Wnt7 signaling agonists

Abstract

The present invention relates to agonist molecules of the Wnt7 signaling pathway, wherein the agonists comprise one or more LRP binding domains (e.g., 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 for use in medical therapy, more specifically, in the treatment of neurological and / or ophthalmic diseases and / or conditions.

Description

Technical Field

[0001] This invention relates to agonist molecules of the Wnt7 signaling pathway, more particularly to bispecific binders targeting two members of the Wnt7 receptor complex, and even more specifically to bispecific antibodies or fragments of bispecific antibodies. These agonists are developed for use in medical treatments, such as the treatment of neurological and / or ophthalmic diseases and / or conditions. Background Technology

[0002] Endothelial Wnt / β-catenin signaling functions as a key regulator of blood-brain barrier (BBB) ​​physiology in response to neurogenic Wnt7a / b ligands. Wnt / β-catenin signaling initiates the BBB differentiation cascade in the initial stages of central nervous system (CNS) vascular invasion and then sustains BBB function into adulthood. Recent evidence suggests that conditional knockout of β-catenin signaling in brain vascular endothelial cells (ECs) to inhibit Wnt signaling leads to BBB disruption and accelerates disease progression in mouse models of stroke, glioblastoma, and multiple sclerosis. Conversely, recombinant constitutive active forms of β-catenin in the CNS endothelium exhibit protective effects in brain cancer and stroke models.

[0003] Wnt7a / b is an endogenous ligand that regulates β-catenin-dependent BBB maturation, and therefore theoretically a rational therapeutic agent for repairing dysfunctional BBB. However, Wnt signaling is activated via Wnt7 ligand in both healthy and disease states, exhibiting pleiotropic effects in multiple tissues and organs. Furthermore, the structural pattern of Wnt / Frizzled (FZD) interactions makes natural Wnt ligands unsuitable as safe therapeutic agents. Interestingly, Wnt7 ligand activates Wnt signaling through two different types of membrane receptor complexes. The first receptor complex, composed of Frizzled (FZD) family receptor members and LRP5 or LRP6, has a wide tissue distribution, non-selectively binds to Wnt7a / b, and leads to systemic activation of Wnt signaling. The second is a GPR124 / RECK / FZD / LRP5 or LRP6 complex, which is enriched in brain endothelial cells expressing GPR124 and / or RECK.

[0004] The observation that Wnt signaling requires different receptor complexes in the BBB and CNS compared to other cell types and tissues presents an opportunity to develop CNS-specific Wnt agonists. However, developing chemicals or biologics that bind to and stimulate quaternary receptor complexes is challenging. For example, WO 2019 / 180204 proposes many compounds but does not disclose specific data or experimental evidence demonstrating the availability of such molecules or their effectiveness in modulating Wnt signaling in the CNS. Simply proposing a scheme without sufficient implementable disclosure does not provide a viable pathway for developing such therapeutics.

[0005] This invention provides a solution based on the unexpected observation that the binding of a compound to only two specific members of the receptor complex is sufficient to activate atypical GPR124 / RECK / FZD / LRP5 or LRP6-mediated Wnt signaling. Summary of the Invention

[0006] A first aspect of the invention provides an agonist of the Wnt7 signaling pathway, preferably mediated by Frizzled (Fzd), a lipoprotein receptor-associated protein (LRP) (e.g., LRP5 or LRP6), a G-protein-coupled receptor (Gpr)124, and a reversibly inducible cysteine-rich protein (Reck) with a Kazal motif, wherein the agonist comprises one or more LRP-binding domains (e.g., an LRP5-binding domain and / or an LRP6-binding domain) and one or more Gpr124 or Reck-binding domains. In one embodiment, the agonist is a selective or specific agonist. In another or further embodiment, the one or more LRP domains bind to an amino acid sequence having at least 75% sequence identity with an amino acid sequence selected from the list consisting of SEQ ID NO: 3-5. In another or further embodiment, one or more Gpr124 or Reck-binding domains bind to an amino acid sequence having at least 75% sequence identity with an amino acid sequence selected from SEQ ID NO: 6-8. In another or further embodiment, the binder comprises an antibody or antibody fragment, more particularly an immunoglobulin single variable domain (ISVD) or VHH, or is composed of therefrom. 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, which are LRP5 and / or LRP6 binding domains, and wherein the LRP6 binding domain comprises the CDR3 region shown in SEQ ID NO: 85, 86, 87, or 88 or consists of an amino acid sequence consisting of at most two different amino acids from SEQ ID NO: 85, 86, 87, or 88. In another or further embodiment, the antibody or antibody fragment comprises one or more LRP binding domains, which are LRP5 and / or LRP6 binding domains, and wherein the LRP6 binding domain comprises the CDR2 region shown in SEQ ID NO: 81, 82, 83 or 84 and / or the CDR1 region shown in SEQ ID NO: 77, 78, 79 or 80. In another or further embodiment, the antibody or antibody fragment comprises anti-LRP VHH containing the sequence of SEQ ID NO: 59, 60, 61 or 62.In another or further embodiment, the antibody or antibody fragment comprises IgG containing a VH sequence according to SEQ ID NO: 63, 65 or 67 and a VL sequence according to SEQ ID NO: 64, 66 or 68, optionally separated by a linker sequence; preferably, the VH sequence according to SEQ ID NO: 63 and the VL sequence according to SEQ ID NO: 64, the VH sequence according to SEQ ID NO: 65 and the VL sequence according to SEQ ID NO: 66, or the VH sequence according to SEQ ID NO: 67 and the VL sequence according to SEQ ID NO: 68. In another or further embodiment, the antibody or antibody fragment comprises IgG, said IgG comprising a VH sequence according to SEQ ID NO: 69, 71, 73 or 75, and a VL sequence according to SEQ ID NO: 70, 72, 74 or 76, optionally separated by a linker sequence; preferably, comprising a VH sequence according to SEQ ID NO: 69 and a VL sequence according to SEQ ID NO: 71 and a VL sequence according to SEQ ID NO: 72, a VH sequence according to SEQ ID NO: 71 and a VL sequence according to SEQ ID NO: 72, or a VH sequence according to SEQ ID NO: 75 and a VL sequence according to SEQ ID NO: 76.

[0007] In another or further embodiment, the agonist cannot bind Fzd and / or does not contain an Fzd binding domain. In another or further embodiment, the agonist does not contain any binding domain other than the one or more LRP binding domains and one or more Gpr124 or Reck binding domains. In another or further embodiment, the binding domains of the agonist are selected from one or more LRP binding domains and one or more Gpr124 or Reck binding domains. In another or further embodiment, the binding domains of the agonist are selected from one or more LRP binding domains and one or more Gpr124 binding domains. In another or further embodiment, the binding domains of the agonist are selected from one or more LRP binding domains and one or more Reck binding domains. In another or further embodiment, the agonist is a bispecific antibody having two binding domains, the binding domains comprising an LRP binding domain and a Gpr124 binding domain. In another or further embodiment, the agonist is a bispecific antibody having two binding domains, the binding domains comprising an LRP binding domain and a Reck binding domain.

[0008] It also provides a nucleic acid molecule or nucleic acid sequence encoding any of the agonists disclosed herein. A vector comprising said nucleic acid is also provided.

[0009] The second aspect provides any of the agonists, nucleic acids, or vectors disclosed herein for use as a medicine. This is analogous to saying that the invention provides a treatment method comprising administering any of the agonists, nucleic acids, or vectors disclosed herein to a subject in need. In one embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for gene therapy. In another or further embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for blood-brain barrier endothelial cell-guided gene therapy. In another or further embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for treating blood-brain barrier integrity. In another or further embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for treating neurological disorders. In one specific embodiment, the neurological disorder is selected from the following list: ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation (AVMs), cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, intracranial atherosclerosis and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorder, epilepsy, neurodegenerative disease, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Charcot-Marie Toothdisease, dystonia, infectious brain disease, traumatic brain injury, migraine, neuroinflammation, COVID-19, and chronic traumatic encephalopathy and combinations thereof. In another or further embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for the treatment of blood-retinal barrier (BRB) integrity. In another or further embodiment, any of the agonists, nucleic acids, or vectors disclosed herein are provided for the treatment of ophthalmic diseases or disorders. Preferably, the ophthalmic diseases or disorders are selected from: retinopathy, retinal vascular diseases such as Norrie disease, diabetic retinopathy, macular degeneration, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, retinal vein occlusion, and retinopathy of prematurity. Attached Figure Description

[0010] The following description of specific embodiments of the present invention is merely exemplary and is not intended to limit the teachings, their application, or uses. In all the accompanying drawings, corresponding reference numerals denote the same or corresponding parts and features.

[0011] Figure 1This is a schematic diagram of the Gpr124 / Reck / Fzd / LRP5 or LRP6 receptor complex (left) mediating atypical Wnt7 signaling at the BBB, and the Fzd / LRP5 or LRP6 receptor complex (right) mediating classical Wnt7 signaling.

[0012] Figure 2 This is a schematic diagram of different bispecific antibodies AB0002, AB0005, AB0006 and AB0007.

[0013] Figure 3 The image shows Wnt7 signaling induced by different bispecific antibodies in cells expressing Fzd1, LRP6, Flag-Gpr124, and Reck (STF cells) (dark gray) or Fzd1, LRP6, Gpr124, and Flag-Reck (STF cells) (light gray). Wnt7 signaling was measured based on typical luminescence signals in STF cells and normalized to a Wnt7a control. Antibodies were tested in the presence of IWP-2, a small molecule inhibitor of Wnt processing and secretion.

[0014] Figure 4 The dose-response curve of AB0007 is shown. AB0007 was added to STF cells transiently expressing Fzd1, LRP6, Reck, and Flag-Gpr124, and the resulting luminescent signal was normalized based on the activity of Wnt7a under saturation.

[0015] Figure 5 The images show the luminescent signals in STF cells expressing Fzd1, LRP6, and flag-Gpr124 (light gray) and in STF cells expressing Fzd1, LRP6, and flag-Gpr124 (dark gray) after the addition of AB0002, AB0005, AB0006, AB0007, and Wnt7a as positive controls and an empty vector as a negative control.

[0016] Figure 6 Possible embodiments of the compounds according to the present invention are shown.

[0017] Figure 7 The invention illustrates the testing of the agonist activity of the construct in STF cells expressing Gpr124, Reck, Fzd1, and LRP6 according to an embodiment of the invention.

[0018] Figure 8 The structures of the anti-Gpr124 / LRP6 and anti-Reck / LRP6 bispecific antibodies used in Example 5 are shown, wherein the LRP6 VHH is fused to the N-terminus of the VL domain of the corresponding anti-Gpr124 or anti-Reck IgG.

[0019] Figure 9 The luminescence signal in STF cells expressing Gpr124, Reck, Fzd1, and LRP6 is shown after the addition of the indicated bispecific antibody (50 nM). Wnt7a delivered via transient transfection was used as a positive control, and the empty vector was used as a negative control.

[0020] Figure 10 The dose-response relationships of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in Gpr124, Reck, Fzd1, and LRP6 STF cells are shown. The EC50 values ​​calculated from the curves for 01E12-05A10 and 01B01-06C03 were 1.9 nM and 1.7 nM, respectively.

[0021] Figure 11 The dose-response relationships of the bispecific antibodies 01B01-06A05 (A) and 01E12-06A05 (B) in Gpr124, Reck, Fzd1, and LRP6 STF cells are shown. The EC50 values ​​calculated from the curves for 01B01-06A05 and 01E12-06A05 were 2.8 nM and 3.0 nM, respectively.

[0022] Figure 12 The luminescence signal in STF cells expressing Gpr124, Reck, Fzd1, and LRP5 is shown after the addition of the indicated bispecific antibody (20 nM). Wnt7a delivered via transient transfection was used as a positive control, and the empty vector was used as a negative control.

[0023] Figure 13 The dose-response relationships of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells of Gpr124, Reck, Fzd5, and LRP6 are shown. The EC50 values ​​calculated from the curves for 01E12-05A10 and 01B01-06C03 were 4.2 nM and 1.5 nM, respectively.

[0024] Figure 14 The luminescent signal in STF cells expressing Gpr124, Fzd1, and LRP6 (without Reck) is shown upon addition of the indicated bispecific antibody (20 nM). In the Wnt7a control, Wnt7a was delivered via transient transfection, but the native ligand did not show activity under these conditions, i.e., in the absence of Reck. An empty vector was used as a negative control.

[0025] Figure 15The images show the luminescent signals in STF cells expressing Gpr124, Reck, Fzd4, and LRP6 (A) or Gpr124, Reck, Fzd5, and LRP6 (B) after the addition of the indicated bispecific antibody (50 nM). Wnt7a delivered via transient transfection was used as a positive control, and an empty vector was used as a negative control.

[0026] Figure 16 The dose-response relationships of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells of Gpr124, Reck, Fzd4, and LRP6 are shown. The EC50 values ​​calculated from the curves for 01E12-05A10 and 01B01-06C03 were 3.7 nM and 1.3 nM, respectively.

[0027] Figure 17 The signal transduction activities of the 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) were demonstrated. Recombinant Wnt3a (13.4 nM) was used as a positive control. Activation of the Wnt pathway was assessed by monitoring the level of Axin2 mRNA using qRT-PCR.

[0028] Figure 18 The dose-response relationship of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hRMEC cells is shown. Wnt pathway activation was assessed by monitoring Axin2 mRNA levels using qRT-PCR. The EC50 values ​​calculated from the curves were 66 pM for 01E12-05A10 and 32 pM for 01B01-06C03.

[0029] Figure 19 Treatment of hRMEC cells with the bispecific antibody 01B01-06C03 (10 nM) for 6 hours resulted in a detectable increase in the mRNA expression of downstream mediators of the Wnt / β-catenin signaling pathway, namely Lef1 (lymphocyte enhancer-binding protein 1) and NKD1 (Naked cuticle 1). The mRNA levels of Lef1 and NKD1 were monitored by qRT-PCR.

[0030] Figure 20The dose-response relationship of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hBMEC cells is shown. Wnt pathway activation was assessed by monitoring Axin2 mRNA levels using qRT-PCR. The EC50 values ​​calculated from the curves were 17 pM for 01E12-05A10 and 61 pM for 01B01-06C03.

[0031] Figure 21 The bEnd.3 cells involved were treated with (i) the bispecific antibody 01E12-05A10 (1 nM); (ii) the control antibody (1 nM) obtained by combining irrelevant VHH with anti-Gpr124 IgG 05A10; (iii) the control antibody (1 nM) obtained by combining anti-LRP6 VHH 01E12 with irrelevant IgG; or (iv) the response to treatment with two controls (1 nM each). Treatment was applied for 24 hours, and Wnt pathway activation was assessed by monitoring the levels of Axin2, PLVAP, Lef1, and MFSD2AmRNA using qRT-PCR. Figure 21 The mRNA levels of Axin2 (A), Lef1 (B), PLVAP (C), and MFSD2A (D) in treatment (i) with construct 01E12-05A10 are shown.

[0032] Figure 22 The dose-response of the bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in bEnd.3 (mouse brain endothelial cells) cells is shown. Wnt pathway activation was assessed by monitoring Axin2 mRNA levels using qRT-PCR. The EC50 values ​​calculated from the curves were 25 pM for 01E12-05A10 and 61 pM for 01B01-06C03. As a positive control, cells were treated with different concentrations of recombinant Wnt3a (C). The EC50 values ​​calculated from this curve... 50 The value is 6.6 nM.

[0033] Figure 23 The dose-response relationship of the bispecific antibodies 01E12-06C03 (A) and 01F11-06C03 (B) in bEnd.3 (mouse brain endothelial cells) is shown. Wnt pathway activation was assessed by monitoring Axin2 mRNA levels using qRT-PCR. EC50 was calculated from the curve. 50 The value for both antibodies was 18 pM. As a positive control, cells were treated with different concentrations of recombinant Wnt3a (C). EC50 was calculated from this curve. 50 The value is 6.6 nM.

[0034] Figure 24 Treatment of bEnd.3 cells with the bispecific antibody 01E12-05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 (“CHIR”, 10 μM) for 24 hours resulted in detectable alterations in the levels of Lef1 (lymphocyte enhancer-binding protein 1) and Mfsd2a (major promoter superfamily domain containing protein 2a) mRNA in the downstream Wnt / β-catenin signaling pathway, as well as the level of PLVAP (plasma membrane vesicle-associated protein) mRNA, as monitored by qRT-PCR.

[0035] Figure 25 Treatment of bEnd.3 cells with the bispecific antibody 01B01-06C03 (10 nM) for 24 hours resulted in detectable changes in the levels of Lef1 (lymphocyte enhancer-binding protein 1) and Mfsd2a (major promoter superfamily containing protein 2a) mRNA in the downstream Wnt / β-catenin signaling pathway, as well as the level of PLVAP (plasma membrane vesicle-associated protein) mRNA, as monitored by qRT-PCR.

[0036] Figure 26 The luminescence signal in STF cells expressing Gpr124, Reck, Fzd1, and LRP6 is shown when the indicated bispecific antibody (50 nM) is added. Wnt7a delivered via transient transfection was used as a positive control, and the empty vector was used as a negative control.

[0037] Figure 27 The dose-response relationships of the bispecific antibodies 01B01-01F11 (A), 01E12-01F11 (B), 01E12-03A04 (C), and 01B01-03A04 (D) in STF cells expressing Gpr124, Reck, Fzd1, and LRP6 are shown. The EC50 values ​​calculated from the curves are 4.2 nM for 01B01-01F11, 1.3 nM for 01E12-01F11, 4.3 nM for 01E12-03A04, and 6.4 nM for 01B01-03A04.

[0038] Figure 28 The luminescent signal in STF cells expressing human Gpr124, human Reck, human Fzd1, and mouse LRP5 is shown after the addition of the indicated bispecific antibody (50 nM). Transiently transfected Wnt7a was used as a positive control, and an empty vector was used as a negative control.

[0039] Figure 29The results show the luminescent signals in STF cells expressing Gpr124, Reck, Fzd4, and LRP6 (A) or Gpr124, Reck, Fzd5, and LRP6 (B) upon addition of the indicated bispecific antibody (50 nM). Transiently transfected Wnt7a was used as a positive control, and an empty vector was used as a negative control.

[0040] Figure 30 The dose-response relationships of the bispecific antibodies 01B01-01F11 (A), 01E12-03A04 (B), and 01B01-03A04 (C) in STF cells expressing Gpr124, Reck, Fzd4, and LRP6 are shown. The EC50 values ​​calculated from the curves are 5.5 nM for 01B01-01F11, 5.9 nM for 01E12-03A04, and 1.8 nM for 01B01-03A04.

[0041] Figure 31 The study showed the regulation of downstream mediators of the Wnt / β-catenin signaling pathway, including Axin2, Lef1, and Mfsd2a mRNA levels, and the level of PLVAP mRNA, a classic Wnt downregulated protein, in bEnd.3 cells after 24 hours of exposure to the Reck-LRP6 bispecific antibody (10 nM), as monitored by qRT-PCR.

[0042] Figure 32 The dose-response of the bispecific antibodies 01B01-01A08 (A), 01E12-03A04 (B), and 01E12-01F11 (C) in bEnd.3 cells is shown. Wnt pathway activation was assessed by monitoring Axin2 mRNA levels using qRT-PCR. The EC50 values ​​calculated from the curves were 10 pM for 01B01-01A08, 7 pM for 01E12-03A04, and 7 pM for 01E12-01F11.

[0043] Figure 33 The images show the expression of BRB markers (Plvap and Claudin-5) in the retinas of Ndp WT and Ndp KO mice treated with bispecific antibodies (5 mg / kg) or as a control vector, as well as mouse immunoglobulin leakage (mIgG). The images are representative of 2–3 animals from each experimental group.

[0044] Figure 34The study shows the luminescent signals in STF cells expressing Gpr124, Reck, Fzd1, and LRP6 upon the addition of either the tetravalent (VHH-linked IgG) or the bivalent (VHH-linked Fab) (20 nM) bispecific antibody 01E12-05A10 or 01B01-06C03. Wnt7a delivered via transient transfection served as a positive control. Detailed Implementation

[0045] This invention relates to novel ways and means of activating or stimulating the Wnt7 signaling pathway, and more particularly to atypical Wnt7 signaling pathways mediated or controlled by a tetragonal receptor complex comprising a frizzled receptor, LRP5 or LRP6, Gpr124, and Reck. Even more specifically, the Wnt7 agonists provided herein are specific or selective agonists of the Wnt7 signaling pathway coordinated by the GPR124 / RECK / FZD / LRP5 / 6 complex. The adhesives disclosed herein do not significantly stimulate classical Wnt7 signaling mediated solely by Fzd / LRP5 or Fzd / LRP6.

[0046] General definition

[0047] To facilitate understanding of this specification, certain terms are defined first. Additional definitions are provided throughout the detailed description. The invention has been described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto; rather, it is defined solely by the claims.

[0048] It should be noted that the term "a" or "one" entity refers to one or more of that entity; for example, "nucleotide sequence" is understood to mean one or more nucleotide sequences. Therefore, the terms "a," "one or more," and "at least one" can be used interchangeably herein. For example, "one or more" can refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. Furthermore, the term "and / or" as used herein indicates a specific disclosure of each of two specified features or components, with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" means including "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover 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). When referring to singular nouns such as "a" or "an," or "the," this includes the plural form of that noun, unless specifically stated otherwise. Furthermore, unless otherwise stated, the terms first, second, third, etc., in the specification and claims are used only to distinguish similar elements and are not necessarily used to describe order or chronological order. It should be understood that the terms are interchangeable where appropriate, and the embodiments of the invention described herein can be implemented in an order different from that described or illustrated herein.

[0049] It should be understood that wherever the language "comprising" is used herein to describe various aspects or embodiments, other similar aspects or embodiments described using "consisting of" and / or "consisting essentially of" are also provided. Wherein, the use of the term "comprising" in this specification and claims does not exclude other elements or steps. Unless specifically defined herein, all terms used herein have the same meaning as those skilled in the art.

