLYRPQGLRCACPIGFELISDMK TCIVPEAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDFDVTDNRIYWTDISLKTISRAF MNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNRIEVSKLDGQHRQVLVWKDLDSP RALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTLVPNVGRANGLTIDYAKRRLYWTDLD TNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIYWTDWSRRSIERANKTSGQNRTIIQGHL

DYVMDILVFHSSRQSGWNECASSNGHCSHLCLAVPVGGFVCGCPAHYSLNADNRTCSAPTT FLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVRAIDYDPLDKQLYWIDSRQNMIRKAQEDGS QGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYWTCEATNVINVTRLDGRSVGVVLKGEQDRPR AIVVNPEKGYMYFTNLQERSPKIERAALDGTEREVLFFSGLSKPIALALDSRLGKLFWADSDLR RIESSDLSGANRIVLEDSNILQPVGLTVFENWLYWIDKQQQMIEKIDMTGREGRTKVQARIA QLSDIHAVKELNLQEYRQHPCAQDNGGCSHICLVKGDGTTRCSCPMHLVLLQDELSCGESQ FQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGKHKKCDHNVDCS DKSDELDCYPTEEPAPQATNTVGSVIGVIVTIFVSGTVYFICQRMLCPRMKGDGETMTNDYVV HGPASVPLGYVPHPSSLSGSLPGMSRGKSMISSLSIMGGSSGPPYDRAHVTGASSSSSSSTK GTYFPAILNPPPSPATERSHYTMEFGYSSNSPSTHRSYSYRPYSYRHFAPPTTPCSTDVCDSDY APSRRMTSVATAKGYTSDLNYDSEPVPPPPTPRSQYLSAEENYESCPPSPYTERSYSHHLYPPP PSPCTDSS

SEQ ID N°:6 (human Reck)

MATVRASLRGALLLLLAVAGVAEVAGGLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSES RLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACKQASS KNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVE NYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLI EGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLY SMSWGNTQSWQEFDRFCEYNPVEVSMLTCLADVREPCQLGCRNLTYCTNFNNRPTELFRSC NAQSDQGAMNDMKLWEKGSIKMPFINIPVLDIKKCQPEMWKAIACSLQIKPCHSKSRGSIIC KSDCVEILKKCGDQNKFPEDHTAESICELLSPTDDLKNCIPLDTYLRPSTLGNIVEEVTHPCNP

NPCPANELCEVNRKGCPSGDPCLPYFCVQGCKLGEASDFIVRQGTLIQVPSSAGEVGCYKICS CGQSGLLENCMEMHCIDLQKSCIVGGKRKSHGTSFSIDCNVCSCFAGNLVCSTRLCLSEHSS EDDRRTFTGLPCNCADQFVPVCGQNGRTYPSACIARCVGLQDHQFEFGSCMSKDPCNPNPC QKNQRCIPKPQVCLTTFDKFGCSQYECVPRQLACDQVQDPVCDTDHMEHNNLCTLYQRGKS LSYKGPCQPFCRATEPVCGHNGETYSSVCAAYSDRVAVDYYGDCQAVGVLSEHSSVAECAS VKCPSLLAAGCKPIIPPGACCPLCAGMLRVLFDKEKLDTIAKVTNKKPITVLEILQKIRMHVSVP QCDVFGYFSIESEIVILIIPVDHYPKALQIEACNKEAEKIESLINSDSPTLASHVPLSALIISQVQ VSSSVPSAGVRARPSCHSLLLPLSLGLALHLLWTYN

SEQ ID N°:7 (human Gprl24) / Adhesion G protein-coupled receptor A2

(ADGRA2)

MGAGGRRMRGAPARLLLPLLPWLLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGPA RRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKITGLRNGSFLGLSLLEKLDLRNNIISTVQPG AFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDELPALKVVDLGTEF LTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSALHAQALGSLQEAQLCCEGALELHTHHLIPSL RQVVFQGDRLPFQCSASYLGNDTRIRWYHNRAPVEGDEQAGILLAESLIHDCTFITSELTLSH IGVWASGEWECTVSMAQGNASKKVEIVVLETSASYCPAERVANNRGDFRWPRTLAGITAYQ SCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEPGDYSHCLYTNDITRVLYTFVLMPINASNA LTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKFLGYVDQIKELVEVMVDMASNLMLVDEHLL

WLAQREDKACSRIVGALERIGGAALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRRE GGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSS LPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKRRGVATPVIFAGTSGCGVGNL TEPVAVSLRHWAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVA VLMELSAFPREVGGAGAGLHPVVYPCTALLLLCLFATIITYILNHSSIRVSRKGWHMLLNLCFHI