[0050] Unless otherwise defined, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, general dictionaries of many terms used in this disclosure are provided in Juo Pei-Show's *Concise Dictionary of Biomedical and Molecular Biology*, 2nd edition, CRC Press, 2002; *The Dictionary of Cell and Molecular Biology*, 3rd edition, Academic Press, 1999; and *Oxford Dictionary of Biochemistry and Molecular Biology*, revised edition, Oxford University Press, 2000. Those skilled in the art will see in particular Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); Ausubel et al., *Current Protocols in Molecular Biology* (Supplement 100), John Wiley & Sons, New York (2012), for the description of definitions and terminology in the art. The definitions provided herein should not be construed as having a narrower scope than that understood by one of ordinary skill in the art.

[0051] Units, prefixes, and symbols are represented in their International System of Units (SI) accepted form. Numerical ranges expressed in terms of endpoints include all numbers and fractions contained within that range, as well as the endpoints. Unless otherwise indicated, nucleotide sequences are written in a 5′ to 3′ direction from left to right. Amino acid sequences are written in a left-to-right direction from amino to carboxyl. The headings provided herein do not limit the scope of the various aspects of this disclosure, which are available by referring to the entire specification. Therefore, terms defined below are defined more fully by reference to the entire specification.

[0052] The term "approximately" is used in this document to mean approximately, roughly, around, or within its range. When used with a numerical range, the term "approximately" modifies the range by extending the boundaries above and below the stated value. Generally, the term "approximately" can modify numerical deviations above and below the stated value.

[0053] This application relates to binding agents such as multispecific antibodies, which contain at least two binding domains, one for binding LRP5 or LRP6 and the other for binding Gpr124 or Reck.

[0054] As used herein, the term "antibody" refers to an immunoglobulin (Ig) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen. An antibody can be a complete immunoglobulin derived from natural or recombinant sources, and can be an immunoreactive portion of a complete immunoglobulin. The term "immunoglobulin (Ig) domain" as used herein refers to a globular region of an antibody chain, or a polypeptide consisting essentially of such globular regions. Immunoglobulin domains are characterized by retaining the immunoglobulin folding (Ig folding, as referred to herein) property of the antibody molecule, consisting of a bilayer sandwich of approximately seven to nine antiparallel β chains arranged in two β-sheets, optionally stabilized by conserved disulfide bonds. The term "immunoglobulin (Ig) domain" includes both "immunoglobulin constant domain" and "immunoglobulin variable domain" (abbreviated as "IVD"), the latter referring to an immunoglobulin domain consisting essentially of four frame regions, referred to in the art and herein as "frame region 1" or "FR1", "frame region 2" or "FR2", "frame region 3" or "FR3", and "frame region 4" or "FR4", respectively. These frame regions are interrupted by three complementarity-determining regions (CDRs), referred to in the art and herein as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. By carrying antigen-binding sites, the immunoglobulin variable domain confers antibody specificity for the antigen.

[0055] The term "immunoglobulin domain" in this application also includes "immunoglobulin single variable domain" (abbreviated as "ISVD"), which is synonymous with the term "single variable domain" and defines a molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes the immunoglobulin single variable domain from conventional immunoglobulins or their fragmental regions, where two immunoglobulin domains, specifically two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) interacts with the light chain variable domain (VL) to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL contribute to the formation of the antigen-binding site; that is, a total of six CDRs will participate in the formation of the antigen-binding site. Given the above definition, the antigen-binding domains of conventional four-chain antibodies (e.g., IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or Fab fragments, F(ab')2 fragments, Fv fragments (e.g., disulfide-linked Fv or scFv fragments), or bispecific antibodies (diabody) derived from these conventional four-chain antibodies (all of which are known in the art) are generally not considered immunoglobulin monovariable domains because, in these cases, binding to the epitopes of each antigen occurs through a single immunoglobulin domain rather than through a pair of associated immunoglobulin domains (e.g., light chain and heavy chain variable domains), i.e., through VH-VL binding of the immunoglobulin domains to the epitopes of their respective antigens. Conversely, immunoglobulin monovariable domains can specifically bind to the epitopes of an antigen without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin monovariable domain is formed by a single VH / VHH or VL domain. Therefore, the antigen-binding site of an immunoglobulin monovariable domain is formed by no more than three CDRs. Therefore, a single variable domain can be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or a VHH sequence) or a suitable fragment thereof; as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially 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, an immunoglobulin single variable domain is a heavy chain variable domain sequence (e.g., a VH-sequence); more particularly, an immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. For example, an immunoglobulin single variable domain can be (a single-domain antibody) (or an amino acid sequence suitable for use as a (single-domain antibody)), a dAb or dAb (or an amino acid sequence suitable for use as a dAb) or a nanobody (as defined herein, and including but not limited to VHH); other single variable domains, or any suitable fragment thereof.Specifically, the immunoglobulin single variable domain can be a nanobody (as defined herein) or a suitable fragment thereof. Note: Nanobody. TM Nanobodies TM and Nanoclone TM It is a registered trademark of Ablynx NV. For a general description of nanobodies, see the further description below and the prior art referenced herein, such as that described in WO 2008 / 020079.

[0056] The immunoglobulin domains in this article also include the “VHH domain,” which is also referred to as VHH, VHH domain, VHH antibody fragment, and VHH antibody, originally described as the antigen-binding immunoglobulin (Ig) (variable) domain of “heavy chain antibodies” (i.e., “antibodies without light chains”; Hamers-Casterman et al. (1993) Nature 363:446-448). The term “VHH domain” was chosen to distinguish these variable domains from the heavy chain variable domains present in conventional four-chain antibodies (referred to herein as the “VH domain”) and from the light chain variable domains present in conventional four-chain antibodies (referred herein as the “VL domain”). For a further description of VHH and nanobodies, see Muyldermans' article (Reviews in Molecular Biotechnology 74: 277-302, 2001), and the following patent applications mentioned as general background: WO94 / 04678, WO95 / 04079, and WO96 / 34103 from the Vrije Universiteit Brussel; WO94 / 25591, WO99 / 37681, WO00 / 40968, WO00 / 43507, WO00 / 65057, WO01 / 40310, WO01 / 44301, EP1134231, and WO02 / 48193 from Unilever; and Vlaams Instituutvoor Biotechnologie. (VIB)) WO97 / 49805, WO01 / 21817, WO03 / 035694, WO03 / 054016 and WO03 / 055527; Algonomics N.V. WO03 / 050531. And Ablynx NV's WO 01 / 90190; by the National Research Council of Canada's WO 03 / 025020; and by Ablynx NV's 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. And further published patent applications by Ablynx NV as described in these documents indicate that nanobodies (particularly VHH sequences and partially humanized nanobodies) can be characterized, in particular, by the presence of one or more "marker residues" in one or more of one or more of one or more framework sequences.Further descriptions of nanobodies, including humanization and / or camelification of nanobodies and other modifications, portions or fragments, derivatives or “nanobody fusions”, multivalent constructs (including some non-limiting examples of linker sequences) and various modifications and preparations thereof to increase the half-life of nanobodies, can be found, for example, in WO 08 / 101985 and WO 08 / 142164.

[0057] "Domain antibodies," also known as "Dab," "domain antibody," and "dAb" (the terms "domain antibody" and "dAb" are used as trademarks by the GlaxoSmithKline Group), have been described in, for example, EP 0368684, Ward et al. (Nature 341:544-546, 1989), Holt et al. (Trends in Biotechnology 21:484-490, 2003), and WO 03 / 002609, as well as other published patent applications such as WO 04 / 068820, WO 06 / 030220, WO 06 / 003388, and Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-cameloid mammals, particularly human four-chain antibodies. In order to bind an epitope as a single antigen-binding domain, i.e., not separately to a VL or VH domain, specific selection of this antigen-binding property is required, for example, by using a library of single human VH or VL domain sequences. Similar to VHH, domain antibodies have a molecular weight of approximately 13 to approximately 16 kDa and, if derived from a fully human sequence, do not require humanization, for example, for therapeutic purposes in humans. It should also be noted that a single variable domain can be derived from certain species of sharks (e.g., the so-called "IgNAR domain," see, for example, WO 05 / 18629).

[0058] Immunoglobulin monovariable domains, such as domain antibodies and nanobodies (including VHH domains and humanized VHH domains), represent macromolecules that mature in vivo after their production, but can be further maturated for affinity by introducing one or more changes in the amino acid sequence of one or more CDRs. These changes result in an increased affinity of the resulting immunoglobulin monovariable domain for its respective antigen compared to its parent molecule. The affinity maturation immunoglobulin single variable domain molecule of the present invention can be prepared by methods known in the prior art, such as those 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. MoI. Biol. 226: 889 896, 1992), and Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). The process of designing / selecting and / or preparing peptides from immunoglobulin monovariable domains, such as domain antibodies or nanobodies, is also referred to herein as the “formatting” of the immunoglobulin monovariable domain, and an immunoglobulin monovariable domain that becomes part of a peptide is referred to as “formatted” or “in the format of the peptide.” Based on this disclosure, examples of immunoglobulin monovariable domains that can be formatted, and examples of such formats, such as those used to avoid glycosylation, will be apparent to those skilled in the art.

[0059] Immunoglobulin single variable domains, such as domain antibodies and nanobodies (including VHH domains), can be humanized, i.e., their sequence identity with the closest human germline sequence is increased. Specifically, humanized immunoglobulin single variable domains, such as nanobodies (including VHH domains), can be immunoglobulin single variable domains as generally defined in the preceding paragraph, but with at least one amino acid residue present (and specifically, at least one framework residue) that corresponds to a humanization substitution (as defined herein). Potentially useful homology substitutions can be determined by comparing the framework region sequence of a native VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences. One or more such potentially useful homology substitutions (or combinations thereof) can then be introduced into the VHH sequence (in any manner known per se, as further described herein), and the resulting homology VHH sequence can be tested for affinity, stability, ease of expression, and level and / or other desired properties against a target. Thus, through limited trial and error, those skilled in the art can determine other suitable humanization substitutions (or suitable combinations thereof). Furthermore, based on the foregoing, immunoglobulin single variable domains (the framework regions) such as nanobodies (including the VHH domain) can be partially or fully humanized. It should be noted that, in its broadest sense, immunoglobulin single variable domains and the antigen-binding chimeric proteins of the present invention are not limited to specific biological sources or specific preparation methods. For example, but not for limiting purposes, immunoglobulin single variable domains, particularly the antigen-binding chimeric protein of the present invention, can generally be obtained by: (1) isolating the VHH domain of a naturally occurring heavy chain antibody and further engineering the sequence to obtain the antigen-binding chimeric protein; (2) expressing the nucleic acid sequence encoding the naturally occurring VHH domain in a host cell in a form fused with the backbone protein of the antigen-binding chimeric protein; (3) “humanizing” the naturally occurring VHH domain and / or backbone protein, and / or expressing the nucleic acid encoding such humanized VHH domain and / or backbone protein and / or antigen-binding chimeric protein; (4) “mutating” the naturally occurring VHH domain to reduce binding to a pre-existing antibody, or engineering the backbone protein fusion site to obtain the antigen-binding chimeric protein of the present invention having reduced binding to a pre-existing antibody compared to the natural VHH; or (5) preparing a protein, polypeptide, or other amino acid sequence, which is known itself, using synthetic or semi-synthetic techniques.

[0060] As used herein, “epitope” refers to a polypeptide antigenic determinant that constitutes a binding site or binding pocket on a target molecule (e.g., an immunoglobulin or a portion thereof, an antibody, a VHH, or an ISVD to which the protein binds). “Binding” refers to any interaction, whether direct or indirect. Direct interaction means contact between two binding partners (e.g., physical or chemical). Indirect interaction is any interaction in which the interacting partners interact in a complex of more than two molecules. Interactions can occur entirely indirectly (e.g., two molecules are part of the same complex in the presence of one or more bridging molecules, but do not bind in the absence of those bridging molecules). Interactions can be partially direct or partially indirect: direct contact still exists between two interacting partners, but this contact is, for example, unstable and stabilized by interaction with one or more other molecules. The terms “binding pocket,” “binding domain,” or “binding site” refer to a region of a molecule or molecular complex that is associated with another chemical entity, compound, protein, peptide, antibody, single-domain antibody, or ISVD or VHH due to its shape and charge.

[0061] Epitopes can have a spatial conformation comprising one, two, or three amino acids, and this spatial conformation is unique to the epitope. Generally, epitopes consist of at least four, five, six, or seven such amino acids, and more commonly, at least eight, nine, or ten such amino acids. Methods for determining the spatial conformation of amino acids are known in the art and include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance. As used herein, a “conformal epitope” refers to an epitope with a three-dimensional configuration whose spatial conformation is specific to the three-dimensional conformation of polypeptide folding. Generally, conformal epitopes consist of amino acids that are discontinuous in a linear sequence but aggregated together in the folded structure of a protein. However, conformal epitopes can also consist of a linear sequence of amino acids that takes a conformation specific to the three-dimensional conformation of polypeptide folding (and not present in a denatured state). In protein complexes, conformal epitopes consist of amino acids that are discontinuous in the linear sequence of one or more polypeptides that aggregate together during the folding of different polypeptides and their binding in a unique quaternary structure. Similarly, conformational epitopes can also consist of a linear sequence of amino acids from one or more polypeptides that aggregate and adopt a conformation unique to their quaternary structure. The term "conformation" or "conformational state" for proteins generally refers to the range of structures a protein can adopt at any given time. Those skilled in the art will recognize that the determinants of conformation or conformational state include the protein's primary structure and the environment surrounding it, as reflected in the protein's amino acid sequence (including modified amino acids). Protein conformation or conformational state also involves structural features such as secondary structure (e.g., α-helices, β-sheets, etc.), tertiary structure (e.g., three-dimensional folding of the polypeptide chain), and quaternary structure (e.g., interactions between the polypeptide chain and other protein subunits). Post-translational modifications and other modifications to the polypeptide chain, such as ligand binding, phosphorylation, sulfation, glycosylation, or the attachment of other hydrophobic groups, can affect protein conformation. Furthermore, environmental factors such as the pH, salt concentration, ionic strength, and osmotic pressure of the surrounding solution, as well as interactions with other proteins and cofactors, can influence protein conformation. The conformational state of a protein can be determined by functional assays of its activity or by measurements of its binding to another molecule, or by physical methods such as X-ray crystallography, NMR, or spin labeling. For a general discussion of protein conformation and conformational state, see Cantor and Schimmel, *Biophysical Chemistry, Part I: The Conformation of Biological*, W.H. Freeman and Company, 1980, and Creighton, *Proteins: Structures and Molecular Properties*, W.H. Freeman and Company, 1993.

[0062] As used herein, "epitope" refers to an antigen-binding site, specifically the portion of an antibody that recognizes and binds to an antigen. Therefore, the epitopes of the bispecific binders disclosed herein include the binding amino acid residues that bind to LRP5 or LRP6 protein epitopes and the binding amino acid residues that bind to Gpr124 or Reck protein epitopes.

[0063] Antibodies or binding agents can typically bind to one or more epitopes of one or more targets.

[0064] As used in this article, "bispecific" refers to the ability of a binder to simultaneously bind to two different epitopes or two different antigens. "Multispecific" refers to the ability to simultaneously bind to two or more different binding sites or antigens. Therefore, multispecific binders include bispecific antibodies, but can also refer to antibodies capable of binding to three or more targets.

[0065] Regardless of the number of targets the binder can bind to, the binder can contain multiple binding sites. The number of binding sites is called the antibody valency. A "bivalent" antibody is a typical antibody structure with two binding sites. The common structure of natural antibodies includes two identical binding sites that bind to two identical antigenic determinants or epitopes. However, a bivalent antibody can also bind to two different epitopes or two different targets. A "tetravalent" antibody is an antibody with four binding sites. It can bind to four identical or different antigenic determinants or epitopes, or it can bind to two different epitopes or two different targets, each bound by two binding sites.

[0066] The term "affinity" generally refers to the degree to which an antibody or other binding protein (as further defined herein) binds to a target protein, causing the equilibrium between the target and binding proteins to shift towards the presence of the complex they form. Thus, for example, in a combination of antigen and antibody (fragments) at approximately equal concentrations, a high-affinity antibody (fragment) will bind to the available antigen, shifting the equilibrium towards a higher concentration of the complex. The equilibrium dissociation constant KD (or K0) is commonly used to describe the affinity between a ligand and a target protein or between an antibody and its antigen. KD is a calculated ratio of koff / kin between the antibody and its antigen, and thus measures the tendency of the complex to dissociate into its component molecules. The binding constant (k0) on The dissociation constant (kk) is used to characterize how quickly an antibody binds to its target. off The k-off (also known as kdis, Kdis, Kd, ​​or kd) is used to measure how quickly an antibody dissociates from its target and represents the number of units dissociated from the target per second. Therefore, a lower k-off indicates a lower rate of dissociation. off This means a high affinity for the target.off Thus, KD is inversely proportional to affinity. High-affinity interactions are characterized by low KD values, rapid recognition (high KD), and strong stability of the formed complex (low KD). It should be understood that, within the scope of this application, the term "affinity" is used in the context of antibodies or antibody fragments that bind to epitopes of LRP5, LRP6, Gpr124, and / or Reck proteins, and more particularly the "function" of said antibodies or antibody fragments to bind their targets via the CDR region of their immunoglobulin (Ig) domain.

[0067] As used in this article, "amino acid" refers to the structural units (monomers) that make up proteins. These monomers combine to form short polymer chains called peptides, or long chains called polypeptides or proteins. These chains are linear and unbranched, in which each amino acid residue within the chain is attached to two adjacent amino acids. The 20 amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are referred to as protein amino acids or native amino acids. Natural amino acids, or naturally occurring amino acids, are glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), isoleucine (Ile 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 (Gln 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 glutamate (Glu or E).

[0068] The terms “nucleic acid,” “nucleic acid sequence,” or “nucleic acid molecule” as used herein are used interchangeably and refer to any form of nucleotide polymer, namely deoxyribonucleotides or ribonucleotides or analogues of any length. Nucleic acids can have any three-dimensional structure and can perform known or unknown functions. Non-limiting examples of nucleic acids include genes, gene fragments, 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. Nucleic acid molecules can be linear or circular. Nucleic acids can include promoters, introns, enhancer regions, polyadenylation sites, translation initiation sites, 5′ or 3′ untranslated regions, reporter genes, selectable markers, etc. Nucleic acids can include single-stranded or double-stranded DNA or RNA. Nucleic acids can include modified bases or modified backbones. Nucleic acids up to approximately 100 nucleotides in length are often also referred to as oligonucleotides. As used herein, “nucleotide” refers to the building blocks of oligonucleotides and polynucleotides, and for the purposes of this disclosure, includes both naturally occurring and non-naturally occurring nucleotides. Naturally, nucleotides such as DNA and RNA nucleotides comprise a ribose moiety, a base moiety, and one or more phosphate groups (which are absent in nucleosides). A nucleotide without a phosphate group is called a “nucleoside,” and is therefore a compound comprising a nucleobase moiety and a sugar moiety. As used herein, “nucleobase” refers to a set of atoms that can be linked to a sugar moiety to form a nucleoside capable of incorporating an oligonucleotide, and wherein this set of atoms can bond to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Naturally occurring nucleobases of RNA or DNA include purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0069] As used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymer of nucleotides of any length, namely ribonucleotides or deoxyribonucleotides. The term refers only to the main structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA, complementary DNA, and RNA. It also includes known types of modifications, such as methylation, “cap” substitution (replacing one or more naturally occurring nucleotides with analogs), and so on. By “nucleic acid construct,” a constructed nucleic acid sequence is defined as containing one or more functional units that are not naturally present together. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from λ phage), viral genomes containing non-natural nucleic acid sequences, and so on. A “coding sequence” is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide under the control of appropriate regulatory sequences. The boundaries of a coding sequence are defined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences can include, but are not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and in some cases, introns may also be present.

[0070] As used herein, "expression cassette" refers to any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, operatively linked to a promoter of the expression cassette. The expression cassette is generally a DNA construct, preferably comprising (from 5′ to 3′ in the transcriptional direction): a promoter region, a polynucleotide sequence, its homologs, variants, or fragments, operatively linked to a transcription initiation region, and a termination sequence comprising a termination signal for RNA polymerase and a polyadenylation signal. It should be understood that each of these regions is operable in a biological cell, such as the prokaryotic or eukaryotic cell to be transformed. The promoter region may be naturally present in the biological cell to be transformed or may be obtained from an alternative source, wherein the region is functional in the biological cell and contains a transcription initiation region, preferably containing an RNA polymerase binding site and a polyadenylation signal. Such a cassette can be constructed as a "vector". The term "vector," or alternatively "vector construct," "expression vector," or "gene transfer vector," refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked thereto, and includes any vector known to those skilled in the art, including any suitable type, but not limited to, for example, plasmid vectors, granular vectors, phage vectors such as λ phage, viral vectors such as adenovirus vectors, AAV vectors, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Expression vectors include plasmids and viral vectors, and typically contain the desired coding sequence and an appropriate DNA sequence for operatively linking the coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to engineer and amplify specific desired DNA fragments and may lack the active sequence required for expression of the desired DNA fragment. The construction of expression vectors for transfecting cells is also well known in the art and can therefore be achieved using standard techniques (see, for example, Sambrook, Fritsch and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press Inc., Clifton, NJ), and the Ambion 1998 catalog (Ambion, Austin, TX).

[0071] In the context of two or more nucleic acid or amino acid sequences, the term "identical" or percentage "identity" means that, when compared and aligned (with gaps introduced where necessary) to obtain the maximum correspondence, without considering any conserved amino acid substitutions as part of sequence identity, two or more sequences each have the same nucleotide or amino acid residues, or have a specified percentage of the same nucleotide or amino acid residues. Percentage identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of nucleotide or amino acid sequences.

[0072] The terms "sequence identity %" or "% sequence identity" or "percentage identity" or "% identity" between two polynucleotide or polypeptide sequences refer to the number of identical matching positions shared by the sequences within a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced to achieve optimal alignment of the two sequences. A matching position is any location in both the target and reference sequences where the same nucleotide or amino acid is present. Since gaps are not nucleotides or amino acids, gaps occurring in the target sequence are not counted. Similarly, gaps occurring in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, rather than those from the reference sequence.

[0073] A non-limiting example of such a sequence alignment algorithm is found in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87: 2264-2268, as modified by Karlin et al. in Proc. Natl. Acad. Sci., 1993: 5873-5877, and incorporated into the NBLAST and XBLAST procedures (Altschul et al., 1991, Nucleic Acids Res., 25: 3389-3402). In some respects, interval 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 other publicly available software programs that can be used for sequence alignment. In some respects, the GAP program in the GCG software package is used to determine the percentage identity between two nucleotide sequences (e.g., using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6). In some alternative aspects, the GAP procedure in the GCG software package can be used to determine the percentage identity between two amino acid sequences (e.g., using a BLOSUM 62 matrix or a PAM250 matrix with nick weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5), which incorporates the Needleman and Wunsch algorithm (J. Mol. Biol. (48): 444-453 (1970)). Alternatively, in some aspects, the Myers and Miller algorithm (CABIOS, 4: 11-17 (1989)) is used to determine the percentage identity between nucleotide or amino acid sequences. For example, percentage homology can be determined using the ALIGN procedure (version 2.0) with a PAM120 matrix having a residue table, 12 nick length penalties, and 4 nick penalties. Those skilled in the art can determine the appropriate parameters for maximum alignment using specific alignment software. In some aspects, the default parameters of the alignment software are used.

[0074] Those skilled in the art will understand that the generation of sequence alignments used to calculate percentage sequence identity is not limited to binary sequence-sequence comparisons driven solely by master sequence data. Sequence alignments can be derived from multiple sequence alignments. A suitable program for generating multiple sequence alignments is ClustalW2, which is available at www.clustal.org. Another suitable program is MUSCLE, available at www.drive5.com / muscle / . Alternatively, CLUSTAL W2 and MUSCLE can be obtained from the European Bioinformatics Institute (EBI).

[0075] In some respects, the percentage identity “X” between the first and second nucleotide sequences is calculated as 100x (Y / Z), where Y is the number of identical matching nucleotide residues in the alignment of the first and second sequences (by visual inspection or a specific sequence alignment procedure), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percentage homology between the first and second sequences will be higher than the percentage homology between the second and first sequences. Different regions within a single polynucleotide target sequence aligned with a polynucleotide reference sequence can each have their own percentage of sequence identity. It should be noted that sequence identity values ​​are 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 should also be noted that length values ​​are always integers.

[0076] According to this disclosure, the degree of identity between a given reference nucleotide sequence and a homologous nucleotide sequence of the given nucleotide sequence is preferably 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 homology is preferably given for a nucleic acid region that occupies 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 total length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of homology is preferably given as 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 consecutive nucleotides. In one specific embodiment, the degree / percentage of similarity or identity is given for the entire length of the reference nucleic acid sequence.

[0077] The term “amino acid identity” as used in this paper refers to the degree to which sequences are identical amino acid-wise within a comparison window. Therefore, the “percentage of sequence identity” is calculated as follows: comparing two best-aligned sequences within a comparison window; determining the number of matching positions by the number of identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appearing in both sequences; dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. According to this disclosure, the degree of identity between a given reference amino acid sequence and a homolog of the given amino acid sequence is preferably 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 homology is preferably given for an amino acid region that occupies 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 total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, it is preferable to provide 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 the degree of identity of consecutive amino acids. In one specific embodiment, the degree / percentage of similarity or identity is given over the entire length of the reference amino acid sequence.

[0078] The term “homology” or “synonyms” encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the unmodified protein and have similar biological and functional activities to the unmodified protein from which they are derived.

[0079] The term "by SEQ ID N" as used in this article o :X-qualified or "as in SEQ ID N" o The ":X" indicates that it is composed of SEQ ID NO. oA biological sequence consisting of the amino acid or nucleotide sequence given in SEQ ID NO: X. For example, a protein defined in SEQ ID NO: X consists of the amino acid sequence given in SEQ ID NO: X. Another example is an amino acid sequence containing SEQ ID NO: X, which refers to an amino acid sequence that is longer than the amino acid sequence given in SEQ ID NO: X but completely contains the amino acid sequence given in SEQ ID NO: X (wherein the amino acid sequence given in SEQ ID NO: X may be located at the N-terminus or C-terminus of the longer amino acid sequence, or may be embedded in the longer amino acid sequence), or an amino acid sequence consisting of the amino acid sequence given in SEQ ID NO: X.

[0080] As used herein, "agonist" refers to a molecule (e.g., a binder, ligand, or antibody) that, through its interaction with a receptor or another target in a signaling pathway, activates downstream signaling, thereby potentially triggering a biological response. In the context of the Wnt / β-catenin signaling pathway, agonists act as activators of this pathway, leading to β-catenin stabilization and aggregation, and subsequently, transcriptional activation of Wnt target genes. Agonists need not necessarily exhibit a strong binding affinity for the receptor, but can still effectively promote pathway activation. In this context, the term "agonist" is used interchangeably with the term "activator," preferably an activator of the Wnt / β-catenin signaling pathway, and more preferably an activator of the Wnt7 / β-catenin signaling pathway.

[0081] Conversely, an "antagonist" refers to a molecule that interferes with or inhibits the activation of downstream signaling pathways, thereby preventing such biological responses. In the context of the Wnt / β-catenin signaling pathway, anagonists act by blocking or inhibiting the pathway, thereby preventing the accumulation of β-catenin and the transcriptional activation of Wnt target genes. Anagonists do not activate the pathway and can act by preventing the initiation of signaling cascade reactions by agonists or natural ligands. In this context, the term "antagonist" may be used interchangeably with the term "inhibitor," preferably an inhibitor of the Wnt / β-catenin signaling pathway, more preferably an inhibitor of the Wnt7 / β-catenin signaling pathway.

[0082] The terms “treatment,” “manipulation,” or “application” are used interchangeably and are defined as: slowing, interrupting, preventing, controlling, stopping, reducing, or reversing the development or severity of symptoms, signs, disorders, conditions, lesions, or diseases through a therapeutic intervention, but not necessarily involving the complete elimination of all disease-related symptoms, signs, conditions, or disorders. The population requiring treatment includes those already diagnosed with the condition, as well as those who are susceptible to or prone to developing the condition, or those who need to prevent the condition. For example, in the treatment of tumors (e.g., cancer), a therapeutic agent can directly reduce the pathological characteristics of tumor cells or make tumor cells more susceptible to treatment by other therapeutic agents or the subject's own immune system.

[0083] As used herein, the terms “diagnosis,” “prognosis,” and / or “prediction” include the diagnosis, prognosis, and / or prediction of a particular disease and / or disorder, thereby predicting the occurrence and / or presence of the particular disease and / or disorder, and / or predicting the progression and / or duration of the particular disease and / or disorder, and / or predicting the response of a patient with the particular disease and / or disorder to treatment.

[0084] The term "statistically significant difference" is well known to those skilled in the art. Statistical significance plays a crucial 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: nothing has happened or changed. To reject the null hypothesis, the observation must be statistically significant, i.e., the observed p-value is less than a pre-specified significance level α. The p-value p is the probability of obtaining at least the same extreme result when the null hypothesis is true. In one embodiment, α is 0.05. In a more specific embodiment, α is 0.01. In an even more specific embodiment, α is 0.001.

[0085] Peptides, polypeptides, or proteins can be naturally occurring, for example, present in nature or obtainable from nature, such as being naturally or endogenously produced or expressed by cells or tissues, and optionally isolated therefrom. Peptides, polypeptides, or proteins can be recombinant, i.e., produced by recombinant DNA technology, and / or can be partially or wholly chemically or biochemically synthesized. Not limited thereto, peptides, polypeptides, or proteins can be recombinantly produced by suitable host or host cell expression systems, and optionally isolated therefrom (e.g., suitable bacterial, yeast, fungal, plant, or animal host or host cell expression systems), or recombinantly produced by cell-free transcription or cell-free transcription and translation, or by abiotic peptide, polypeptide, or protein synthesis.

[0086] Conservative amino acid substitution is the substitution of one amino acid by another amino acid with similar characteristics. Conservative amino acid substitutions include substitutions from 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. Nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (i.e., basic) amino acids include arginine, lysine, and histidine. Negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above polar, basic, or acidic groups by another member of the same group can be considered a conservative substitution. Conversely, nonconservative substitution is the substitution of one amino acid by another amino acid with dissimilar characteristics.

[0087] The term “biologically active” is interchangeable with terms such as “functionally active” or “functional”, indicating that the protein disclosed herein retains at least part of the biological activity or intended function of the corresponding or related peptide, polypeptide, or protein. References to the “activity” of a peptide, polypeptide, or protein may generally encompass any one or more aspects of its biological activity, such as, but not limited to, any one or more aspects of its biochemical activity, enzymatic activity, signal transduction activity, interaction activity, ligand activity, and / or structural activity, for example, in cells, tissues, organs, or organisms.

[0088] Preferably, compared with the corresponding protein, the functionally active protein retains 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%), further 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 expected biological activity or function.

[0089] In some embodiments, the functionally active protein may even exhibit higher biological activity or function compared to the corresponding peptide, polypeptide, or protein, for example, it may exhibit 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 expected biological activity or function compared to the corresponding protein. For example, where the activity of a given protein can be easily measured by a quantitative output assay (e.g., an enzyme assay, a signal transduction assay, or a binding assay that produces a quantifiable signal transduction), the signal transduction produced by the functionally active fragment or variant of the peptide, polypeptide, or protein is at least about 20%, at least about 25%, at least 30%, at least about 40%, at least about 50%, at least 60%, more preferably at least about 70%, at least 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500%.

[0090] In this specification, references to any peptide, polypeptide, protein, or nucleic acid indicate the corresponding peptide, polypeptide, protein, or nucleic acid commonly known under the various names described in the art. More specifically, references to “Wnt,” particularly “Wnt7,” “G protein-coupled receptor 124” (GPR124), “reversion-inducing cysteine-rich protein with Kazal motifs” (RECK), “Frizzled” (FZD), or “lipoprotein receptor-associated protein” (LRP) indicate the corresponding peptide, polypeptide, protein, or nucleic acid as is evident from the context and commonly known under the names described in the art.

[0091] In the context of this invention, "Wnt7" refers to "Wnt7a" and / or "Wnt7b," which are part of the Wnt protein family. In this specification, the terms "Wnt7" and "WNT7" are used interchangeably. "Wnt," "Wingless-associated integration site," "Wingless and Int-1," or "Wingless-Int1" refers to gene families and proteins that include homologous "Wingless" or "Wg" and "integration site" or "Int." In one specific embodiment, Wnt is the Wnt7a protein having the amino acid sequence shown in SEQ ID NO: 1. In another specific embodiment, Wnt is the Wnt7b protein having the amino acid sequence shown in SEQ ID NO: 2.

[0092] The terms "Gpr124 / RECK / FZD / LRP receptor complex," "Gpr124 / RECK / FZD / LRP co-receptor complex," "Gpr124 / RECK / FZD / LRP complex," or "Gpr124 / RECK / FZD / LRP protein complex" broadly refer to protein complexes comprising at least one Gpr124 protein, at least one RECK protein, at least one FZD protein, and at least one LRP protein, particularly membrane-associated protein complexes, and more particularly plasma membrane-associated protein complexes. When located at the plasma membrane of a cell, the GPR124 / RECK / FZD / LRP receptor complex can activate Wnt / β-catenin signaling in the cell in response to an exogenously provided Wnt7 ligand.

[0093] The terms "FZD / LRP receptor complex," "FZD / LRP co-receptor complex," "FZD / LRP complex," or "FZD / LRP protein complex" broadly refer to protein complexes, particularly membrane-associated protein complexes, and more specifically, plasma membrane-associated protein complexes, which contain at least one FZD protein and at least one LRP protein. When the FZD / LRP receptor complex is located at the plasma membrane of a cell, it is able to activate Wnt / β-catenin signaling in the cell in response to extracellularly provided Wnt ligands (e.g., but not limited to Wnt7 ligand).

[0094] In this article, "Wnt signaling" and "Wnt / β-catenin signaling" can be used interchangeably.

[0095] As used herein, “Frizzled,” “Fz,” “FZ,” “Fzd,” or “FZD” refers to at least ten families of atypical G protein-coupled receptors (GPCRs) that act as receptors in the Wnt signaling pathway and other signaling pathways. Upon activation, Frizzled leads to the activation of Dishevelled in the cytoplasm. In some particularly preferred embodiments, the terms refer to Fzd1, Fzd4, Fzd5, and / or Fzd8.

[0096] In one specific embodiment, Fzd is Fzd1, wherein Fzd1 is a protein having the amino acid sequence shown in SEQ ID NO: 9.

[0097] The term “LRP” or “lipoprotein receptor-related protein” encompasses any and all lipoprotein receptor-related proteins, also referred to in the art as low-density lipoprotein receptor-related proteins or prolow-density lipoprotein receptor-related proteins. In some particularly preferred embodiments, the term refers to LRP5, LRP6, or LRP5 and LRP6 (LRP5 / 6). In one specific embodiment, LRP is LRP5 having the amino acid sequence shown in SEQ ID No: 3. In another specific embodiment, LRP is LRP6 having the amino acid sequence shown in SEQ ID NO: 4 or 5. Since both LRP5 and LRP6 can exist as multiple isoforms, those skilled in the art will understand that these isoforms are also covered herein.

[0098] The interaction between Wnt ligand, Frizzled receptor, and LRP is essential for activation of the classical Wnt pathway, which has broad 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 β-catenin. In the nucleus, β-catenin functions as a transcriptional coactivator, regulating the expression of target genes involved in various cellular processes.

[0099] “Reck,” or “RECK,” is a GPI-anchored membrane protein that stands for “a cysteine-rich protein that is reverse-transcribed with a Kazal domain.” In one specific embodiment, Reck is a protein having the amino acid sequence shown in SEQ ID No: 6.

[0100] “Gpr124” or “GPR124” is an orphan GPCR expressed in endothelial cells. Gpr124 is also known as ADGRA2 or adhesive G protein-coupled receptor A2. In one particular embodiment, Gpr124 is a protein having the amino acid sequence shown in SEQ ID No: 7 or 8.

[0101] As used in this specification, the terms “binding,” “interaction,” “specific binding,” or “specific interaction” mean that a reagent binds to or influences one or more desired molecules or analytes, substantially excluding other random or unrelated molecules, and optionally substantially excluding other structurally related molecules. This term does not necessarily require that the reagent bind only to its intended target. For example, if the reagent, under binding conditions, has an affinity for the intended target that is at least about 2 times higher, preferably at least about 5 times higher, more preferably at least about 10 times higher, even more preferably at least about 25 times higher, still more preferably at least about 50 times higher, and even more preferably at least about 100 times higher (e.g., at least about 1000 times higher, at least about 1 x 10⁻⁶) than its affinity for non-target molecules. 4 More than 1x10, or at least about 1x10 5 If the reagent binds to the target specifically (more than twice the amount of the target), then the reagent can be said to bind to the target specifically.

[0102] As used herein, the terms “gene therapy” and / or “RNA therapy” refer to the introduction of exogenous polynucleotides into host cells for therapeutic or preventative purposes, regardless of the method used for such introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (via, for example, viral infection / transfection, or a variety of other protein- or lipid-based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains a replication origin compatible with the host cell or integrates into a replicon of the host cell, such as an extrachromosomal replicon (e.g., a plasmid) or integrates into a nuclear or mitochondrial chromosome. Various vectors are known in the art capable of mediating gene transfer into mammalian cells. The RNA molecule can be any type of RNA molecule. For example, the RNA molecule can be cytoplasmic RNA, nuclear RNA, mRNA, antisense RNA, or non-coding RNA, preferably mRNA.

[0103] The terms "host cell" and "host organism" may suitably include cells or organisms of prokaryotes (e.g., bacteria) and eukaryotes (e.g., yeast, fungi, protozoa, plants, and animals). Host cells that may be considered include, but are not limited to, single-celled organisms such as bacteria (e.g., *Escherichia coli*). E. coli Salmonella typhimurium ( Salmonella typhimurium Serratia marcescens ( ) Serratia marcescens ) or Bacillus subtilis ( Bacillus subtilis )), yeast (e.g., brewer's yeast ( Saccharomyces cerevisiae ) or Pichia pastoris ( Pichia pastoris )), (cultured) plant cells (e.g., from Arabidopsis thaliana) Arabidopsis thaliana ) or safflower tobacco ( Nicotiana tabacum And (cultured) animal cells (e.g., vertebrate cells, mammalian cells, primate cells, human cells, or insect cells). Potential host organisms include, but are not limited to, multicellular organisms such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrates, even more preferably mammals, and even more preferably primates; particularly considered are non-human organisms such as animals and animal species.

[0104] Unless otherwise stated, the terms "subject" or "patient" are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, and even more preferably primates, and specifically include human patients as well as non-human mammals and primates. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects requiring treatment, and more particularly subjects who will benefit from treatment of a given condition, especially a neurological disorder, neurovascular disease, or a central nervous system (CNS) disease involving neurovascular dysfunction; or for the treatment of blood-retinal barrier (BRB) integrity, or ophthalmic diseases or disorders such as retinal vascular disorders or retinal degeneration disorders. Such subjects may include, but are not limited to: individuals diagnosed with the said condition, individuals susceptible to the said condition, and / or individuals requiring prevention of the said condition.

[0105] As used herein, the term "therapeutic effective amount" refers to the amount of an active compound or agent sought by a surgeon, researcher, veterinarian, physician, or other clinician to elicit a biological or medical response in a subject, which may include, but is not limited to, alleviating symptoms of the treated disease or condition. The term "preventive effective amount" refers to the amount of an active compound or agent sought by a researcher, veterinarian, physician, or other clinician to inhibit or delay the onset of a disease in a subject. In the context of this invention, "therapeutic effective amount" is used to refer to both the aforementioned "therapeutic effective amount" and "preventive effective amount," unless the context clearly distinguishes the two. Methods for determining the therapeutic and / or preventive effective doses of compounds, proteins, nucleic acids encoding such compounds / proteins, nucleic acid expression cassettes, or pharmaceutical compositions as taught herein are known in the art. As used herein, the term "therapeutic effective dose" refers to the amount of a compound, protein, nucleic acid encoding such compound / protein, nucleic acid expression cassette, or pharmaceutical composition as taught herein that elicits a positive therapeutic response when administered to a patient suffering from a specific disease or disorder. Detailed Implementation

[0106] The inventors here demonstrate the design of agonists capable of selectively activating Wnt7 signaling in cells expressing RECK and / or GPR124; for example, in cells expressing GPR124, or in cells expressing both GP124 and RECK, i.e., in brain endothelial cells. Such agonists are also disclosed as therapeutic agents, for example, particularly for the treatment of neurological disorders, neurovascular disorders, or central nervous system (CNS) disorders, including neurovascular dysfunction, or ophthalmic diseases or disorders, particularly retinal vascular disorders or retinal degeneration disorders. Therefore, the present invention allows for the provision, among other things, of agonists capable of stimulating Wnt / β-catenin signaling in brain endothelial cells (also referred to as “targeting activity”), which have only low cross-reactivity with other Fz pathways (i.e., “unwanted” or “off-target activity”), and can be used as therapeutic agents, particularly for neurological disorders, neurovascular disorders, or central nervous system (CNS) disorders (including neurovascular dysfunction).

[0107] Surprisingly, the inventors have found that the direct recruitment or binding of agonists with LRPs such as LRP5 or LRP6 and at least Gpr124 or RECK is sufficient to specifically stimulate Wnt7 signaling via the Gpr124 / RECK / FZD / LRP receptor complex.

[0108] In the following paragraphs, different aspects or embodiments of the invention are defined in more detail. Each of these aspects or embodiments may be combined with any other aspect or embodiment unless expressly stated to the contrary. Specifically, any feature indicated as preferred or advantageous may be combined with any other feature indicated as preferred or advantageous.

[0109] Therefore, in a first aspect, the present invention provides Wnt7 signaling pathway agonists, more particularly the Wnt7 signaling pathway mediated by the Gpr124 / RECK / Frizzled / LRP receptor complex, and even more particularly the Wnt7 signaling pathway mediated by the Gpr124 / RECK / Frizzled / LRP (LRP5 or LRP6) receptor complex.

[0110] In one embodiment, the agonist is a selective or specific agonist of the Gpr124- and Reck-dependent Wnt / β-catenin pathway. The terms "selective agonist of the Gpr124- and Reck-dependent Wnt / β-catenin pathway" or "specific agonist of the Gpr124- and Reck-dependent Wnt / β-catenin pathway" should be understood as an agonist that activates the pathway with high selectivity and specificity (targeting activity) without, or with statistical significance, or with only minimal activation of other Wnt signaling pathways (off-target activity). In embodiments, the agonists disclosed herein are agonists of the Gpr124- and Reck-dependent Wnt / β-catenin pathway exhibiting less than 20% off-target activity, more particularly less than 15%, 10%, 5%, or 1%, as measured by a cell culture Super TOP-Flash assay (as known in the art and described in the Examples section below).

[0111] In embodiments, the agonist disclosed herein is an agonist of the GPR124 / RECK / Frizzled / LRP complex, wherein the agonist is capable of binding LRPs, such as LRP5 or LRP6, to the GPR124 protein or to the RECK protein. In embodiments, the agonist is incapable of binding or activating the coiled receptor protein or has impaired ability to bind or activate the coiled receptor protein.

[0112] In one embodiment, the agonist is not a Wnt7 protein or polypeptide, such as Wnt7a or Wnt7b protein or polypeptide, or a simple variant of its sequence having one or more mutations, such as a simple variant having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mutations.