AMTSAVFAGGITLTNYQMVCQAVGITLHYSSLSTLLWMGVKARVLHKELTWRAPPPQEGDPA LPTPSPMLRFYLIAGGIPLIICGITAAVNIHNYRDHSPYCWLVWRPSLGAFYIPVALILLITWIYF LCAGLRLRGPLAQNPKAGNSRASLEAGEELRGSTRLRGSGPLLSDSGSLLATGSARVGTPGP PEDGDSLYSPGVQLGALVTTHFLYLAMWACGALAVSQRWLPRWCSCLYGVAASALGLFVFT HHCARRRDVRASWRACCPPASPAAPHAPPRALPAAAEDGSPVFGEGPPSLKSSPSGSSGHPL ALGPCKLTNLQLAQSQVCEAGAAAGGEGEPEPAGTRGNLAHRHPNNVHHGRRAHKSRAKG HRAGEACGKNRLKALRGGAAGALELLSSESGSLHNSPTDSYLGSSRNSPGAGLQLEGEPMLT PSEGSDTSAAPLSEAGRAGQRRSASRDSLKGGGALEKESHRRSYPLNAASLNGAPKGGKYD DVTLMGAEVASGGCMKTGLWKSETTV

SEQ ID N°:8 (human Gprl24) / Adhesion G protein-coupled receptor A2 (ADGRA2)

MGAGGRRMRGAPARLLLPLLPWLLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGPA RRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKITGLRNGSFLGLSLLEKLDLRNNIISTVQPG AFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDELPALKVVDLGTEF LTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSALHAQALGSLQEAQLCCEGALELHTHHLIPSL RQVVFQGDRLPFQCSASYLGNDTRIRWYHNRAPVEGDEQAGILLAESLIHDCTFITSELTLSH IGVWASGEWECTVSMAQGNASKKVEIVVLETSASYCPAERVANNRGDFRWPRTLAGITAYQ SCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEPGDYSHCLYTNDITRVLYTFVLMPINASNA

LTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKFLGYVDQIKELVEVMVDMASNLMLVDEHLL WLAQREDKACSRIVGALERIGGAALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRRE GGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSS LPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKRRGVATPVIFAGTSGCGVGNL TEPVAVSLRHWAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVA VLMELSAFPREVGGAGAGLHPVVYPCTALLLLCLFATIITYILNHSSIRVSRKGWHMLLNLCFHI AMTSAVFAGGITLTNYQMVCQAVGITLHYSSLSTLLWMGVKARVLHKELTWRAPPPQEGDPA LPTPSPMLRFYLIAGGIPLIICGITAAVNIHNYRDHSPYCWLVWRPSLGAFYIPVALILLITWIYF

LCAGLRLRGPLAQNPKAGNSRASLEAGEELRGSTRLRGSGPLLSDSGSLLATGSARVGTPGP PEDGDSLYSPGVQLGALVTTHFLYLAMWACGALAVSQRWLPRWCSCLYGVAASALGLFVFT HHCARRRDVRASWRACCPPASPAAPHAPPRALPAAAEDGSPVFGEGPPSLKSSPSGSSGHPL ALGPCKLTNLQLAQSQVCEAGAAAGGEGEPEPAGTRGNLAHRHPNNVHHGRRAHKSRAKG HRAGEACGKNRLKALRGGAAGALELLSSESGSLHNSPTDSYLGSSRNSPGAGLQLEGEPMLT PSEGSDTSAAPLSEAGRAGQRRSASRDSLKGGGALEKESHRRSYPLNAASLNGAPKGGKYD DVTLMGAEVASGGCMKTGLWKSETTV

SEQ ID N°:9 (human Fzdl)

MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWL LEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIP LCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALP PCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEF WTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYG LMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAG FLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMK

WGHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFV YLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVIACYFYEQAFRD QWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSG KTLNSWRKFYTRLTNSKQGETTV

Table 1. CDR1, CDR2 and CDR3 sequences of VHHs targeting LRP6

Table 2. CDR1, CDR2 and CDR3 sequences of VHHs targeting LRP5

SEQ ID N°:18 (anti-LRP6 VHH)

DVQLVESGGGLVQPGGSLRLSCAASGSIFMINTMAWYRQAPGNQRELVATIRPVVSETTYAD

AVKGRFTISRDNAKNTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

SEQ ID N°:19 (anti-LRP6 VHH)

DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYAD

SVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS

SEQ ID N°:20 (anti-LRP6 VHH)

EVQLVESGGGLVQAGGSLRVSCAASGGTFSRYHMGWFRQAPGKEREFVSAITWSGGRTYY ADFVKGRFTISRDDARNTVYLQMSSLKPEDTAVYYCALTWAPTPTNRRSDYAYWGQGTQVT VSS

SEQ ID N°:21 (anti-LRP6 VHH)

EVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDMAWYRQAPGNQRELVATIRPVVSETTYAD

AVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

SEQ ID N°:22 (anti-LRP6 VHH) AVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRQAPGNQRELVATIRPVVSETTYAD

AVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

SEQ ID N°:23 (anti-LRP6 VHH)

AVQLVDSGGGLVQAGGSLRLSCAVSGRTFSMYDMGWFRQAPGKEREFVASIRWSSGNTWY ADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYANIYYTRRAPEEYWGQGTLVTVSSS

SEQ ID N°:34 (anti-LRP5 VHH)

EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYYCYARTQRMGVVNSYWGQGTLVTVSS

SEQ ID N°:35 (anti-LRP5 VHH)

QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG

SLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYFCNAVTYDGYTIRGQGTLVTVSS

SEQ ID N°:36 (anti-LRP5 VHH)

EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAD SVQGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAQFRNDYGLRYQSTNNYWGQGTLVTV SS

SEQ ID N°:37 (anti-LRP5 VHH)

QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTVYLQMNSLKPEDSAVYYCNANYRGNRYWGQGTLVTVSS

SEQ ID N°: 44 (zGprl24 (25-742))

AGCPELFSSGCSCTEDRSKAHPTPGTRRKVSCGGKELTETPEVSLLPNRTVSLNLSNNRIRML

KNGSFAGLSSLEKLDLRNNLISTIMPGAFLGLTALRKLDLSSNRIGCLTPEMFQGLTNLTKLNI

SGNIFSSLDPNVFMELHSLKLVNFHSEFLSCDCGLRWVPSFFRSGSARLGDETLCAYPRRLQN

KPLRLLRESDLSCEGPLELHTLSLLPSQRQVVFKGDRLPFHCTASLVDKITALHWRQNGQPVT

SDPTKGIHLEESVQHDCTFITSELILSNVHVEASGEWECVVSTGRGNTSCSVEIVVLENSASF

CPEQKVNNNRGEFRWPRTLAGITSYQHCLQLRYPSLTLGGGVEQKKASRNCDRSGRWEEAD

YSQCLYTNDITRILHTFILMPVNASNAVTLAHQVRSYTLEAAGFTDTVDVLYVAQMMHKFMDY

VTELRELSEVLVEMGSNLMQVDDQILARAQREERACSSIVYTLETLAWPQLHSHAQDLSRYS

RNIVMEAHLIRPAHFTGISCTVYQRREGAAGSQVHDGADLSLEQQLRFRCTTGTHNTSLNAF HLKNAVALATVSLPATLFPPNAPPDCKLQFVAFRNGRFFPFTSNFTGHSDLARRRGISTPVIYA GLDGCSMWNQSDPIIVSLRHTSPGHDPVAAHWNSQALGHHGSWSLDGCQLIHSDVSISTL

RCSVLSNYAVLQEIPDFPGSPSIPVEVLHP

SEQ ID N°: 45 (hADAMTSlO (823-1103))

SLPPYSWHYAPWTKCSAQCAGGSQVQAVECRNQLDSSAVAPHYCSAHSKLPKRQRACNTE

PCPPDWVVGNWSLCSRSCDAGVRSRSVVCQRRVSAAEEKALDDSACPQPRPPVLEACHGPT CPPEWAALDWSECTPSCGPGLRHRVVLCKSADHRATLPPAHCSPAAKPPATMRCNLRRCPPA RWVAGEWGECSAQCGVGQRQRSVRCTSHTGQASHECTEALRPPTTQQCEAKCDSPTPGD

GPEECKDVNKVAYCPLVLKFQFCSRAYFRQMCCKTCHGH SEQ ID N°: 46 (hWnt7a linker (171-199))

VEPVRASRNKRPTFLKIKKPLSYRKPMDT

SEQ ID N°: 47 (hReck CKl-5 (27-340))

GLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNS

SLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGS

VCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVENYCASISPQLIHCVNNYTQSYPMRNPT

DSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHP

GVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVS MLTCLA

SEQ ID N°: 48 (Wnt7b linker)

VEVVRASRLRQPTFLRLKQLRSYQKPMET

SEQ ID N°: 49 (CK4 motif RECK)

CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC

SEQ ID N°: 50 (CK5 motif RECK)

CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC

SEQ ID N°: 51 (RECK binding domain)

HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC

SEQ ID N°: 52 (RECK binding domain)

YEWRASRLRQPTFLRIKQLRSYQKPMET

SEQ ID N°: 53 (ANTI-LRP5 VHH)

DVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG

SLKGRFTMSTESAKNTMYLQMNSLKPEDTAVYYCYVKLRDDDYVYRGQGTQVTVSS

SEQ ID N°: 54 (RECK binding domain)

XXXVXAXRXXXXXFLXIXXXXXYXKXXXX

SEQ ID N°: 55 (RECK binding domain)

VXAXRXXXXXFLXIXXXXXYXK

SEQ ID N°: 56 (RECK binding domain) XXXVXAXRXXXXXFLXXXXXXXXXKXXXX

SEQ ID N°: 57 (RECK binding domain)

VXAXRXXXXXFLXXXXXXXXXK

SEQ ID N°: 58 (N-Terminal Domain Gprl42)

APGCPLSIRSCKCSGERPKGLSGGVPGPARRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKI TGLRNGSFLGLSLLEKLDLRNNIISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRL NISGNIFSSLQPGVFDELPALKVVDLGTEFLTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSAL HAQALGSLQEAQLCCEGALELHTHHLIPSLRQVVFQGDRLPFQCSASYLGNDTRIRWYHNRA PVEGDEQAGILLAESLIHDCTFITSELTLSHIGVWASGEWECTVSMAQGNASKKVEIVVLETS ASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTSVPLGGGAPGTRASRRCDRAGRWE PGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKF LGYVDQIKELVEVMVDMASNLMLVDEHLLWLAQREDKACSRIVGALERIGGAALSPHAQHIS VNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTT GRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSR PG AAG PG KRRG VATPVI FAGTSGCG VG N LTEP VAVS LRH WAEG AE PVAA WWSQEG PG E AG

GWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELSAFPREVGGAGAGLHP

LRP6 Sequences

GPR124 sequences

Reck sequences

SEQ ID N°: 172 (5-mer linker)

GSGGS

Claims

1. An agonist of the Wnt7 signaling pathway wherein the agonist is a soluble antibody or antibody fragment, and wherein said agonist comprises one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

2. The agonist according to claim 1, wherein said LRP binding domains are able to bind to LRP5 and/or LRP6.

3. The agonist according to claim 1 or 2, wherein said agonist is bivalent or tetravalent.

4. The agonist according to claim 3, wherein said agonist is tetravalent, said agonist comprises: i) two regions that each specifically bind to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes; and ii) two regions that each specifically bind to a set of one or more Reck or Gprl24 epitopes.

5. The agonist according to claim 3, wherein said agonist is bivalent, said agonist comprises: i) one region that specifically binds to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and/or LRP6 epitopes; and ii) one region that specifically binds to a set of one or more Reck or Gprl24 epitopes.

6. The agonist according to any one of the previous claims, wherein each one or more LRP binding domains is fused in tandem to either one or more Reck binding domains or one or more Gprl24 binding domains, and wherein said domains are optionally separated by a linker sequence.

7. The agonist according to any one of the previous claims, wherein the one or more LRP binding domains bind an amino acid sequence having at least 75% sequence identity to a fragment of the LRP5 and/or LRP6 proteins as depicted in SEQ ID N°: 3, 4 or 5.

8. The agonist according to any one of the previous claims, wherein the one or more Gprl24 or Reck binding domains bind an amino acid sequence having at least 75% sequence identity to a fragment of the Gprl24 or Reck proteins as depicted in SEQ ID N°: 6, 7 or 8.

9. The agonist according to any of the preceding claims, wherein at least one of the one or more LRP binding domains and/or the one or more Gprl24 or Reck binding domains is an antibody, an antibody fragment, an immunoglobulin single variable domain (ISVD) or VHH.

10. The agonist according to any one of the previous claims, wherein the agonist is a bispecific antibody, a bispecific antibody fragment, a bispecific ISVD, or a VHH.

11. The agonist according to any one of the previous claims, wherein the one or more LRP binding domains are LR.P5 and/or LR.P6 binding domains, and wherein said LR.P6 binding domains comprise a CDR.3 region as depicted in SEQ ID N°: 85, 86, 87, or 88 or consists of an amino acid sequence with maximally two amino acids different from SEQ ID N°: 85, 86, 87, or 88.