[0113] In some embodiments, one or more, preferably all, of Wnt7, GPR124, RECK, FZD and LRP as used herein are of animal origin, preferably of warm-blooded animal origin, more preferably of vertebrate origin, and even more preferably of mammalian origin, including human and non-human mammalian origins, and even more preferably of human origin.

[0114] Those skilled in the art will understand that any sequence represented in a sequence database or this specification may belong to the precursor of the corresponding peptide, polypeptide, protein, or nucleic acid, and may include portions processed from mature molecules; or, these sequences may represent sequences of mature molecules while precursor sequences are also considered part of this concept.

[0115] The term covers peptides, polypeptides, proteins, or nucleic acids when they form part of a living organism, organ, tissue, or cell; when they form part of a biological sample; and when they are at least partially isolated from such sources. The term also covers peptides, polypeptides, proteins, or nucleic acids when they are produced by recombinant or synthetic means.

[0116] Unless otherwise apparent from the context, any peptide, polypeptide, protein, or nucleic acid mentioned herein also encompasses modified forms of said peptide, polypeptide, protein, or nucleic acid, such as those with post-expression modifications, including, for example, phosphorylation, glycosylation, esterification, methylation, cysteine ​​modification, sulfonation, glutathioneization, acetylation, ubiquitination, oxidation of methionine to methionine sulfoxide or methionine sulfone, cleavage of the signal peptide, removal of the N-terminal Met, and conversion of a prozymogen or precursor hormone to its active form. The above provides a broad definition.

[0117] The agonists disclosed herein can have any of a variety of different structural formats or configurations. The agonists may include a polypeptide and / or a binding moiety other than a polypeptide, such as a small molecule. In certain embodiments, the agonists may include both a polypeptide region and a non-polypeptide binding moiety. In some embodiments, the agonists may comprise a single polypeptide, or they may comprise two or more, three or more, or four or more polypeptides. In some embodiments, the agonists may comprise one, two, three, or four polypeptides, for example, linked, bound, or fused together.

[0118] When the agonist comprises a single peptide, it can 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 can be directly fused or they can be linked via linkers, such as peptide or chemical linkers, including but not limited to any of those disclosed herein.

[0119] When an agonist comprises two or more polypeptides, the polypeptides can be linked by covalent bonds, such as disulfide bonds, and / or non-covalent interactions. For example, the heavy chains of human immunoglobulin IgG interact directly at their CFI3 domain level, while at their CFI2 domain level, they interact through carbohydrates linked to asparagine (Asn)N84.4 in the DE ring.

[0120] In one implementation, the agonist described herein can be designed.

[0121] In another embodiment, the agonists described herein may be water-soluble. By "water-soluble," or simply "soluble," it means a composition that is soluble in an aqueous buffer solution without the presence of detergents, typically at concentrations that provide a biologically effective dose of the peptide. The agonists disclosed herein typically form substantially homogeneous aqueous solutions with concentrations of at least 25 μM and higher, such as at least 25 μM, 40 μM, or 50 μM, typically at least 60 μM, 70 μM, 80 μM, or 90 μM, and sometimes up to 100 μM, 120 μM, or 150 μM. In other words, the agonists disclosed herein typically form substantially homogeneous aqueous solutions with 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.

[0122] The agonists described herein can be designed to promote the binding between two peptides. For example, amino acid modifications to the pore can be introduced into two different peptides to promote their binding. Knobs-into-hole mutations of amino acids (AAs) are a rationally designed strategy developed in antibody engineering for heavy chain heterodimerization, used in the production of bispecific IgG antibodies. AA modifications are made to create a protrusion from the CFI3 heavy chain of the first antibody and a pore from the CFI3 heavy chain of the second antibody. The protrusion can be represented by tyrosine (Y), which belongs to the 'very large' IMGT volume class of AAs; while the pore can be represented by threonine (T), which belongs to the 'small' IMGT volume class. Other ways to introduce modifications into peptides to promote their binding are known and available in the art. For example, specific amino acids, such as cysteine, can be introduced and used for cross-linking to form intermolecular disulfide bonds.

[0123] In a particular embodiment, the agonist comprises at least one binding region derived from an antibody or antigen-binding fragment, such as an antibody heavy chain or antibody light chain or a fragment thereof.

[0124] In one embodiment, the agonist disclosed herein can bind simultaneously to GPR124 or RECK protein and LRP protein—more specifically LRP5 or LRP6.

[0125] In one embodiment, the Wnt7 signaling pathway agonist includes one or more LRP binding domains and one or more Gpr124 or Reck binding domains. In a further embodiment, the agonist includes one, two, three, four, or five LRP binding domains and one, two, three, four, or five Gpr124 or Reck binding domains. In a preferred embodiment, the agonist includes one or two LRP binding domains and one or two Gpr124 or Reck binding domains, more preferably two LRP binding domains and two Gpr124 or Reck binding domains.

[0126] In one embodiment, the LRP binding domain or binding region is an LRP5 and / or an LRP6 binding domain or binding region, and / or the LRP binding domain or binding region is capable of binding with LRP5 and / or LRP6.

[0127] In one embodiment, the agonist described herein may be tetravalent or divalent.

[0128] In one embodiment, the agonist described herein is tetravalent.

[0129] In one particular embodiment, the agonist comprises: i) Two regions, each specifically incorporating a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes (e.g., LRP5 and / or LRP6); and ii) Two regions, each specifically binding to one or more Reck or Gpr124 epitope groups.

[0130] In one implementation, each or more LRP binding domains, such as LRP5 / 6 binding domains, are fused in series with one or more Reck binding domains and / or one or more Gpr124 binding domains, and wherein the domains are optionally separated by a connector sequence.

[0131] The connector can be any connector known in the art or described herein. As a non-limiting example, the connector is a 5-element connector, such as GSGGS (SEQ ID NO: 172).

[0132] In another embodiment, the agonist described herein is divalent.

[0133] In one specific embodiment, the agonist includes: i) A region that specifically binds to one or more LRP epitope groups, preferably one or more LRP epitopes, such as a group of LRP5 and / or LRP6 epitopes; and ii) A region that specifically binds to one or more Reck or Gpr124 epitope groups.

[0134] In one implementation, an LRP binding domain, such as an LRP5 / 6 binding domain, is fused in series with a Reck or Gpr124 binding domain, and wherein the domains are optionally separated by a connector sequence.

[0135] The connector can be any connector known in the art or described herein. As a non-limiting example, the connector is a 5-mer connector, such as GSGGS (SEQ ID NO: 172).

[0136] In one specific embodiment, the agonist does not contain an Fzd binding domain. According to certain embodiments, the agonists disclosed herein cannot bind FZD.

[0137] In other or further embodiments, the agonists disclosed herein cannot stimulate the Wnt7 signaling pathway in GRP124 / RECK negative cells. In other embodiments, GRP124 / RECK negative means cells that do not have or express low levels of GPR124 and / or RECK proteins, cells that do not have or express low levels of GPR124 and / or RECK proteins, cells that do not have or have low levels of GPR124 and / or RECK proteins, or cells that do not have or have low levels of GPR124 and / or RECK proteins on their cell surface.

[0138] In other embodiments, GRP124 / RECK positive refers to the expression of GPR124 and / or RECK in cells, the presence of GPR124 and / or RECK proteins in cells, and / or the presence of GPR124 and / or RECK proteins on the cell surface.

[0139] In other or additional embodiments, FZD / LRP positivity may mean the expression of FZD and / or LRP in cells, the presence of FZD and / or LRP proteins in cells, and / or the presence of FZD and / or LRP proteins on the cell surface.

[0140] In a particular embodiment, cells expressing GPR124, RECK, FZD, and LRP proteins at their plasma membrane, more specifically LRP5 or LRP6 cells are cells that naturally express all GPR124, RECK, FZD, and LRP proteins on their cell surface, such as brain endothelial cells.

[0141] In some embodiments, "presence of RECK and GPR124" may refer to the presence of RECK and GPR124 proteins at or near the cell membrane, preferably in close proximity to Frizzled and LRP proteins, more specifically, LRP5 or LRP6. This close proximity promotes the formation of the GPR124 / RECK / Frizzled / LRP receptor complex under conditions favorable to its formation, such as when the agonists taught herein are externally supplied to the cell. In other or further embodiments, when used in gene or RNA therapy, the agonists taught herein may be expressed from the target cell population. On the other hand, the phrase "lack of RECK and / or GPR124" may refer to the absence or absence of RECK and / or GPR124 proteins at the cell membrane. When cells do not express, transcribe, or properly transport RECK and / or GPR124, RECK and / or GPR124 are not present at or near the cell membrane. The absence of RECK and / or GPR124 does not necessarily mean the complete absence of RECK and / or GPR124 protein on the cell membrane, but may, for example, refer to an amount of RECK and / or GPR124 protein that cannot be detected by conventional protein detection or quantification methods known to those skilled in the art (e.g., immunoblotting, immunohistochemistry, or immunofluorescence) or an amount of RECK and / or GPR124 protein below the sensitivity range of such conventional protein detection or quantification methods.

[0142] In other or further embodiments, the agonist disclosed herein is capable of activating GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling, wherein, in the absence of RECK and / or GPR124, the agonist does not activate Frizzled / LRP-mediated Wnt signaling.

[0143] In a particular embodiment, the ability of the agonist disclosed herein to activate GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling but not Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124 refers to the ability of the agonist to activate Wnt7 signaling in cells positive for GPR124, RECK, FZD, and LRP (more specifically, cells positive for LRP5 or LRP6), but not in cells positive for FZD and LRP and negative for GPR124 and / or RECK, wherein cells positive for GPR124, RECK, FZD, and LRP (more specifically, cells positive for LRP5 or LRP6) and cells positive for FZD and LRP and negative for GPR124 and / or RECK are substantially the same.

[0144] In a particular implementation, the ability to activate GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling in the absence of RECK and / or GPR124 without activating classical Frizzled / LRP-mediated Wnt signaling represents the ability to activate atypical Wnt signaling in the presence of RECK and GPR124 but not in the absence of RECK and / or GPR124.

[0145] The ability to activate Wnt7 signaling refers to the ability of the agonists disclosed herein to mimic, reproduce, or approximate the signal transduction effects and / or activities of the natural Wnt7 ligand bound to the Gpr124 / RECK / FZD / LRP complex.

[0146] Activation of Wnt7 signaling can be suitably determined and / or quantified by measuring the expression of one or more Wnt7 target genes, TCF reporter gene expression, β-catenin stabilization, LRP phosphorylation, and / or Axin translocation from the cytoplasm to the cell membrane, as is known in the art. For example, activation of Wnt signaling can be suitably determined and / or quantified by measuring the expression of TCF genes (e.g., by RT-PCR or any other transcript detection method), which is the primary output of Wnt signaling. For example, TCF reporter gene assays (also known as TOP / FOP or TOP scintillation) can be used to assess changes in gene transcription controlled by TCF / LEF. TCF reporter gene assays can be luciferase reporter gene assays. For example, activation of Wnt signaling can be suitably determined and / or quantified by measuring the expression of c-myc, n-myc, LEF1, or c-jun. Alternatively, activation of Wnt signaling can be determined by measuring the location, level, and / or phosphorylation status of β-catenin. A non-limiting example of such an assay is the "β-catenin redistribution assay" (ThermoScientific) that provides recombinant U20S cells stably expressing human β-catenin fused with enhanced green fluorescent protein (C-terminal EGFP). This assay allows observation and monitoring of the translocation of the GFP-β-catenin fusion protein from the membrane to the nucleus. Another way to determine Wnt signaling activation is, for example, to observe Axin translocation using the GFP-Axin fusion protein.

[0147] protein

[0148] In certain embodiments, the agonists disclosed herein are considered capable of activating central nervous system (CNS) Wnt7 signaling or Gpr124 / RECK / FZD / LRP5 or LRP6-dependent Wnt7 signaling if the agonist enhances Wnt / β-catenin signaling baseline or background induced by neutral substances or negative controls by at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, or at least 1 × 10⁻⁶ times. 4 times or at least 1×10 5 Multiples, for example, as measured in determinations described elsewhere in this article.

[0149] In certain embodiments, the agonists disclosed herein may be considered not to activate (classical) Wnt signaling if the compound enhances Wnt / β-catenin signaling by less than 10-fold, for example, particularly by a maximum of 5-fold or 2.5-fold, or if the protein does not enhance or even decrease (e.g., by 2-fold or 5-fold or 10-fold less) Wnt / β-catenin signaling compared to the baseline or background of Wnt / β-catenin signaling induced by a neutral substance or negative control, as determined elsewhere herein.

[0150] In certain embodiments, the agonists disclosed herein are considered to activate GPR124 / RECK / Frizzled / LRP5 or LRP6-mediated Wnt7 signaling (referred to as "targeting activity") but not Frizzled / LRP-mediated Wnt signaling (referred to as "off-target activity") if the targeting signaling activity is induced by the agonist by at least 30% and the off-target activity is induced by it by less than 20%. In embodiments, the agonist can activate GPR124 / RECK / Frizzled / LRP5 or LRP6-mediated Wnt7 signaling by 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 thereof, preferably more than 70%, compared to the control, for example, in the absence of the agonist.

[0151] In an implementation, in the absence of RECK and / or GPR124, the agonist can activate Frizzled / LRP-mediated Wnt at concentrations below 20%, below 18%, below 16%, below 14%, below 12%, below 10%, below 8%, below 6%, below 4%, below 2%, below 1%, and all ranges and subranges thereof, preferably below 10% compared to a control, for example in the presence of Wnt7. Before comparing the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity (representing "on-target activity") with the Frizzled / LRP-mediated Wnt signaling activity in the absence of RECK and / or GPR124 (representing "off-target activity" in this paragraph), the agonist-induced on-target and off-target activities can be normalized to the on-target activity induced by wild-type Wnt7a or b, the latter being, for example, set to represent 100% activity.

[0152] In one embodiment, the agonist of the present invention acts via Wnt7-specific RECK / GPR124 / Frizzled / LRP5 or LRP6-mediated signal transduction, wherein the agonist specifically binds to LRP5 or LRP6 and RECK in a FZD and Gpr124-independent manner. Thus, the agonist recruits Gpr124 to the FZD / LRP complex via RECK, thereby assembling the Wnt7-ligand-specific RECK / GPR124 / FZD / LRP signal transduction complex and activating classical Wnt7 signaling. According to other embodiments, the agonist of this disclosure thus induces heterodimerization of RECK with LRP5 or LRP6 proteins in the presence of Frizzled and GPR124.

[0153] In another embodiment, the agonist of the present invention acts via Wnt7-specific RECK / GPR124 / coil receptor / LRP-mediated signal transduction, wherein the agonist specifically binds to LRP5 or LRP6 and Gpr124 in a RECK and FZD-independent manner. Thus, the agonist recruits RECK to the FZD / LRP complex via Gpr124, thereby assembling the Wnt7-ligand-specific RECK / GPR124 / FZD / LRP signal transducer and activating classical Wnt7 signaling. According to other embodiments, the agonist of this disclosure thus induces heterodimerization of Gpr124 with LRP5 or LRP6 proteins in the presence of frizzled and RECK.

[0154] Therefore, in certain embodiments, the agonists disclosed herein are capable of binding to LRP5 and / or LRP6 proteins, as well as proteins selected from Gpr124 and RECK, preferably at the cell membrane.

[0155] Those skilled in the art will also understand that the GPR124 / RECK / FZD / LRP receptor complex envisioned herein may further include other components, which may or may not functionally modulate the complex. For example, the complex may include hairless (Dvl) cells that form an intracellular cytoskeleton capable of bridging GPR124 and Fz.

[0156] In a particular embodiment, the agonist of the present invention disclosed herein induces 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 full-length wild-type Wnt7 protein GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity. In a preferred embodiment, the agonist disclosed herein has at least 70% of the full-length wild-type Wnt7 protein GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity. In a particular embodiment, in the presence of all four co-receptors, the agonist of the present invention disclosed herein activates more than 30% of the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. For example, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 99%, and all ranges and subranges thereof.

[0157] In certain embodiments, the agonists of the present invention disclosed herein can activate Wnt signaling in the absence of one of the four co-receptors (i.e., RECK, Fzd, LRP, or GPR124) to a degree less than 20% of the 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 thereof.

[0158] In one embodiment, the agonist of the present invention is a binding agent comprising one or more LRP binding domains (e.g., LRP5 or LRP6 binding domains) and one or more Gpr124 or Reck binding domains. In another embodiment, the binding agent is a fusion protein comprising a first polypeptide capable of binding to at least one LRP protein, such as LRP5 or LRP6, and a second polypeptide capable of binding to at least one Gpr124 or Reck protein.

[0159] In one embodiment, the binding domain or peptide can be separated from each other via a linker or spacer sequence, such as a flexible GS or (Gly) linker. Other suitable linker sequences will be known to those skilled in the art.

[0160] In embodiments, the agonists disclosed herein can bind to LRP proteins, particularly LRP5 or LRP6, for example, by binding to the extracellular domains of LRP proteins, their DKK binding sites, and / or Wnt binding sites. In more specific embodiments, the agonists disclosed herein are capable of binding to the DKK1 binding sites of LRP5 and / or LRP6 proteins. In other specific embodiments, the agonists disclosed herein are capable of binding to β-wing domains 1 and 2 (P1E1P2E2) and / or β-wing domains 3 and 4 (P3E3P4E4) of LRP proteins, more particularly to β-wing domains 1 and 2 (P1E1P2E2) and / or β-wing domains 3 and 4 (P3E3P4E4) of LRP5 and / or LRP6 proteins.

[0161] In certain embodiments, the agonists disclosed herein may be selective for 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, or at least 1×10⁻⁶ compared to other less preferred LRP proteins. 4 times or at least 1×10 5 times.

[0162] In various embodiments, the binder of this application comprises a targeting portion having an antigen recognition domain that recognizes epitopes present on the LRP (e.g., on LRP5 and / or LRP6). In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on the LRP, such as LRP5 and / or LRP6. As used herein, a linear epitope refers to any continuous sequence of amino acids present on an LRP, such as LRP5 and / or LRP6. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the LRP, such as LRP5 and / or LRP6. As used herein, a conformational epitope refers to one or more amino acid moieties (which may be discontinuous) forming a three-dimensional surface with characteristic and / or shape and / or tertiary structure, said three-dimensional surface being recognizable by the antigen recognition domain.

[0163] In one particular embodiment, one or more LRP binding domains or targeting portions, such as one or more LRP5 and / or one or more LRP6 binding domains or targeting portions, bind to 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 the LRP5 or LRP6 protein fragments shown in SEQ ID NO: 3, 4, and 5. In another specific embodiment, the amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids.

[0164] In another specific embodiment, the LRP5 protein fragment is the amino acid sequence between positions 32-614 or 644-1254 of SEQ ID NO: 3. In another further specific embodiment, the LRP6 protein fragment is the amino acid sequence between positions 20 and 628 or 631 and 1244 of SEQ ID NO: 4.

[0165] In some embodiments, one or more LRP5 and / or LRP6 binding domains comprise one or more antigen-binding fragments of the antibody. In some embodiments, the one or more antigen-binding fragments are selected from: IgG, scFv, Fab, and VHH or sdAb. In some embodiments, the one or more antigen-binding fragments are humanized.

[0166] In other embodiments, the binder of this application further comprises a targeting portion having an antigen recognition domain that recognizes epitopes present on Reck or Gpr124. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes or one or more conformational epitopes present on Reck or Gpr124.

[0167] In one embodiment, the agonist disclosed herein can bind to the RECK protein involved in Wnt signaling. RECK consists of five N-terminal cysteine ​​knot (CK) domains or regions (i.e., CK1, CK2, CK3, CK4, and CK5), a cysteine-rich domain (CRD), and three Kazal domains located prior to the glycosylphosphatidylinositol (GPI) anchor site. The CK domains, CRD, and Kazal domains are located extracellularly. Therefore, in embodiments, the RECK protein-binding agonist disclosed herein can bind to the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and / or one or more Kazal motifs of the RECK protein, preferably the agonist binding to the CK4 and / or CK5 regions of the RECK protein.

[0168] Therefore, in certain embodiments, the reagents disclosed herein are capable of binding to the CK4 and / or CK5 regions of the RECK peptide. The CK4 motif spans from amino acids C216 to C263, while the CK5 motif spans from amino acids C292 to C338, as disclosed in the amino acid sequence of the human RECK protein annotated under NCB Genbank accession number NP 066934.1. Thus, the CK4 motif of human RECK comprises, and is substantially composed of, or consists of, the amino acid sequence CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID NO: 49), and the CK5 motif of human RECK comprises, and is substantially composed of, or consists of, the amino acid sequence CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC (SEQ ID NO: 50). In certain embodiments, the reagents disclosed herein are capable of binding to the amino acid sequence of SEQ ID NO: 49 and / or the amino acid sequence of SEQ ID NO: 50.

[0169] In another or further embodiment, one or more Reck binding domains or targeting portions are bound to the Reck protein fragment shown in SEQ ID NO: 6 with an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one particular embodiment, the 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 embodiment, the fragment of the Reck protein is the amino acid sequence between positions 23 and 789, 37 and 338, 627 and 673, 698 and 752, or 753 and 789 of SEQ ID No: 6.

[0170] In one embodiment, the RECK binding domain comprises a RECK-specific antibody or a RECK-binding region comprising one or more RECK-specific antibodies, preferably a variable region sequence or CDR. Preferably, the RECK-specific antibody targets the extracellular portion of the RECK peptide, such as the CK4 and / or CK5 regions of the RECK peptide. Non-limiting examples of RECK antibodies include antibodies known in the art and commercially available, such as those from Abcam (e.g., ab88249 and ab89915 specifically targeting the full-length human RECK), Cell Signaling Technology (e.g., #3433 specifically targeting human, mouse, rat, and monkey RECK (D8C7)), and Santa Cruz (e.g., sc-373929 specifically targeting the C-terminus of human RECK).

[0171] In a particular embodiment, the RECK-binding domain comprises at least one, at least two, at least three, at least four, at least five, or at least six CDRs, 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 corresponding CDR of the RECK-specific antibody.

[0172] In a particular implementation, the RECK-binding domain is a RECK-specific scFv, for example, the scFv contains one or more (preferably all six) CDRs of a RECK-specific antibody.

[0173] In a particular implementation, the RECK binding domain is a RECK-specific VHH, such as a VHH containing one or more (preferably all three) CDRs of a RECK-specific heavy chain antibody.

[0174] In certain embodiments, the RECK-binding domain of the reagents disclosed herein may comprise two or more CDRs, each CDR 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 corresponding CDRs of two or more different RECK-specific antibodies. In certain embodiments, the RECK-binding domain of the reagents disclosed herein comprises, is substantially composed of, is derived from, or is derived from (e.g., a biologically active fragment and / or variant of a RECK-binding polypeptide such as a Wnt ligand or a Wnt polypeptide). Preferably, the RECK-binding domain is derived from a Wnt7 polypeptide (e.g., Wnt7a or Wnt7b) described elsewhere herein, more preferably from a human or mouse Wnt7 polypeptide, and even more preferably from a human Wnt7 polypeptide (e.g., human Wnt7a or human Wnt7b). For example, in some embodiments, the RECK-binding domain may comprise, consist substantially of, or be composed of a RECK-binding fragment of Wnt7, preferably human or mouse Wnt7, more preferably human Wnt7 (e.g., human Wnt7a or human Wnt7b) or a variant thereof. In another example, in some embodiments, the RECK-binding domain may comprise, consist substantially of, or be composed 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 NO: 46) or VEVVRASRLRQPTFLRLKQLRSYQKPMET (SEQ ID NO: 48).

[0175] In a particular embodiment, the RECK-binding domain is derived from the Wnt7 polypeptide. In a particular embodiment, the RECK-binding domain comprises, is substantially composed of, or is composed of the following amino acid sequences: HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC (SEQ ID NO:51), VEPVRASRNKRPTFLKIKKPLSYRKPMDT (SEQ ID NO:46), or YEWRASRLRQPTFLRIKQLRSYQKPMET (SEQ ID NO: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 with SEQ ID NO:51, SEQ ID NO:46; or SEQ ID NO:52. In a particular embodiment, the amino acid sequence of the RECK binding domain derived from the Wnt7 polypeptide includes, is substantially composed of, SEQ ID NO: 51, SEQ ID NO: 46; or SEQ ID NO: 52.

[0176] In a particular embodiment, the RECK-binding domain comprises, and is composed of substantially the amino acid sequence XXXVXAXRXXXXXFLXIXXXXXYXKXXXX (SEQ ID NO: 54), VXAXRXXXXXFLXIXXXXXYXK (SEQ ID NO: 55), XXXVXAXRXXXXXFLXXXXXXXXXKXXXX (SEQ ID NO: 56), or VXAXRXXXXXFLXXXXXXXXXK (SEQ ID NO: 57), wherein X is any amino acid, preferably wherein the amino acid sequence has 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 with any one of SEQ ID NO: 51, 52, or SEQ ID NO: 46.

[0177] In one embodiment, the RECK-binding domain is selected from the RECK-binding domain group of the protein ADAMTS10, such as SEQ ID N: 45 (AA823-1103 of hADAMTS10) or a portion thereof; the Wnt7a linker region shown in SEQ ID N: 46 or multiples thereof (e.g., 3-fold Wnt7a linker); or the extracellular domain of Gpr124 (or a portion thereof). In one embodiment, the RECK-binding domain is derived from the N-terminal extracellular domain of Gpr124 and has a sequence identity of 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%, with respect to SEQ ID NO: 58.

[0178] In one embodiment, the Reck binding domain may comprise or consist of an amino acid sequence of 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 identical to the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 58.

[0179] In another or further embodiment, one or more Gpr124 binding domains are bound to 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 the Gpr124 protein fragment shown in SEQ ID NO: 7 and 8. In another embodiment, the 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 yet another more specific embodiment, the Gpr124 protein fragment is an amino acid sequence between positions 34 and 344 of SEQ ID NO: 7, or between positions 82 and 106, or between positions 107 and 130, or between positions 131 and 154, or between positions 156 and 178, or between positions 190 and 240, or between positions 247 and 344.

[0180] In another or further embodiment, the binder of this application comprises a full-length multimeric protein containing two heavy chains and two light chains. Each heavy chain includes a variable region (e.g., VH) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain includes a variable region (VL) and a constant region (CL). As described in the definition section, the variable region determines the antibody specificity and contains three hypervariable regions, also known as complementarity-determining regions (CDRs), which contribute to antibody binding specificity.

[0181] In one embodiment, the Gpr124 binding domain is derived from RECK, preferably a human RECK sequence. It has been reported that the N-terminal portion of RECK binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124. In one embodiment, the Gpr124 binding domain comprises one or more cyclic cysteine ​​knot (CK) domains derived from RECK, preferably human RECK. In one embodiment, the binder comprises CK1 to CK5 of RECK, preferably human RECK (see SEQ IDN: 47). In one embodiment, the GPR124 binding domain comprises a portion of CK1-5 of RECK, preferably hRECK. In one embodiment, the Gpr124 binding domain may comprise, substantially comprise, or comprise 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 given in SEQ ID NO: 47.

[0182] In some embodiments, the binders disclosed herein comprise an LRP-targeting portion, such as an LRP5 or LRP6-targeting portion, and a Gpr124 or Reck-targeting portion, wherein at least one targeting portion is an antibody fragment. The term "antibody fragment" refers to any antibody or part of an antibody-like structure that has a high affinity for an antigenic determinant or epitope and contains one or more CDRs representing this specificity. In some specific embodiments, at least one of the targeting portions of the binder of this application is a single-domain antibody, immunoglobulin monovariable domain, heavy chain-only antibody (VHH), single-chain antibody (scFv), shark heavy chain-only antibody (VNAR), cysteine ​​knot protein (knottin), DARPin, Tetranectin, Affibody, Affimer, Transbody, Anticalin, AdNectin, Affilin, Microbody, peptide aptamer, alterase, plastic antibody, phylomer, stradobodies, maxibodies, evibody, fynomer, armadillo repeat protein, Kunitz domain, ammer, atrimer, probody, immunobody, triomab, troybody, pepbody, vaccibody, UniBody, DuoBody, Fv, Fab, Fab', F(ab')2, peptide mimic molecule, or synthetic molecule, such as U.S. Patent No. or Patent Publication No. US. 7,417,130, US2004 / 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, US2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446 and / or US The entire contents of 7,166,697 are incorporated herein by reference. See Storz 2011 MAbs 3: 310-317.

[0183] In certain embodiments, at least one of the targeting portions of the binder of this 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 camel, a shark, or the VHH may be a engineered VHH. A VHH is an antibody-derived therapeutic protein containing the unique structural and functional properties of naturally occurring heavy-chain antibodies (see the definition section above). In some embodiments, the single-domain antibody described herein is an immunoglobulin single variable domain or ISVD. In most specific embodiments, at least one of the targeting portions of the binder is a VHH. In a further specific embodiment, the VHH comprises a single amino acid chain having four “architectural regions” and three “complementarity-determining regions” or CDRs. As used herein, a “architectural region” refers to a region located within a variable domain between CDRs. As used herein, a “complementarity-determining region” or “CDR” refers to a variable region within the VHH that contains an amino acid sequence capable of specifically binding to an antigen target. In various embodiments, the binder of this application comprises a VHH or ISVD targeting LRP5 or LRP6, said VHH or ISVD having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. The CDR3 sequence represents an essential characteristic of the ISVD family, more specifically, the VHH. The ISVD family as defined herein refers to a group of ISVD amino acid sequences having highly similar or even identical CDR3 sequences. By default, ISVDs belong to the same family when binding to the same target epitope. Variations within the ISVD family can be interesting if the expression / stability / affinity / crystallinity of the representative of that family is poor, as small deviations, such as single amino acid mutations, occurring within a family can improve these properties.

[0184] In one specific embodiment, the ISVD or VHH targeting LRP5 comprises a CDR3 region selected from the list consisting of SEQ ID NO: 38-41 or has an amino acid sequence that differs from SEQ ID NO: 38-41 by up to two amino acids or from SEQ ID NO: 38-41 by up to one amino acid.

[0185] In another specific embodiment, the ISVD or VHH includes the CDR2 region shown in SEQ ID NO: 42 and / or the CDR1 region shown in SEQ ID NO: 43.

[0186] In one most specific embodiment, the anti-LRP5ISVD or VHH comprises an amino acid sequence having 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%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 34, 35, 36, 37, or 53. In a particular embodiment, the difference in amino acid sequence between the homolog and SEQ ID NO: 34, 35, 36, 37, or 53 exists in the framework region.

[0187] In another specific embodiment, the ISVD or VHH targets LRP6 and contains CDR3 as shown in SEQ ID NO: 15, 24, 25 or 26, or has an amino acid sequence that differs from SEQ ID NO: 15, 24, 25 or 26 by at most two amino acids, or has an amino acid sequence that differs from SEQ ID NO: 15, 24, 25 or 26 by at most one amino acid.

[0188] In another specific embodiment, the ISVD or VHH includes a CDR2 region as shown in SEQ ID N: 16, 27, 28 or 29 and / or a CDR1 region as shown in SEQ ID N: 17, 30, 31, 32 or 33.

[0189] In one most specific embodiment, the anti-LRP6ISVD or VHH comprises an amino acid sequence that is 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%, or at least 99% homologous to any one of SEQ ID NO: 14, 18-23.

[0190] In a particular embodiment, the amino acid sequence difference between the homolog and any of SEQ ID NO: 14, 18-23 exists in the framework region.

[0191] In another specific embodiment, the binder of this application targeting LRP5 or LRP6 also includes ISVD or VHH targeting Reck.

[0192] In another, more specific embodiment, the binder of this application that targets LRPs such as LRP5 or LRP6 also includes one that targets ISVD or VHH.

[0193] In one embodiment, the LRP-binding domain of the binder of this application, such as the LRP5 or LRP6 binding domain, or the first polypeptide binding to at least one LRP, such as LRP5 or LRP6, of the fusion protein of this application, comprises the antibody or any antibody fragment, such as ISVD or VHH, described herein, which binds LRP, such as LRP5 or LRP6. In one specific embodiment, the antibody, antibody fragment, ISVD, or VHH binding LRP6 comprises or consists of the amino acid sequence shown in SEQ ID NO: 14. In another specific embodiment, the antibody or antibody fragment binding LRP6 comprises or consists of the amino acid sequences shown in SEQ ID NO: 12 and / or 13.

[0194] In another embodiment, the Gpr124 or Reck binding domain of the binding agent or second polypeptide of this application binds to at least one Gpr124 or Reck, said binding agent or second polypeptide being the Gpr124 or Reck binding domain of a fusion protein containing the antibody or any antibody fragment described herein, such as ISVD or VHH.

[0195] In another specific embodiment, the binder of this application is a bispecific antibody, antibody fragment, or ISVH or VHH, which preferably targets LRP such as LRP5 or LRP6 and Gpr124 or Reck.

[0196] In another or further embodiment, the antibody, antibody fragment ISVD, or VHH is tetravalent.

[0197] In one most specific embodiment, the binder of this application comprises a VHH targeting LRP6, wherein the VHH comprises or consists of the amino acid sequence shown in SEQ ID NO: 14.

[0198] As described above, in one embodiment, the agonist disclosed herein does not contain an Fzd-binding domain. In another or further embodiment, the agonist cannot bind Fzd or is incapable of binding Fzd. This is particularly noteworthy because, based on prior knowledge, it is not obvious that molecules that cannot bind Fzd and / or do not have an Fzd-binding domain and / or are therefore not cross-linked with Fzd can exhibit agonist activity, i.e., be able to activate the Wnt / β-catenin signaling pathway and thus act as activators of the signaling pathway. However, the inventors of this invention have demonstrated that agonists without an Fzd-binding domain and / or unable to bind Fzd can activate the signaling pathway.

[0199] In one embodiment, the agonist does not contain any other binding domains beyond the one or more LRP binding domains and one or more Gpr124 or Reck binding domains.

[0200] In one embodiment, the binding domain of the agonist is selected from one or more LRP binding domains and one or more Gpr124 or Reck binding domains.

[0201] In a preferred embodiment, the agonist binding domain is selected from one or more LRP binding domains and one or more Gpr124 binding domains. In another preferred embodiment, the agonist binding domain is selected from one or more LRP binding domains and one or more Reck binding domains.

[0202] In one embodiment, the agonist is a bispecific antibody having two binding domains. In a preferred embodiment, the agonist is a bispecific antibody having two binding domains, the binding domains comprising an LRP binding domain and a Gpr124 binding domain. In another preferred embodiment, the agonist is a bispecific antibody having two binding domains, the binding domains comprising an LRP binding domain and a Reck binding domain.

[0203] Similar to the aforementioned agonists that do not have an Fzd binding domain and / or cannot bind Fzd, based on previously known information, it is not expected that a molecule possessing only one or more LRP binding domains and one or more Gpr124 or Reck binding domains as binding domains, or possessing only two binding domains selected from the LRP binding domain and optionally the Reck or Gpr124 binding domain, could exhibit agonist activity, i.e., be able to activate the Wnt / β-catenin signaling pathway and thus act as an activator of the signaling pathway.

[0204] In another or further embodiment, in the absence of Reck, the agonists described herein are capable of activating the Wnt / β-catenin signaling pathway and can therefore act as activators of said signaling pathway.

[0205] In another or further embodiment, the agonist described herein comprises an anti-LRP VHH containing the sequence of SEQ ID NO: 59, 60, 61 or 62.

[0206]

[0207] In another or further embodiment, the agonist described herein comprises IgG, and the method includes: - According to SEQ ID N o VH sequences of 63, 65, or 67, and - Based on the VL sequence of SEQ ID NO: 64, 66 or 68, - VH and VL can optionally be separated by a connector sequence.

[0208] Preferably, the agonist described herein comprises IgG, and the method includes: - Based on the VH sequence of SEQ ID NO: 63 and the VL sequence of SEQ ID NO: 64, - Based on the VH sequence of SEQ ID NO: 65 and the VL sequence of SEQ ID NO: 66, or - Based on the VH sequence of SEQ ID NO: 67 and the VL sequence of SEQ ID NO: 68.

[0209]

[0210] In another or further embodiment, the agonist described herein comprises IgG, and the method comprises: - According to SEQ ID N o VH sequences of 69, 71, 73 or 75, and - According to SEQ ID N o VL sequences of 70, 72, 74, or 76, - VH and VL can optionally be separated by a connector sequence.

[0211] Preferably, the agonist described herein comprises IgG, and the method includes: - Based on the VH sequence of SEQ ID NO: 69 and the VL sequence of SEQ ID NO: 70, - According to SEQ ID N o The VH sequence of SEQ ID 71 and the VL sequence according to SEQ ID 72, - Based on the VH sequence of SEQ ID NO: 73 and the VL sequence of SEQ ID NO: 74, or - Based on the VH sequence of SEQ ID NO: 75 and the VL sequence of SEQ ID NO: 76.

[0212]

[0213] In a preferred embodiment, the agonist described herein includes: Anti-LRP VHH, which contains according to SEQ ID N o Sequences of 59, 60, 61 or 62, and Any one: IgG, the method comprising: - Based on the VH sequence of SEQ ID NO: 63, 65 or 67, and - According to SEQ ID N o VL sequences of 64, 66, or 68, - VH and VL can optionally be separated by a connector sequence.

[0214] Alternatively: IgG, the method comprising: - According to SEQ ID N o VH sequences of 69, 71, 73 or 75, and - According to SEQ ID N o VL sequences of 70, 72, 74, or 76, - VH and VL can optionally be separated by a connector sequence.

[0215] Based on the above, this article discloses any combination of VHH and IgG. The most preferred combination is shown in the Examples section.

[0216] In a preferred embodiment, VHH is fused to the N-terminus of VL of IgG, preferably using a linker (e.g., a 5-mer linker) having the sequence GSGGS (SEQ ID NO: 172). Preferably, there is no linker between VL of IgG and VH of IgG.

[0217] Humanization

[0218] In one embodiment, the binder of this application comprises one or more immunoglobulin single variable domains or VHHs, such as “humanized” VHHs targeting LRPs such as LRP5 or LRP6, i.e., one or more amino acid residues in the amino acid sequence of a VHH obtained through immunization are replaced by one or more amino acid residues appearing at corresponding positions in the VH domain of a conventional human four-chain antibody to increase sequence identity with the closest human lineage sequence. Potentially useful homology substitutions can be determined by comparing the sequence of the scaffold region of a native VHH sequence with the corresponding scaffold sequence of one or more closely related human VH sequences. Thereafter, one or more such potentially useful homology substitutions (or combinations thereof) can be introduced into the VHH sequence (in any manner known in the art, as further described herein), and the resulting homologous VHH sequences can be tested for their affinity for the target, stability, ease of expression and level, and / or other desired properties. Thus, through limited trial and error, those skilled in the art can identify other suitable humanization alternatives (or suitable combinations thereof). Furthermore, based on the aforementioned, immunoglobulin single variable domains (framework regions), such as the VHH domain, can be partially or fully humanized.

[0219] Therefore, in various embodiments, the binder of this application comprises a targeting portion comprising an amino acid sequence having one or more amino acid mutations relative to SEQ ID NO: 12, 13, 14, 18, 19, 20, 21, 22, 23, 34, 35, 36, 53 and / or 37. In various embodiments, the binder comprises a targeting portion comprising an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, fifteen or twenty amino acid mutations relative to SEQ ID NO: 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 substitution, insertion, deletion and truncation. In some embodiments, the amino acid mutation is an amino acid substitution and may include conserved and / or non-conserved substitutions. In a particular embodiment, the one or more amino acid mutations may be located within the CDR of the targeting portion (e.g., CDR1, CDR2 or CDR3 region). In other specific embodiments, one or more amino acid mutations may be located within the framework regions of the targeting portion (e.g., FR1, FR2, FR3, or FR4 regions). In most specific embodiments, the one or more amino acid mutations are present only in the framework region FRα of the binder.

[0220] For example, conservative substitutions can be performed based on the similarity between the polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the involved amino acid residues. The 20 natural amino acids can be divided into the following six groups of standard amino acids: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, “conservative substitution” is defined as the substitution of one amino acid by another amino acid within the same group of the above six standard amino acid groups. For example, the substitution of Asp by Glu preserves a negative charge in the thus modified polypeptide. Furthermore, glycine and proline can substitute for each other based on their ability to disrupt the α-helix.

[0221] As used in this article, “non-conservative substitution” is defined as exchanging an amino acid with another amino acid listed in different groups of the six standard amino acid groups (1)-(6) above.

[0222] In various embodiments, substitutions may also include unconventional amino acids (e.g., selenocysteine, pyrrolidone, N-formylmethionine β-alanine, GABA and δ-aminovaleric acid, 4-aminobenzoic acid (PABA), D isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, leucine, valine, hydroxyproline, carnosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, engineered amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general).

[0223] Humanization can be performed using humanization techniques known in the art. In some embodiments, possible humanization substitutions or combinations of humanization substitutions can be determined by methods known in the art, for example, without limitation, by comparing the VHH sequence with a naturally occurring VH domain sequence. In some embodiments, humanization substitutions are chosen such that the resulting humanized VHH retains advantageous functional properties. Generally, due to humanization, the VHH of this application can become more "human-like" than the corresponding naturally occurring VHH domain, while still possessing advantageous properties such as reduced immunogenicity. In various embodiments, the chimeric VHH of this application can be obtained by any suitable method known in the art, and is therefore not strictly limited to peptides obtained using naturally occurring VHH domains as starting materials. In fact, modification of the amino acid sequence can be achieved using techniques known in the art, such as site-directed mutagenesis or PCR-based mutagenesis. For example, these techniques are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0224] In various embodiments, mutations introduced, for example, for humanizing the binder, do not substantially reduce the ability of the binder of the present invention to specifically bind human LRP5 or LRP6 and Gpr124 or Reck, and to activate Gpr124 / Reck / Fzd / LRP5 or LRP6-mediated Wnt7 signaling.

[0225] Production methods

[0226] The binders of this application, particularly antibodies, and more particularly the ISVD or VHH of this application, are not limited to specific biological sources or preparation methods. Methods for preparing the binders of this application are described herein. For example, the DNA sequence encoding the binder of this application can be readily prepared using techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc., and various isolation techniques are those known and commonly used by those skilled in the art. Many standard techniques are described in Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed) (1993), and Ausubel et al. (1992). Alternatively, the DNA sequence encoding the binder of this application can be chemically synthesized using methods known in the art. The synthesized DNA sequence can be ligated with other suitable nucleotide sequences, such as expression control sequences, to produce a gene expression construct encoding the desired FRα binder.

[0227] Therefore, in various embodiments, this application provides isolated nucleic acids comprising a nucleotide sequence encoding an agonist of any of the binding agents described in this application. One embodiment also discloses an expression cassette comprising said nucleic acid molecules. More specific embodiments disclose expression cassettes in which elements for cell-specific or tissue-specific expression are present. Further embodiments involve vectors comprising said expression cassettes or said nucleic acid molecules. More specifically, said vectors may be viral vectors or lentiviral vectors, preferably viral vectors specifically targeting the central and / or peripheral nervous systems (e.g., brain-specific viral vectors). In other specific embodiments, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant. In a preferred embodiment, the viral vector is a blood-brain barrier endothelial cell-specific viral vector. In other preferred embodiments, the viral vector is a blood-brain barrier endothelial cell-specific capsid adeno-associated virus serotype 2 (AAV2) mutant.

[0228] Important factors for selecting a particular vector include, but are not limited to: the selection of recipient cells; the ease with which recipient cells containing the vector can be identified and selected from those that do not contain the vector; the desired vector copy number in a particular recipient cell; whether it is desired that the vector integrates into the chromosome or remains outside the chromosome of the recipient cell; and whether it is desired that the vector can be moved between recipient cells of different species.