12. The agonist according to any one of the previous claims, wherein the one or more LRP binding domains are LRP5 and/or LRP6 binding domains, and wherein said LRP6 binding domain comprises a CDR2 region as depicted in SEQ ID N°: 81, 82, 83, or 84and/or a CDR1 region as depicted in SEQ ID N°: 77, 78, 79, or 80.

13. The agonist according to any one of the previous claims, wherein said agonist comprises an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62.

14. The agonist according to claim 13, wherein said agonist comprises an IgG comprising a VH sequence according to SEQ ID N° 63, 65, or 67, and a VL sequence according to SEQ ID N° 64, 66, or 68, optionally separated by a linker sequence; preferably a VH sequence according to SEQ ID N° 63 and a VL sequence according to SEQ ID N° 64, VH sequence according to SEQ ID N°65 and a VL sequence according to SEQ ID N° 66, or a VH sequence according to SEQ ID N° 67 and a VL sequence according to SEQ ID N° 68.

15. The agonist according to claim 13, wherein said agonist comprises an IgG comprising a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and a VL sequence according to SEQ ID N° 70, 72, 74, or 76, optionally separated by a linker sequence; preferably comprising a VH sequence according to SEQ ID N° 69 and a VL sequence according to SEQ ID N° 70, a VH sequence according to SEQ ID N° 71 and a VL sequence according to SEQ ID 72, a VH sequence according to SEQ ID N° 73 and a VL sequence according to SEQ ID N° 74, or a VH sequence according to SEQ ID N° 75 and a VL sequence according to SEQ ID N° 76.

16. The agonist according to any one of the previous claims, wherein said agonist does not comprise a Fzd binding domain.

17. The agonist according to any one of the previous claims, wherein said agonist is not able to bind to Fzd.

18. The agonist according to any one or the previous claims, wherein said agonist does not comprise further binding domains over said one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

19. The agonist according to any one of the previous claims, wherein the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

20. The agonist according to claim 19, wherein the binding domains of said agonist are selected from one or more LRP binding domains and one or more Gprl24 binding domains.

21. The agonist according to claim 20, wherein said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Gprl24 binding domain.

22. The agonist according to claim 19, wherein the binding domains of said agonist are selected from one or more LRP binding domains and one or more Reck binding domains.

23. The agonist according to claim 22, wherein said agonist is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Reck binding domain.

24. A nucleic acid encoding the agonist according to any of the preceding claims 1 to 23 or a vector comprising said nucleic acid.

25. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent, or carrier, and the agonist according to any of claims 1 to 23 or the nucleic acid or vector according to claim 24.

26. The agonist according to any of claims 1-23, the nucleic acid according or the vector according to claim 24 for use as a medicament.

27. The agonist according to any of claims 1-23, the nucleic acid according or the vector according to claim 24 for use in blood-brain barrier endothelial cell- directed gene therapy, or for use in the treatment of disorders linked to the blood-brain barrier integrity or for use in the treatment of the blood-brain barrier integrity.

28. The agonist according to any of claims 1-23, the nucleic acid or the vector according to claim 24 for use in a treatment of a neurological disorder, said neurological disorder is preferably selected from the group of: retinopathy, ischemic stroke, hemorrhagic stroke, ischemia/reperfusion injury, brain aneurysms, arteriovenous malformations (AVMs), cavernous malformations, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformations, carotid artery stenosis, Moyamoya disease intracranial atherosclerosis, and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorders, epilepsy, neurodegenerative disorders, dementia, vascular dementia, HIV-l-associated dementia, Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, Charcot-Marie Tooth disease, dystonia, infectious brain diseases, traumatic brain injuries, migraine, neuroinflammation, COVID- 19, and chronic traumatic encephalopathy and combinations thereof.

29. The agonist according to any of claims 1-23, the nucleic acid according or the vector according to claim 24 for use in blood-retina barrier endothelial cell- directed gene therapy, or for use in the treatment of disorders linked to the blood-retina barrier integrity or for use in the treatment of the blood-retina barrier integrity.

30. The agonist according to any of claims 1-23, the nucleic acid or the vector according to claim 24 for use in a treatment of a ophthalmological disease or disorder, said ophthalmological disease or disorder is preferably selected from the group of: retinopathy, retinal vascular disorders such as Norrie disease, diabetic retinopathy, macular degeneration, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, retinal vein occlusion and retinopathy of prematurity.