[0229] Expression vectors can be autonomous or integrative. Nucleic acids can be introduced into cells using expression vectors (e.g., plasmids, bacteriophages, transposons, granules, or viral particles). Recombinant nucleic acids can be maintained extrachromosomally or integrated into the cell's chromosomal DNA. Expression vectors can contain selectable marker genes that encode proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme essential for uracil biosynthesis, or LEU2, which encodes an enzyme essential for leucine biosynthesis, or TRP1, which encodes an enzyme essential for tryptophan biosynthesis), to allow detection and / or selection of cells transformed with the target nucleic acid. Expression vectors can also contain autonomous replication sequences (ARS). ARS can contain a centromere (CEN) and an origin of replication (ORI). For example, an ARS can be ARS18 or ARS68.

[0230] An integration vector typically comprises a sequence of at least a first insertable DNA fragment, a selection marker gene, and a second insertable DNA fragment arranged sequentially. The first and second insertable DNA fragments are each about 200 nucleotides in length (e.g., about 250, 300, 350, 400, 450, 500, or 1000 or more) and have nucleotide sequences partially homologous to the genomic DNA of the cell species to be transformed. A nucleotide sequence containing the target nucleic acid is inserted into the vector between the first and second insertable DNA fragments, either before or after the marker gene. The integration vector may be linearized prior to transformation to facilitate the integration of the target nucleotide sequence into the cell genome. Before introducing the vector into the target cell, the vector may be grown (e.g., amplified) in bacterial cells (e.g., *Escherichia coli*). The vector DNA can be isolated from the bacterial cells by any method known in the art, resulting in the purification of the vector DNA from the bacterial environment. The purified vector DNA can be thoroughly extracted with phenol, chloroform, and ether to ensure that the plasmid DNA preparation is free of E. coli proteins, as these proteins may be toxic to mammalian cells.

[0231] To generate the binder of this application, an expression vector containing a nucleic acid sequence encoding the binder can be subsequently introduced into a host cell via transfection, transformation, or transduction techniques. Therefore, in various embodiments, this application provides a host cell containing a nucleic acid encoding one of the binders of this application. For example, the nucleic acid encoding the binder of this application can be introduced into a host cell via retroviral transfection. Exemplary 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, mouse ascites fibroblast (BHK) cells, monkey kidney cells (COS), human liver cancer cells (e.g., HepG2), and myeloma cells. Transformed host cells can be cultured under conditions that allow the host cells to express the gene encoding the binder of this invention.

[0232] Based on the above expression, the binder can be harvested and purified using techniques known in the art, such as affinity tags like glutathione-transferase (GST) and histidine (His) tags, or by chromatography. Specific expression and purification conditions will vary depending on the expression system used. 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 of a suitable bacterial promoter, such as Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in a eukaryotic host cell, such as CHO cells, it is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, secretory signal, enhancer, and various introns. In one embodiment, the binder of the application comprises a His tag, a FLAG-tag, and / or a Myc tag. In one embodiment, the binder of this application comprises a His tag and a proteolytic site to allow cleavage of the His tag.

[0233] Therefore, this application also provides a host cell comprising one of the binding agents described herein. This application also provides a host cell comprising one of the nucleic acid molecules, expression cassettes, or vectors of this application. The host cell may be prokaryotic or eukaryotic. Representative host cells that can be used in this invention include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells. Bacterial host cells suitable for use with this 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. Suitable yeast host cells for use in this invention include strains of *Saccharomyces*, *Schizosaccharomyces*, *Kluyveromyces*, *Pichia* (e.g., *Pichia pastoris*), *Hansenula* (e.g., *Hansenula polymorpha*), *Yarowia*, *Schwaniomyces*, *Schizosaccharomyces*, *Zygosaccharomyces*, and similar species. *S. cerevisiae*, *S. carlsbergensis*, and *K. lactis* are the most commonly used yeast hosts and convenient fungal hosts. Suitable animal host cells for use in this invention include insect cells and mammalian cells (most particularly derived from Chinese hamsters (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 thaliana* cells, tomato cells, maize cells, algal cells, and the like. The host cells can be provided in the form of suspension, bottle culture, tissue culture, organ culture, etc. Alternatively, the host cells can also be transgenic animals.

[0234] Animal or mammalian host cells suitable for accommodating, expressing, and producing one of the FRα binders of this application include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al. 1986 SomCell Mol Genet 12: 555-556; Kolkekar et al. 1997 Biochemistry 36: 10901-10909), CHO-K1Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT) designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), CHO clone B (GEIMG, Genova, IT) designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), and other designated ECACC clones. CHO-K1 / SF of 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 of ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub & Chasin 1980 PNAS 77:4216) and dp12.CHO cells (US Patent No. 5,721,121); monkey kidney CV1 cells transformed with SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells used for suspension culture clones, Graham et al. 1977 J GenVirol 36:59, or GnTI KO HEK293S cells, Reeves et al. 2002 PNAS 99:13419); hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse supporting cells (TM4, Mather 1980 Biol Reprod 23:243-251); human cervical cancer cells (HELA, ATCC). CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human liver cancer cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383: 44-68); MCR 5 cells; FS4 cells. According to one specific embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

[0235] The host cells described above can be transfected transiently or stably. For example, transfection of DNA from nucleic acid molecules, expression cassettes, or expression vectors into prokaryotic and eukaryotic cells can be achieved by any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation, liposome-mediated, DEAE-glucan-mediated, multivalent cation-mediated, or virus-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*, 2009). ndEd. (RIFreshney. 1987. Liss, Inc. New York, NY). The host cell can also be a recombinant host cell, which involves cells that have been genetically modified to contain the isolated DNA molecule, nucleic acid molecule, or expression construct or vector of the present invention. DNA can be introduced by methods known in the art suitable for specific cell types, including but not limited to transformation, liposome transfection, electroporation, or virus-mediated transfection.

[0236] Furthermore, in alternative embodiments, a vector providing the nucleic acid molecules, expression cassettes, or binding agents, particularly antibodies, antibody fragments, ISVDs, or VHHs, described herein is used to produce said binding agents, antibodies, antibody fragments, ISVDs, or VHHs. In one specific embodiment, the use is for the production of in vivo antibodies. Intracellular antibodies, or “endobodies,” are antibodies or fragments thereof heterologously expressed in a designated intracellular compartment, made possible by in-frame integration of intracellular transport signals. Endobodies can be expressed in any shape or form, such as intact IgG molecules or Fab fragments, more specifically as genetically engineered antibody fragments, such as single-domain endobodies or VHHs. See Zhu and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. Ed. Springer-Verlag Berlin, Heidelberg).

[0237] Medical applications

[0238] As previously mentioned, atypical Wnt7 signaling mediated by the Gpr124 / Reck / Fzd / LRP5 or LRP6 complexes is crucial for initiating blood-brain barrier differentiation and maintaining BBB function, as well as blood-retinal barrier differentiation and maintaining BRB function. Furthermore, stimulation of Wnt7 signaling in brain / retinal endothelial cells with impaired BBB / BRB can restore BBB / BRB and improve related disease effects.

[0239] Therefore, in another aspect of the invention, any agonist, binder, or fusion protein disclosed herein, as well as any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or carrier described herein, are provided for use as a medicament. This is equivalent to providing a treatment method comprising the step of administering to a subject or patient in need any agonist, binder, or fusion protein disclosed herein, or any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or carrier described herein.

[0240] In a particular embodiment, the treatment method is a method for treating or preventing a disease in which abnormal Wnt signaling is present in cells, tissues, and / or organs expressing RECK and / or GPR124.

[0241] In one specific embodiment, the agonist, binder, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector is used for gene therapy, and more particularly for treating blood-brain barrier integrity.

[0242] In another specific embodiment, the agonist, binder, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector is used for gene therapy, and more particularly for blood-brain barrier endothelial cell-guided gene therapy.

[0243] Therefore, a method for gene therapy is also provided herein, particularly a method for central and / or peripheral nervous system-directed gene therapy for a subject requiring said gene therapy, comprising: introducing a nucleic acid expression cassette or vector described herein into the subject, particularly into the subject's central and / or peripheral nervous system; and expressing, in the subject, particularly in the subject's central and / or peripheral nervous system, a therapeutically effective amount of a Wnt7 agonist encoded by said nucleic acid, as taught herein.

[0244] In certain implementations, agonists or nucleic acids encoding the antibodies taught herein are used in mRNA therapy, particularly in blood-brain barrier endothelial cell-directed mRNA therapy.

[0245] Therefore, an RNA therapy, preferably an mRNA therapy, particularly a central and / or peripheral nervous system-directed mRNA therapy, is also provided herein for a subject requiring said mRNA therapy, comprising: introducing a nucleic acid encoding any of the agonists disclosed herein into the subject, particularly into the subject's central and / or peripheral nervous system; and expressing in the subject, particularly in the subject's central and / or peripheral nervous system, a therapeutically effective amount of the agonist encoded by the nucleic acid taught herein.

[0246] The advantage of using RNA therapy is that RNA is generally believed not to integrate into the genome, and therefore there is no risk of insertional mutations.

[0247] This article may use any other known methods for introducing nucleic acids into animal cells. Most simply, nucleic acids can be directly injected into target cells / tissues. Other methods include fusion of recipient cells with bacterial protoplasm containing nucleic acids, using combinations such as calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids, or liposomes, or similar receptor-mediated endocytosis, bio-ejector particle bombardment (“gene gun” method), infection with viral vectors (i.e., derived from lentiviruses, adeno-associated viruses (AAVs), adenoviruses, retroviruses, or antivirals), electroporation, etc. Other techniques or methods suitable for delivering nucleic acid (NA) molecules to target cells include continuous delivery of NA molecules from poly(lactic acid-co-glycolic acid) polymer microspheres or direct injection of protected (stable) NA molecules into a micropump of the delivery product. Another possibility is the use of implantable drug-release biodegradable microspheres. Encapsulation of NA or delivery of NA in various types of liposomes (immunoliposomes, PEGylated (immunoliposomes), cationic lipids and polymers, nanoparticles or dendritic molecules, poly(lactic-co-glycolic acid) polymeric microspheres, implantable drug-release biodegradable microspheres; and injection of NA with a protective agent such as the nuclease inhibitor uracil nucleoside carboxylic acid. It should be understood that combinations of different delivery modalities or methods described above can also be used.

[0248] In certain embodiments, the Wnt7 agonist is provided as a carrier, such as liposomes, lipid nanoparticles, structured lipid carriers, nanoemulsions, polymer nanoparticles, polymer micelles, or dendrimers. In a preferred embodiment, the carrier is a liposome or lipid nanoparticle (LNP).

[0249] In one embodiment, the carrier is a lipid-based carrier. The lipid-based carrier comprises one or more lipids. The one or more lipids may be in solid and / or liquid form. The lipid-based carrier may be LNPs, liposome complexes, liposomes, phospholipid microcapsules, solid lipid nanoparticles, structured lipid carriers, or nanoemulsions. Lipid-based carriers useful according to the invention include, for example, cationic lipids, liposomes, particularly cationic liposomes, as well as micelles and nanoparticles. Cationic lipids can form complexes with negatively charged nucleic acids. Any cationic lipid can be used according to the invention. Liposomes are self-assembled phospholipid and cholesterol bilayers surrounding a water core, in which hydrophilic molecules can be incorporated. Hydrophobic compounds can also be incorporated into the lipid bilayer. Liposomes can be classified according to their size and monolayer nature into (i) small monolayer vesicles (SUVs), (ii) large monolayer vesicles (LUVs), and (iii) multilayer vesicles (MLVs). Solid lipid nanoparticles (SLNs) have a spherical shape with an average diameter of 10-1000 nm. They are used as colloidal NP drug delivery systems in which lipid drug carriers solidify at room temperature and at body temperature. Different solid lipids can be used to generate SLNs, such as tripalmitoylglycerol, cetyl alcohol, cetearyl stearate, glyceryl monostearate, trimyristate, tristearate, stearic acid, etc. SLNs comprise solid lipids such as triacylglycerols, fatty acids, waxes, partially glycerol esters, and polyethylene glycol-modified lipids; emulsifiers such as sorbitol esters, poloxamer, and lecithin; and water. Nanostructured lipid carriers (NLCs) comprise mixtures of solid and liquid lipids, resulting in partially crystalline lipid systems, and many offer advantages such as enhanced drug loading capacity, flexibility in drug release regulation, and improved stability.

[0250] In one embodiment, the lipid-based carrier is a lipid nanoparticle. Solid lipid nanoparticles (SLNs, sLNPs), or lipid nanoparticles (LNPs), are nanoparticles composed of lipids suitable for use as drug delivery carriers for pharmaceutical compounds, particularly polynucleotides such as RNA or DNA.

[0251] In another embodiment, any agonist, binder, or fusion protein disclosed herein, as well as any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector described herein, are provided for the treatment of neurological and / or ophthalmic disorders.

[0252] Methods for treating blood-brain barrier dysfunction or neurological conditions, or blood-retinal barrier dysfunction or ophthalmic conditions such as retinal vascular diseases or retinal degeneration, are also provided, comprising the steps of administering to a desired subject or patient any agonist, binder, or fusion protein disclosed herein, or any nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or vector described herein. In one specific embodiment, the administration is performed via gene therapy, more particularly via blood-brain barrier endothelial cell-guided gene therapy or blood-retinal barrier endothelial cell-guided gene therapy.

[0253] A method for stimulating, activating, or activating Gpr124 / Reck / Fzd / LRP5 or LRP6-mediated Wnt7 signaling in cells is also provided, the method comprising administering to the cells a therapeutically effective amount of any of the agonists, binders, fusion proteins, nucleic acids, nucleic acid sequences, nucleic acid molecules, expression vectors, or carriers described herein, wherein the cells express at least Gpr124, Reck, Fzd, more specifically Fzd1, and an LRP protein selected from LRP5 and LRP6. In another embodiment, in the absence of RECK and / or GPR124, the agonist, binder, fusion protein, nucleic acid, nucleic acid sequence, nucleic acid molecule, expression vector, or carrier cannot activate classical Wnt signaling or Wnt7 signaling.

[0254] In one specific implementation, the neurological disorder used herein is a disorder selected from a list consisting of ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation (AVMs), cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, intracranial atherosclerosis, and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorder, epilepsy, neurodegenerative diseases, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Charcot-Marie Tooth disease, dystonia, infectious brain disease, traumatic brain injury, migraine, neuroinflammation, COVID-19, chronic traumatic encephalopathy, and combinations thereof.

[0255] "Neuroinflammatory disease" can refer to neurovasculitis. Furthermore, neuroinflammatory disease can be triggered by various factors. For example, in a non-limiting embodiment, neuroinflammatory disease is caused by injuries such as traumatic brain injury and / or spinal cord injury, infections such as viral, bacterial, and fungal infections, exposure to toxins or due to toxic metabolites, neurodegenerative diseases, autoimmune diseases, smoking or passive smoking, aging, or any combination thereof.

[0256] Neuroinflammatory diseases can be caused by infectious diseases. These infections can be bacterial, fungal, parasitic, or viral in origin. In some cases, the infectious disease is a viral infection. In some cases, viral infections include those caused by one or more of the following: Herpesviruses (Herpes Zoster Virus (VZV) infection, Herpes simplex virus (HSV-1 or HSV-2), Cytomegalovirus (CMV)), Paramyxoviridae, Coronaviruses (SARS-CoV-1 or MERS-CoV or SARS-CoV-2, HCoVNL63, HCoV-229E, HCoV-OC43, or HKU1 or their variants), Influenza viruses (Influenza A, Group 1 (H1N1), 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 Papillomavirus (HPV), Human T-cell Leukemia Virus Type 1 (HTLV-1) and Kaposi's sarcoma herpesvirus (KSHV).

[0257] Neuroinflammation can be associated with lung conditions such as acute lung injury (ALI) and / or acute respiratory distress syndrome (ARDS), which may be associated with or result from the use of mechanical ventilation in the subject, viral infection, sepsis, or systemic bacterial infection. The lung condition may be caused by an infectious disease resulting from bacterial, fungal, parasitic, or viral infection. Preferably, the infection is an infection causing respiratory problems or a respiratory tract infection. Preferably, the infection is a viral infection.

[0258] Neuroinflammation can be associated with bacterial infection. This inflammation is primarily caused by blood-borne bacteria that cross the blood-brain barrier (BBB) ​​and eventually invade the brain parenchyma. Pathogens such as Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae are the main causes of bacterial meningitis.

[0259] In this context, prevention and / or treatment of neuroinflammation may include encephalitis. In some cases, encephalitis is viral encephalitis, such as encephalitis associated with COVID-19. Neuroinflammation may also be associated with inflammation of the optic nerve. Neuroinflammation can also be associated with acute disseminated encephalomyelitis, such as acute disseminated encephalomyelitis associated with coronavirus disease 2019. Neuroinflammation may also be associated with inflammation of vitreoretinal ganglion cells.

[0260] In another or further embodiment, the ophthalmic disease or disorder is a retinal vascular disorder or a retinal degenerative disorder. Preferably, the ophthalmic disease or disorder is selected from: 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.

[0261] In embodiments, the agonists described herein and compositions comprising them are particularly suitable for administration to patients susceptible to or suffering from this condition. In certain embodiments, the agonists or compositions are administered orally or non-gastrointestinally, such as intravenously, intraperitoneally, subcutaneously, or intraocularly.

[0262] In certain embodiments, the pharmaceutical composition described herein (see below) is administered to a subject by injection (e.g., intravenous or intraocular) or transplantation of allogeneic cells transformed with the vector described herein. Upon administration, the injected or transplanted allogeneic cells will transcribe and translate in vivo nucleic acids encoding the Wnt7 agonist, as taught herein.

[0263] The dosage or amount of the Wnt7 agonist taught herein, optionally combined with one or more other active compounds to be administered, depends on the individual and is generally adapted to the individual condition to achieve optimal effect. Therefore, the unit dose and regimen depend on the nature and severity of the disease to be treated, and also on factors such as: the subject's species, sex, age, weight, general health condition, diet, route and timing of administration, immune status, and the individual responsiveness of the person or animal to the compound, the effectiveness, metabolic stability, and duration of action of the compound used, whether the therapy is acute, chronic, or prophylactic, or whether other active compounds have been administered in addition to the Wnt7 agonist described in any of the above embodiments. To optimize therapeutic efficacy, the Wnt7 agonist taught herein may be administered initially according to different dosing regimens. Typically, appropriate screening assays can be used as part of a clinical testing procedure to monitor the level of the Wnt7 agonist in tissues, for example, to determine the effectiveness of a given treatment regimen. The frequency of administration is within the technical and clinical judgment of the medical practitioner (e.g., physician, veterinarian, or nurse). Typically, dosing regimens are established through clinical trials, which can establish optimal dosing parameters. However, practitioners can modify this dosing regimen based on one or more of the factors mentioned above, such as the subject's age, health condition, weight, sex, and medical status. The frequency of dosing can also be varied depending on whether the treatment is preventative or therapeutic.

[0264] The toxicity and therapeutic efficacy of the Wnt7 agonist as described herein, or the pharmaceutical composition comprising the Wnt7 agonist as described in any of the above embodiments, can be determined by known pharmaceutical procedures, such as in cell cultures or laboratory animals. These procedures can be used, for example, to determine the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective to 50% of the population). The dose-to-therapeutic index between toxic and therapeutic effects can be expressed as the ratio of LD50 / ED50. Pharmaceutical compositions exhibiting a high therapeutic index are preferred. While pharmaceutical compositions exhibiting toxic side effects can be used, care should be taken to design delivery systems that target such compounds to the affected tissue sites to minimize potential damage to normal cells (e.g., non-target cells), thereby reducing side effects.

[0265] Data obtained from cell culture assays and animal studies can be used to determine a dose range suitable for appropriate subjects. The dose of such pharmaceutical compositions is typically within a range of circulating concentrations that include an ED50 with little or no toxicity. This dose can vary within this range depending on the dosage form and route of administration used. For pharmaceutical compositions used as described herein, the therapeutically effective dose can first be estimated from cell culture assays. Doses can be developed in animal models to achieve a range of circulating plasma concentrations that include the IC50 (i.e., the concentration of the pharmaceutical composition that achieves maximum symptom inhibition) determined in cell cultures. Such information can be used to more accurately determine the dose that is useful in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0266] In certain embodiments, a continuous delivery system, such as a (partially) implanted continuous delivery system, is used to administer the Wnt7 agonist taught herein. Those skilled in the art will understand that such a continuous delivery system may include reservoirs, pumps, and infusion devices (e.g., tubing systems) for containing the Wnt7 agonist as taught herein. For example, a continuous delivery system may be a microosmotic pump system implanted in the brain.

[0267] In certain embodiments, the agonist disclosed herein is the main active ingredient or the only active ingredient of the pharmaceutical composition.

[0268] In another embodiment, the pharmaceutical composition described in any of the foregoing embodiments can be used in combination with a second therapy, preferably selected from surgery, chemotherapy, radiotherapy or immunotherapy.

[0269] Pharmaceutical compositions and formulations

[0270] This document also discloses pharmaceutical compositions comprising any of the agonists, binders, nucleic acids, nucleic acid expression cassettes, or carriers described in any of the embodiments, and optionally pharmaceutically acceptable loaders, diluents, excipients, or adjuvants.

[0271] As used herein, the term "pharmaceutically acceptable" is consistent with the prior art and means that it is compatible with other components of a pharmaceutical composition and does not cause harm to the recipient.

[0272] As used herein, "carrier" or "excipient" includes any and all solvents, diluents, buffers (e.g., neutral buffered saline or phosphate buffered saline), solubilizers, colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners, reagents for achieving reservoir effects, coating agents, antifungal agents, preservatives, antioxidants, isotonic regulators, absorption delay agents, etc. The use of such media and reagents in pharmaceutically active substances is well known in the art. Unless any conventional media or reagents are incompatible with the active substance, their use in therapeutic compositions may be considered.

[0273] Illustrative, non-limiting carriers used in the formulation of pharmaceutical compositions include, for example, water-in-oil or oil-in-water emulsions, aqueous compositions with or without an organic cosolvent suitable for intravenous (IV) or intraocular use, liposomes or surfactant-containing vesicles, microspheres, microbeads and microparticles, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers that are obvious to those skilled in the art.

[0274] In a specific embodiment, any of the agonists, binders, bispecific antibodies, nucleic acids, nucleic acid expression cassettes, or carriers described herein as part of a pharmaceutical composition are provided in liposomes or lipid nanoparticles.

[0275] The pharmaceutical compositions contemplated herein can be formulated for use essentially in any route of administration, such as, but not limited to, oral administration (e.g., oral or inhalation), intranasal administration (e.g., intranasal inhalation or intranasal mucosal administration), parenteral administration (e.g., subcutaneous, intravenous (IV), intraocular, intramuscular, intraperitoneal, intrathecal, or intracisional injection or infusion), percutaneous or transmucosal administration (e.g., oral, sublingual, intranasal), local administration, rectal, vaginal, or tracheal injection, etc. In this way, the therapeutic effects obtainable by the methods and compositions, depending on the specific needs of a given application, can be, for example, systemic, local, tissue-specific, etc.

[0276] For example, for oral administration, the pharmaceutical composition may be formulated as pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard gelatin capsules and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.

[0277] For example, for oral or nasal aerosol or inhalation administration, the pharmaceutical composition may be formulated for administration as an aerosol or spray, for example, as a solution, suspension, or emulsion of the compound described herein or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable solvent (e.g., ethanol or water, or mixtures of such solvents). If desired, the formulation may also additionally contain other pharmaceutical excipients, such as surfactants, emulsifiers, stabilizers, and propellants.

[0278] Examples of carriers for transmural application depend on the specific route of administration, such as oral, sublingual, intranasal, etc., and are generally known in the art.

[0279] For example, for parenteral administration, the pharmaceutical composition can advantageously be formulated as a solution, suspension, or emulsion, using suitable solvents, diluents, solubilizers, or emulsifiers generally known in the art. Injectable solutions or suspensions can be formulated using suitable non-toxic, parenteral-acceptable diluents or solvents according to known techniques. The compounds of the present invention and their pharmaceutically acceptable salts can also be lyophilized, and the resulting lyophilized products can be used, for example, in the production of injectable or infusion formulations.

[0280] When aqueous formulations are preferred, they may contain one or more surfactants. For example, the composition may be in the form of a micellar dispersion containing at least one suitable surfactant, such as a phospholipid surfactant. Various types of phospholipids are known in the art. Typically, the molar ratio of surfactant to active substance in aqueous formulations is from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, but any effective amount of surfactant may be used in the aqueous formulation to best suit the specific target of interest.

[0281] In a preferred embodiment, the pharmaceutical compositions taught herein containing the agonist of the present invention or encoding the agonist nucleic acid are administered via a parenteral route. More preferably, the pharmaceutical compositions taught herein are administered, for example, via intravenous infusion, intrathecal administration, or intraocular administration.

[0282] When the dosage form for rectal administration is a suppository, these formulations can be prepared by mixing the compounds of the present invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glycerides, or polyethylene glycol, which is solid at normal temperatures but liquefies and / or dissolves in the rectal lumen to release the drug.

[0283] Those skilled in the art will recognize that the above description is illustrative and not exhaustive. In fact, many additional formulation techniques and pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, and the development of dosages and treatment regimens suitable for the use of the specific compositions described herein is also well known.

[0284] Those skilled in the art will recognize that the above description is illustrative and not exhaustive. In fact, many additional formulation techniques and pharmaceutically acceptable excipients and carrier solutions are well known to those skilled in the art, as are the development of dosages and treatment regimens suitable for the use of the specific compositions described herein.

[0285] Example

[0286] The invention will now be further described with reference to the following embodiments. The invention is by no means limited to the given embodiments or the implementations presented in the accompanying drawings.

[0287] Example 1: Bispecific molecular activation of Wnt7 signaling

[0288] Endothelial cells at the brain border (BBB) ​​express both Gpr124 and Reck, which form a quaternary co-receptor complex with one of the ten frizzled receptors and one LRP co-receptor selected from LRP5 and LRP6. Compared to cells outside the CNS, Wnt7 signaling is controlled not only through the classical Fzd / LRP5 or LRP6 pathway but also through atypical signaling pathways mediated by the Gpr124 / Reck / Fzd / LRP5 or LRP6 receptor complexes. The latter is crucial for establishing and maintaining a functional, leak-proof blood-brain barrier. To develop specific agonists capable of repairing the atypical Wnt7 signaling pathways in the dysfunctional BBB of patients with neurological disorders, several binding agents targeting LRP5 or LRP6 on one hand and at least Gpr124 or Reck on the other have been developed.

[0289] To demonstrate the concept, we utilized Super Top Flash (STF) cells, HEK293 cells expressing firefly luciferase under the control of Wnt / β-catenin downstream signaling. STF cells allow for luminescence-based readout, which is directly proportional to Wnt signaling. STF cells were transiently transfected with vectors expressing Fzd1, LRP6, Reck (Reck+ / - flag) with or without a flag tag, and Gpr124 (Gpr124+ / - flag) with or without a flag tag.

[0290] When the Wnt7a construct is expressed, the emission signal is measured and set to 100% for normalization purposes.

[0291] Next, four different antibodies were constructed. Figure 2 ): - AB0002: Anti-LRP6 VHH fused to the N-terminus of each light chain (LC) of anti-flag IgG ( Figure 2 A).

[0292] - AB0005: The VH-CH1 of anti-flag IgG is fused to the N-terminus of the heavy chain (HC) of anti-LRP6 IgG; the LC (VL-CL) of anti-flag and anti-LRP6 IgG, expressed separately, achieves correct pairing in the CL / CH1 pair by introducing a complementary mutant. Figure 2 B).

[0293] - AB0006: The N-terminus of the anti-LRP6 IgG VL fusion and the anti-flag IgG LC fusion are present; the N-terminus of the anti-LRP6 IgG VH fusion and the anti-flag IgG HC fusion are present. Figure 2 C).

[0294] - AB0007: The VL of anti-flag IgG is fused to the N-terminus of the LC of anti-LRP6 IgG; the VH of anti-flag IgG is fused to the N-terminus of the HC of anti-LRP6 IgG. Figure 2 D).

[0295] The sequences of different antibody fragments are as follows: VL (SEQ ID NO:10) that resists flags: DVLMTQIPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIR VH (SEQ ID NO: 11) that resists flags: QVQLQQSAAELARPGASVKMSCKASGYSFTTYTIHWVKQRPGQGLEWIGYINPSSGYAAYNQNFKDETTLTADPSSSTAYMELNSLTSEDSAVYYCAREKFYGYDYWGQGATLTVSS VL (SEQ ID NO:12) resistant to LRP6: DIQMTQSPSSSLSASVGDRVTITCRASQSISLNWYQKKPGKKPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK VH (SEQ ID NO: 13) against LRP6: EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRDAPGKGLEWVASISSTSGSKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAKTYYDFWSGYYTFDYWGDGTLVTVSS Anti-LRP6VHH (SEQ ID NO: 14): DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELLVAMIRPVVTEIDYADSVKGRFTISRNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS The antibody construct was transiently expressed in Fzd1 / LRP6 / Reck / Gpr124 STF cells, where Reck or Gpr124 was either tagged with luciferase or untagged.

[0296] For all antibody constructs, luminescence signals were measured and normalized using Wnt7a construct data. Antibody data were obtained in the presence of the Wnt secretion inhibitor IWP-2. Wnt7a data and control data without Wnt7a were obtained without the use of IWP-2. In control experiments, luminescence signals were measured only in cells expressing Gpr124, Reck, Fzd1, and LRP6.

[0297] according to Figure 3 It is clear that all four antibody constructs binding LRP6 and Gpr124 stimulated the Wnt7 signaling pathway at 50% to 100% of the native Wnt7a ligand. The antibody constructs containing the Flag-tagged Reck protein expressed in STF cells showed lower signal intensity than the control antibody, but still significantly higher.

[0298] These data suggest that atypical Wnt7 signaling mediated by Gpr124 / Reck / Fzd / LRP5 or LRP6 can be stimulated by compounds that bind only two of the four co-receptor proteins, and more particularly by binding LRP5 or LRP6 on one hand and Gpr124 or Reck on the other.

[0299] Example 2: Bispecific antibodies as functional Wnt7 substitutes

[0300] Next, dose-response curves were established for the bispecific antibodies targeting LRP6 and Gpr124. STF cells were transiently transfected with vectors encoding the following proteins: Fzd1, LRP6, Reck, and flag-Gpr124. The purified antibody as described in Example 1 or a control antibody (not shown) was added to the cells at concentrations increasing from 0 to 400 nM. Figure 4 The results for Wnt7a activity against AB0007 at concentrations from 0 to 17 nM, normalized relative to saturation, are shown. This allows for characterization of the antibody based on its signal transduction capabilities. For AB0007, the activity was calculated at 50% of the maximum Wnt7a signal transduction. Figure 4 AB0007 can induce Wnt7 signaling in a dose-dependent manner, EC 0.5. 50 It is 1.7 nM.

[0301] Example 3: Wnt7 signal propagation independent of RACK

[0302] In the next experiment, it was investigated whether the aforementioned antibody required all four members of the receptor complex to induce Wnt7 signaling. STF cells were transiently transfected with vectors encoding the following proteins: Flag-Gpr124, Fzd1, and LRP6. Cells were additionally transfected with Reck or not. Purified Flag / LRP6 bispecific antibody was added to the cells at a concentration of 4 nM. As a control, cells transfected with vectors encoding Flag-Gpr124, Fzd1, and LRP6, or cells transfected with vectors encoding Flag-Gpr124, Fzd1, LRP6, and Reck, were additionally transfected with a vector encoding Wnt7a.

[0303] from Figure 5 As can be seen, the Gpr124 / LRP6 bispecific antibody exerts its agonist activity in a manner independent of Reck.

[0304] Example 4: Therapeutic Bispecific Compound

[0305] Although the data obtained above using Flag-tagged versions of Gpr124 or Reck with bispecific anti-LRP6 / anti-Flag antibodies are compelling and supportive for multispecific binding agents used as agonists of the atypical Wnt7 signaling pathway, novel constructs were prepared to confirm the results published herein.

[0306] First, several bispecific compounds containing LRP5 and Gpr124 binding domains or LRP6 and Gpr124 binding domains were constructed.

[0307] Second, several bispecific compounds containing LRP5 and Reck binding domains or LRP6 and Reck binding domains were constructed.

[0308] The CDR region sequences of anti-LRP5 and anti-LRP6VHH are shown in Tables 1 and 2.

[0309] Instances of such constructs are shown in Figure 6 The protein domains that bind to Gpr124 or Reck (with or without the (G4S)2 linker) fusion with anti-LRP6 VHH form a bispecific tetravalent Fc-fusion form. In the example shown, the protein domain binding to Gpr124 is hReck CK1-5 (27-340). The protein domains binding to Reck are selected from hADAMS10 (823-1103) (SEQ ID NO45); 3×Wnt7a linker (linker-(GSG)-linker-(GSG)-linker) (SEQ ID NO46—Wnt7a linker) or zGpr124-ECD (25-742) (SEQ ID NO: 44).

[0310] Vectors encoding four constructs (VHH anti-LRP6-3xWnt7a adapter-Fc and VHH anti-LRP6-hRECK:ECD-Fc; with or without G4S adapter) were transiently expressed in STF cells expressing Gpr124, Reck, Fzd1, and LRP6. Results are shown in... Figure 7 The tested constructs showed agonist activity.

[0311] The sequences of different binding domains are as follows: zGpr124 (25-742) (SEQ ID NO: 44): AGCPELFSSGCSCTEDRSKAHPTPGTRRKVSCGGKELTETPEVSLLPNRTVSLNLSNNRIRMLKNGSFAGLSSLEKLDLRNNLISTIMPGAFLGLTALRKLDLSSNRIGCLTPEMFQGLTNLTKLNISGNIFSSLDPNVFMELHSLKLVNFHSEFLSCDCGLRWVPSFFRSGSARLGDETLCAYPRRLQNKPLRLLRESDLSCEGPLELHTLSLLPSQRQVVFKGDRLPFHCTASLVDKITALHWRQNGQPVTSDPTKGIHLEESVQHDCTFITSELILSNVHVEASGEWECVVSTGRGNTSCSVEIVVLENSASFCPEQKVNNNRGEFRWPRTLAGITSYQHCLQLRYPSLTLGGGVEQKKASRNCDRSGRWEEADYSQCLYTNDITRILHTFILMPVNASNAVTLAHQVRSYTLEAAGFTDTVDVLYVAQMMHKFMDYVTELRELSEVLVEMGSNLMQVDDQILARAQREERACSSIVYTLETLAWPQLHSHAQDLSRYSRNIVMEAHLIRPAHFTGISCTVYQRREGAAGSQVHDGADLSLEQQLRFRCTTGTHNTSLNAFHLKNAVALATVSLPATLFPPNAPPDCKLQFVAFRNGRFFPFTSNFTGHSDLARRRGISTPVIYAGLDGCSMWNQSDPIIVSLRHTSPGHDPVAAHWNSQALGHHGSWSLDGCQLIHSDVSISTLRCSVLSNYAVLQEIPDFPGSPSIPVEVLHP hADAMTS10 (823-1103) (SEQ ID NO: 45): SLPPYSWHYAPWTKCSAQCAGGSQVQAVECRNQLDSSAVAPHYCSAHSKLPKRQRACNTEPCPPDWVVGNWSLCSRSCDAGVRSRSVVCQRRVSAAEEKALDDSACPQPRPPVLEACHGPTCPPEWAALDWSECTPSCGP GLRHRVVLCKSADHRATLPPAHCSPAAKPPATMRCNLRRCPPARWVAGEWGECSAQCGVGQRQRSVRCTSHTGQASHECTEALRPPTTQQCEAKCDSPTPGDGPEECKDVNKVAYCPLVLKFQFCSRAYFRQMCCKTCHGH hWnt7a connector (171-199) (SEQ ID NO: 46): VEPVRASRNKRPTFLKIKKPLSYRKPMDT hReck CK1-5 (27-340) (SEQ ID NO: 47): GLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAV ENYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTCLA Similar experiments were repeated with constructs having LRP5 binding domains, yielding similar results.

[0312] Materials and Methods

[0313] Expression plasmid construct

[0314] All genes were cloned using the pCS2+ vector as a template. Identification of all constructs was confirmed by Sanger sequencing.

[0315] Super TOP-fluorescent reporter gene assay

[0316] Dual-luciferase assays were performed using the Super TOP-Flash HEK293 luciferase reporter gene cell line, known in the art. Cells were transiently transfected as described in the examples. Assays were performed and activity was measured 48 hours after transfection. The data shown (mean ± standard deviation (SD)) are from three technical replicates.

[0317] Example 5: Generation of antibody-based Wnt7a mimics

[0318] Materials and Methods

[0319] Preparation of recombinant Gpr124, Reck and LRP6 proteins

[0320] - The extracellular domain (residues 34-344) of human and mouse Gpr124 is produced in mammalian expression systems as an Fc fusion protein or with a C-terminal 6-His extension and is purified by conventional chromatography techniques.

[0321] - The extracellular domains (residues 27-340) of human and mouse Reck cells with C-terminal 6-His extensions were produced in mammalian expression systems and purified by conventional chromatographic techniques.

[0322] - Purified human LRP6 extracellular domain (residues 20-630) was purchased from Sino Biological (cat.17052-H08H).

[0323] - The mouse LRP6 extracellular domain (residues 20-628) with the C-terminal 6-His extension was generated in a mammalian expression system and purified by conventional chromatography.

[0324] Production of anti-Gpr124, anti-Reck, and anti-LRP6 antibodies

[0325] - Anti-Gpr124 antibodies and anti-Reck antibodies (IgG) were isolated from the immature Fab full-human phage library through multiple rounds (up to four rounds) of screening on the corresponding antigens. Human and mouse antigens were used alternately during the screening process to facilitate the identification of human / mouse cross-reactive antibodies. HEK293 cells transiently expressing the corresponding full-length human antigen were also used at a specific stage of the phage library screening, typically in the fourth round.

[0326] - Anti-LRP6 nanobodies (VHHs) were isolated from an immature VHH library on phages through multiple rounds (up to four rounds) of screening on recombinant LRP6. Human and mouse antigens were used alternately during the screening process to facilitate the identification of human / mouse cross-reactive VHHs. HEK293 cells transiently expressing the corresponding full-length human LRP6 protein were also used at a specific stage of the phage library screening, typically in round 4.

[0327] Production of anti-Gpr124 / LRP6 and anti-Reck / LRP6 bispecific antibodies

[0328] - Through a 5-residue linker (GSGGS-SEQ ID No: 172), anti-LRP6 VHH is fused to the N-terminus of the VL (variable light chain) domain of anti-Gpr124 or anti-ReckIgG, wherein the IgG is human IgG1 with an Fc-silenced L234A, L235A, P329G “LA-LA-PG” mutation (Lo et al., J. Biol. Chem. 292, 3900-3908, 2017).

[0329] - Constructs exhibiting agonist activity were identified by screening in STF (SuperTopFlash) cells transiently expressing human Gpr124, Reck, Frizzled-1 (Fzd1), and LRP6.

[0330] - Constructs with agonist activity can be identified by binding to some or all of the following LRP6 VHHs using the following anti-Grp124 or anti-Reck IgG: o Gpr124 IgG: 05A10 (SEQ ID No. 63 and 64), 06C03 (SEQ ID No. 65 and 66), 06A05 (SEQ ID No. 67 and 68) o Reck IgG: 01A08 (SEQ ID N) O 69 and 70), 01F11 (SEQ ID N) O 71 and 72), 03A04 (SEQ ID N) O 73 and 74), 07B05 (SEQ ID N) O 75 and 76) o LRP6 VHH:01F11 (SEQ ID N O 61), 01B01 (SEQ ID N) O 59), 01E12 (SEQ ID NO 60), 02G02 (SEQ ID NO 62) Figure 8 The structures of anti-Gpr124 / LRP6 and anti-Reck / LRP6 bispecific antibodies are shown, wherein the LRP6 VHH is fused to the N-terminus of the VL domain of the corresponding anti-Gpr124 or anti-Reck IgG.

[0331] Each construct is tetravalent, meaning it has two binding sites for Gpr124 or Reck and two binding sites for LRP6, unless otherwise explicitly stated.

[0332] 5.1: Gpr124 / LRP6 or Gpr124 / LRP5 bispecific antibody

[0333] Gpr124 / LRP6

[0334] Identification of bispecific constructs with agonist activity

[0335] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP6.

[0336] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0337] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd1, LRP6, and Wnt7a were used as positive controls (“Wnt7a”). Note that the amount of Wnt7a vector used was selected to give the highest possible signal.

[0338] - Bispecific antibodies were added to the cells shown at a concentration of 50 nM.

[0339] - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements

[0340] Results: All tested constructs were able to activate the Wnt / β-catenin pathway to levels exceeding those achieved with the natural ligand Wnt7a, such as... Figure 9 As shown.

[0341] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP6.

[0342] - Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the indicated concentration.

[0343] - Activation of the Wnt / β-catenin pathway was assessed using a fluorescence assay.

[0344] Results: Both bispecific antibodies tested showed agonist activity with low EC50 values ​​(EC50 values ​​of 1.9 nM and 1.7 nM for 01E12-05A10 and 01B01-06C03, respectively). Figure 10 As shown.

[0345] Two other bispecific antibodies were then tested:

[0346] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP6; - Bispecific antibodies 01B01-06A05 (A) or 01E12-06A05 (B) were added to the cells at the indicated concentration. - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements Results: Both bispecific antibodies showed agonist activity with low EC50 values ​​(2.8 nM and 3.0 nM for 01B01-06A05 and 01E12-06A05, respectively). Figure 11 As shown.

[0347] Gpr124 / LRP5

[0348] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP5.

[0349] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0350] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd1, LRP5, and Wnt7a served as positive controls (“Wnt7a”). Notably, the amount of Wnt7a vector used was selected to provide the highest possible signal.

[0351] - The bispecific antibody was added to the cells as instructed, at a concentration of 20 nM.

[0352] - Assess activation of the Wnt / β-catenin pathway using luminescence assays

[0353] Results: Bispecific antibodies 01E12-05A10, 01B01-06C03, and 01E12-06C03 activated the Wnt / β-catenin pathway in STF cells expressing hLRP5 instead of hLRP6, such as Figure 12 As shown. This indicates that: (1) VHH 01B01 and 01E12 exhibit cross-reactivity with hLRP5; and (2) agonist activity can be achieved by binding Gpr124 and LRP6 or Gpr124 and LRP5.

[0354] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP5.

[0355] - Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the specified concentration.

[0356] - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements

[0357] Results: Both bispecific antibodies tested showed agonist activity with low EC50 values ​​(4.2 nM and 1.5 nM for 01E12-05A10 and 01B01-06C03, respectively). Figure 13 As shown.

[0358] Reck independence

[0359] - Transfect STF cells with vectors encoding human Gpr124, Fzd1, and LRP6.

[0360] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0361] - Cells transfected with vectors encoding human Gpr124, Fzd1, LRP6, and Wnt7a served as an additional control (“Wnt7a”). Notably, the amount of Wnt7a vector used was chosen to obtain the highest signal.

[0362] - The bispecific antibody was added to the cells at a concentration of 20 nM as instructed.

[0363] - Assess activation of the Wnt / β-catenin pathway using luminescence assay.

[0364] Results: All tested bispecific antibodies were able to activate the Wnt / β-catenin pathway in a manner independent of Reck, i.e., in the absence of Reck, such as... Figure 14 As shown. In contrast, and as predicted, the natural ligand Wnt7a showed no activity in the absence of Reck.

[0365] Fzd receptor dependence

[0366] - Transfect STF cells with a vector encoding human Gpr124, Reck, Fzd4, and LRP6 (A) or Gpr124, Reck, Fzd5, and LRP6 (B).

[0367] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0368] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd4, LRP6, and Wnt7a (A) or Gpr124, Reck, Fzd5, LRP6, and Wnt7a (B) were used as an additional control (“Wnt7a”). Note that the amount of Wnt7a vector used was selected to obtain the highest possible signal.

[0369] - The bispecific antibody was added to the cells at a concentration of 50 nM as instructed.

[0370] - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements

[0371] Results: All tested constructs were able to activate the Wnt / β-catenin pathway to levels exceeding those achieved with natural ligands in the presence of Gpr124, Reck, LRP6, and either Fzd4 or Fzd5 (and Fzd1, to be discussed above). Figure 15 As shown. Based on the data described herein, the bispecific antibody can acquire activity using any of the 10 Fzd receptors present in humans.

[0372] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd4, and LRP6; - Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the concentrations indicated. - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements Results: Both bispecific antibodies tested showed agonist activity with low EC50 values ​​(3.7 nM and 1.3 nM for 01E12-05A10 and 01B01-06C03, respectively). Figure 16 As shown.

[0373] Bispecific antibodies exhibit agonist activity in hRMEC and hBMEC.

[0374] - Treat hRMECs (human retinal microvascular endothelial cells) (A) and hBMECs (human brain microvascular endothelial cells) (B) with recombinant Wnt3a (13.4 nM) or the antibody shown (10 nM).

[0375] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0376] Results: Both bispecific antibodies activated the Wnt pathway to levels comparable to those achieved with the natural ligand Wnt3a, such as... Figure 17 As shown.

[0377] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly effective agonists in hRMEC.

[0378] - Treat hRMEC (human retinal microvascular endothelial cells) with 01E12-05A10 (A) or 01B01-06C03 (B) at the concentrations shown.

[0379] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0380] Results: Both bispecific antibodies activated the Wnt pathway and exhibited low EC50 values ​​(66 pM and 32 pM for 01E12-05A10 and 01B01-06C03, respectively). Figure 18 As shown.

[0381] 01B01-06C03 regulates Wnt / β- in hRMEC cells cladin Other media of the pathway

[0382] - Treat hRMEC cells with 01B01-06C03 (10nM) for 6 hours

[0383] - Monitoring downstream mediator levels of Lef1 (lymphoid enhancer-binding factor 1) and NKD1 (naked epidermal protein 1) mRNA in the Wnt / β-catenin signaling pathway using qRT-PCR.

[0384] Results: Treatment of hRMEC cells with 01B01-06C03 resulted in a moderate but detectable increase in the expression of Lef1 and NKD1, such as Figure 19 As shown.

[0385] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly effective agonists in hBMEC.

[0386] - Treat hBMECs (human brain microvascular endothelial cells) at the specified concentration using 01E12-05A10 (A) or 01B01-06C03 (B).

[0387] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0388] Results: Both bispecific antibodies activated the Wnt pathway and exhibited low EC50 values ​​(17 pM and 61 pM for 01E12-05A10 and 01B01-06C03, respectively). Figure 20 As shown.

[0389] - Agonist activity requires simultaneous binding of Gpr124 and LRP6. bEnd.3 cells (bEnd.3 cells are mouse brain endothelial cells) were treated with 01E12-05A10, where irrelevant VHHs were linked to 05A10, or 01E12 was linked to irrelevant IgG (1 nM), or with two “irrelevant VHHs”-05A10 and 01E12-“irrelevant IgG” (1 nM each) for 24 hours.

[0390] o 01E12-05A10

[0391] o Unrelated VHH-05A10

[0392] o 01E12-'Irrelevant IgG'

[0393] - Monitoring the levels of Axin2, Lef1, Mfsd2a, and PLVAP mRNA by qRT-PCR

[0394] Result: As Figure 21 As shown, activity was detected only with the construct that can bind to both Gpr124 and LRP6 (i.e., 01E12-05A10).

[0395] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly potent agonists in bEnd.3 cells.

[0396] - Treat bEnd.3 cells (mouse brain endothelial cells) with 01E12-05A10 (A), 01B01-06C03 (B), or recombinant Wnt3a (C) at the concentrations shown.

[0397] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0398] Results: Both bispecific antibodies tested activated the Wnt pathway in this specific cell line, and its EC50... 50 The value was significantly lower than the EC50 value of the natural ligand Wnt3a. 50 (EC of 01E12-05A10 and 01B01-06C03) 50 25pM and 61pM respectively, vs Wnt3a EC 50 (6.56 nM). Compared to Wnt3a, the maximum signal amplitude of the bispecific antibody is also larger, such as... Figure 22 As shown.

[0399] Bispecific antibodies 01E12-06CO3 and 01F11-06CO3 are highly potent agonists in bEnd.3 cells.

[0400] - Treat bEnd.3 cells (mouse brain endothelial cells) with 01E12-06C03 (A), 01F11-06C03 (B), or recombinant Wnt3a (C) at the concentrations shown.

[0401] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0402] Results: Both bispecific antibodies tested activated the Wnt pathway in this specific cell line, and its EC50... 50 The value was significantly lower than the EC50 value of the natural ligand Wnt3a. 50(Both 01E12-06C03 and 01F11-06C03 have a maximum signal amplitude of 18 pM, while Wnt3a has a maximum signal amplitude of 6.1 nM). Compared to Wnt3a, the bispecific antibody also has a larger maximum signal amplitude, such as... Figure 23 As shown.

[0403] 01E12-05A10 regulates Wnt / β- in bEnd.3 cells cladin Other media of the pathway

[0404] - Treat bEnd.3 cells for 24 hours with 01E12-05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 (“CHIR”, 10 μM); - The levels of downstream mediators of Lef1 (lymphocyte enhancer-binding factor 1) and Mfsd2a (protein 2a containing major promoter superfamily domain) mRNA in the Wnt / β-catenin signaling pathway were monitored by qRT-PCR, as well as the level of PLVAP (plasma membrane vesicle-associated protein) mRNA, a protein downregulated by classical Wnt.

[0405] Results: Treatment of bEnd.3 cells with both CHIR 99021 and 01E12-05A10 resulted in increased expression of Lef1 and Mfsd2a and decreased expression of PLVAP. Figure 24 As shown.

[0406] 01B01-06C03 regulates Wnt / β- in bEnd.3 cells cladin Other media of the pathway

[0407] - Treat bEnd.3 cells with 01B01-06C03 (10 nM) for 24 h.

[0408] - The levels of downstream mediators of Lef1 (lymphocyte enhancer-binding factor 1) and Mfsd2a (protein 2a containing major promoter superfamily domain) mRNA in the Wnt / β-catenin signaling pathway, as well as the levels of PLVAP (plasma membrane vesicle-associated protein) mRNA, a classic Wnt downregulated protein, were monitored by qRT-PCR.

[0409] Results: Treatment of bEnd.3 cells with 01B01-06C03 resulted in increased expression of Lef1 and Mfsd2a and decreased expression of PLVAP, such as Figure 25 As shown.

[0410] agonists active This can be achieved through divalent molecules.

[0411] STF cells were transfected with vectors encoding human Gpr124, Reck, Fzd1, and LRP6.

[0412] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0413] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd1, LRP6, and Wnt7a were used as positive controls (“Wnt7a”). Note that the amount of Wnt7a vector used was selected to produce the highest possible signal.

[0414] - The following antibodies were added to the cells at a concentration of 20 nM (see...) Figure 34 B)

[0415] o 01F11-06C03, 01B01-06C03: See other sections; o 01E12-06C03 (VHH-Fab): A VHH-Fab fragment derived from 01F11-06C03 o 01E12-05A10 (VHH-Fab): VHH-Fab fragment derived from 01B01-06C03 - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements Results: All constructs showed similar activity, regardless of whether they were tetravalent (VHH-linked IgG) or bivalent (VHH-Fab), such as Figure 34 As shown in Figure A.

[0416] 5.2: Reck / LRP6 bispecific antibody

[0417] Identification of bispecific constructs with agonist activity

[0418] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP6.

[0419] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0420] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd1, LRP6, and Wnt7a served as positive controls (“Wnt7a”). Notably, the amount of Wnt7a vector used was selected to provide the highest possible signal.

[0421] - The bispecific antibody was added to the cells at the indicated concentration of 50 nM.

[0422] - Use fluorescence measurements to assess activation of the Wnt / β-catenin pathway

[0423] Results: All tested constructs activated the Wnt / β-catenin pathway, achieving activation levels close to those achieved with the natural ligand Wnt7a for at least some constructs, such as... Figure 26 As shown.

[0424] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd1, and LRP6.

[0425] - Bispecific antibodies 01B01-01F11 (A), 01E12-01F11 (B), 01E12-03A04 (C), or 01B01-03A04 (D) are added to the cells at the indicated concentrations.

[0426] - Assess activation of the Wnt / β-catenin pathway using luminescence assays

[0427] Results: All tested bispecific antibodies showed agonist activity with low EC50 values ​​(EC50 values ​​of 4.2 nM, 3.4 nM, 1.3 nM, and 6.4 nM for 01B01-01F11, 01E12-03A04, and 01E12-01F11 and 01B01-03A04, respectively). Figure 27 As shown.

[0428] - Transfect STF cells with vectors encoding human Gpr124, human Reck, human Fzd1 and mouse LRP5; - Cells transfected with the empty vector were used as a negative control (“control group”). - Cells transfected with the vector encoding human Gpr124, human Reck, human Fzd1, mouse LRP5, and human Wnt7a were used as positive controls (“Wnt7a”). Notably, the amount of Wnt7a vector used was selected to obtain the highest signal.

[0429] - The bispecific antibody was added to the cells as instructed, at a concentration of 50 nM.

[0430] - Assess activation of the Wnt / β-catenin pathway using fluorescence measurements

[0431] Results: All tested antibodies were able to activate the Wnt / β-catenin pathway in STF cells expressing mouse LRP5, such as... Figure 28 As shown, (1) VHH 01B01 and 01E12 exhibit at least some degree of cross-reactivity with mouse LRP5, and (2) agonist activity can be achieved by binding Reck and LRP6 or LRP5.

[0432] Fzd receptor dependence (1)

[0433] - Using the code Gpr124, Reck, Fzd4, and LRP6 ( Figure 27 A), or Gpr124, Reck, Fzd5 and LRP6 ( Figure 27 B) Transfection of STF cells with the vector

[0434] - Cells transfected with the empty vector were used as a negative control (“control group”).

[0435] - Cells transfected with vectors encoding human Gpr124, Reck, Fzd4, LRP6, and Wnt7a (A) or Gpr124, Reck, Fzd5, LRP6, and Wnt7a (B) served as an additional control (“Wnt7a”). Notably, the amount of Wnt7a vector used was selected to provide the highest possible signal.

[0436] - The bispecific antibody was added to the cells as instructed, at a concentration of 50 nM.

[0437] - Assess activation of the Wnt / β-catenin pathway using luminescence assays

[0438] Results: All tested constructs were able to activate the Wnt / β-catenin pathway in the presence of Gpr124, Reck, LRP6, and either Fzd4 or Fzd5 (and Fzd1, see above), to levels equal to or exceeding those achieved using the natural ligand Wnt7a. Figure 29 As shown.

[0439] Based on the data, the bispecific antibody described in this paper can be active using any of the 10 Fzd receptors present in humans.

[0440] Fzd receptor dependence (2)

[0441] - Transfect STF cells with vectors encoding human Gpr124, Reck, Fzd4, and LRP6.

[0442] - Bispecific antibodies 01B01-01F11 (A), 01E12-03A04 (B), or 01B01-03A04 (C) were added to the cells at the indicated concentrations.

[0443] - Activation of the Wnt / β-catenin pathway was assessed using fluorescence measurements.

[0444] Result: As Figure 30As shown, all tested bispecific antibodies exhibited agonist activity with low EC50 values ​​(5.5 nM, 5.9 nM, and 1.8 nM for 01B01-01F11, 01E12-03A04, and 01B01-03A04, respectively).

[0445] Reck / LRP6 antibody regulates multiple Wnt / β-catenin target genes in bEnd.3 cells.

[0446] - Treat bEnd.3 cells with the Abs (10 nM) shown for 24 hours.

[0447] - The levels of downstream mediators of the Wnt / β-catenin signaling pathway Axin2 (axon repressor protein 2), Lef1 (lymphoid enhancer binding protein 1), and Mfsd2a (major promoter superfamily domain containing protein 2a) mRNA, as well as the level of classic Wnt downregulated protein PLVAP (plasma membrane vesicle-associated protein) mRNA, were monitored by qRT-PCR.

[0448] Result: As Figure 31 As shown, treatment of bEnd.3 cells with the Reck / LRP6 bispecific antibody resulted in increased expression of Axin2 (A and B), Lef1 (C), and Mfsd2a (D) and decreased expression of PLVAP (E and F).

[0449] Bispecific antibodies 01B01-01A08, 01E12-03A04, and 01E12-01F11 are highly active in bEnd.3 cells. Effective agonists

[0450] - Treat bEnd.3 cells for 24 hours with 01B01-01A08 (A), 01E12-03A04 (B), or 01E12-01F11 (C) at the concentrations stated above.

[0451] - Assess Wnt pathway activation by monitoring Axin2 mRNA levels using qRT-PCR.

[0452] Result: As Figure 32 As shown, 01B01-01A08, 01E12-03A04, and 01E12-01F11 activate the Wnt pathway in this specific cell line, EC 50 The values ​​were 10 pM, 7 pM and 7 pM respectively.

[0453] Example 6: In vivo data

[0454] The antibody was constructed in the manner described above.

[0455] Bispecific antibody 01E12-05A10 improves the blood-retinal barrier (BRB) properties of Ndp KO pups.

[0456] The Ndp gene is responsible for producing the Norrin protein, which plays a crucial role in the development and maintenance of the retina and its blood vessels. In this knockout mouse, a truncated form of Norrin is expressed, which does not activate this pathway. This truncated Norrin expression leads to developmental abnormalities, including incomplete superficial retinal vascularization and leaky BRB. This model is generally used to study diseases such as Norrie disease, which affects the eye and causes blindness, as well as other retinal conditions.

[0457] - As shown in the timeline, Ndp-WT and Ndp-KO pups were administered intraperitoneally between 01E12 and 05A10. Figure 33 A). NaCl was used as a control vector. Representative images of n = 2–3 animals / groups.

[0458] - The retina was carefully dissected and stained to detect leaking vascular markers (Plvap), BRB connexin (Claudin-5), and mouse immunoglobulin (mIgG).

[0459] The results show that, Figure 33 As shown in B: - Pvap expression was reduced in Ndp-KO animals treated with 01E12-05A10. - Claudin-5 was increased in both Ndp-WT and Ndp-KO animals treated with 01E12-05A10. The arrows point to capillaries where Claudin-5 expression was detected (unlike the NaCl control, where Claudin-5 expression was not detected in capillaries). - In Ndp-KO animals, mIgG pathological leakage was reduced in animals receiving 01E12-05A10. No leakage was observed in WT animals.

[0460] Therefore: 01E12-05A10 improves the BRB characteristics of Ndp-KO animals.

[0461] This example was repeated with all the other antibodies described above, and similar results were obtained.

[0462] This invention is not limited to the embodiments described and / or the implementations illustrated in the accompanying drawings. Rather, the method of this invention can be implemented in many different ways without departing from the scope of the invention.

[0463] Sequence Overview

[0464] SEQ ID NO:1 (Mature person Wnt7a)

[0465] LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFGKELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSADIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWTTLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEEDPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTCSERTEMYTCK

[0466] SEQ ID NO:2 (Mature human Wnt7b)

[0467] LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFGQELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSADVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCWTTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCEEDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNT

[0468] SEQ ID NO:3 (Human LRP5)

[0469]

[0470] SEQ ID NO:4 (Human LRP6)

[0471]

[0472] SEQ ID NO:5 (Human LRP6)

[0473]

[0474] SEQ ID NO:6 (Person Reck)

[0475] MATVRASLRGALLLLLAVAGVAEVAGGLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVENYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTCLADVREPCQLGCRNLTYCTNFNNRPTELFRSCNAQSDQGAMNDMKLWEKGSIKMPFINIPVLDIKKCQPEMWKAIACSLQIKPCHSKSRGSIICKSDCVEILKKCGDQNKFPEDHTAESICELLSPTDDLKNCIPLDTYLRPSTLGNIVEEVTHPCNPNPCPANELCEVNRKGCPSGDPCLPYFCVQGCKLGEASDFIVRQGTLIQVPSSAGEVGCYKICSCGQSGLLENCMEMHCIDLQKSCIVGGKRKSHGTSFSIDCNVCSCFAGNLVCSTRLCLSEHSSEDDRRTFTGLPCNCADQFVPVCGQNGRTYPSACIARCVGLQDHQFEFGSCMSKDPCNPNPCQKNQRCIPKPQVCLTTFDKFGCSQYECVPRQLACDQVQDPVCDTDHMEHNNLCTLYQRGKSLSYKGPCQPFCRATEPVCGHNGETYSSVCAAYSDRVAVDYYGDCQAVGVLSEHSSVAECASVKCPSLLAAGCKPIIPPGACCPLCAGMLRVLFDKEKLDTIAKVTNKKPITVLEILQKIRMHVSVPQCDVFGYFSIESEIVILIIPVDHYPKALQIEACNKEAEKIESLINSDSPTLASHVPLSALIISQVQVSSSVPSAGVRARPSCHSLLLPLSLGLALHLLWTYN

[0476] SEQ ID NO:7 (Human Gpr124) / Adhesive G protein-coupled receptor A2 (ADGRA2)

[0477]

[0478] SEQ ID NO:8 (Human Gpr124) / Adhesive G protein-coupled receptor A2 (ADGRA2)

[0479]

[0480] SEQ ID NO:9 (Human Fzd1)

[0481] MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAGFLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMKWGHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFVYLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVIACYFYEQAFRDQWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSGKTLNSWRKFYTRLTNSKQGETTV

[0482] Table 1. CDR1, CDR2 and CDR3 sequences of VHH targeting LRP6

[0483] Table 2. CDR1, CDR2 and CDR3 sequences of VHH targeting LRP5

[0484] SEQ ID NO: 18 (Anti-LRP6 VHH)

[0485] DVQLVESGGGLVQPGGSLRLSCAASGSIFMINTMAWYRQAPGNQRELVATIRPVVSETTYADAVKGRFTISRDNAKNTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0486] SEQ ID NO: 19 (Anti-LRP6 VHH)

[0487] DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYADSVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS

[0488] SEQ ID NO: 20 (Anti-LRP6 VHH)

[0489] EVQLVESGGGLVQAGGSLRVSCAASGGTFSRYHMGWFRQAPGKEREFVSAITWSGGRTYYADFVKGRFTISRDDARNTVYLQMSSLKPEDTAVYYCALTWAPTPTNRRSDYAYWGQGTQVTVSS

[0490] SEQ ID NO: 21 (Anti-LRP6 VHH)

[0491] EVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDMAWYRQAPGNQRELVATIRPVVSETTYADAVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0492] SEQ ID NO: 22 (Anti-LRP6 VHH)

[0493] AVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRQAPGNQRELVATIRPVVSETTYADAVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0494] SEQ ID NO: 23 (Anti-LRP6 VHH)

[0495] AVQLVDSGGGLVQAGGSLRLSCAVSGRTFSMYDMGWFRQAPGKEREFVASIRWSSGNTWYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYANIYYTRRAPEEYWGQGTLVTVSSS

[0496] SEQ ID NO: 34 (Anti-LRP5 VHH)

[0497] EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAGSLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYYCYARTQRMGVVNSYWGQGTLVTVSS

[0498] SEQ ID NO: 35 (Anti-LRP5 VHH)

[0499] QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAGSLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYFCNAVTYDGYTIRGQGTLVTVSS

[0500] SEQ ID NO: 36 (Anti-LRP5 VHH)

[0501] EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYADSVQGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAQFRNDYGLRYQSTNNYWGQGTLVTVSS

[0502] SEQ ID NO: 37 (Anti-LRP5 VHH)

[0503] QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAGSLKGRFTMSTESAKNTVYLQMNSLKPEDSAVYYCNANYRGNRYWGQGTLVTVSS

[0504] SEQ ID NO: 44 (zGpr124 (25 - 742))

[0505] AGCPELFSSGCSCTEDRSKAHPTPGTRRKVSCGGKELTETPEVSLLPNRTVSLNLSNNRIRMLKNGSFAGLSSLEKLDLRNNLISTIMPGAFLGLTALRKLDLSSNRIGCLTPEMFQGLTNLTKLNISGNIFSSLDPNVFMELHSLKLVNFHSEFLSCDCGLRWVPSFFRSGSARLGDETLCAYPRRLQNKPLRLLRESDLSCEGPLELHTLSLLPSQRQVVFKGDRLPFHCTASLVDKITALHWRQNGQPVTSDPTKGIHLEESVQHDCTFITSELILSNVHVEASGEWECVVSTGRGNTSCSVEIVVLENSASFCPEQKVNNNRGEFRWPRTLAGITSYQHCLQLRYPSLTLGGGVEQKKASRNCDRSGRWEEADYSQCLYTNDITRILHTFILMPVNASNAVTLAHQVRSYTLEAAGFTDTVDVLYVAQMMHKFMDYVTELRELSEVLVEMGSNLMQVDDQILARAQREERACSSIVYTLETLAWPQLHSHAQDLSRYSRNIVMEAHLIRPAHFTGISCTVYQRREGAAGSQVHDGADLSLEQQLRFRCTTGTHNTSLNAFHLKNAVALATVSLPATLFPPNAPPDCKLQFVAFRNGRFFPFTSNFTGHSDLARRRGISTPVIYAGLDGCSMWNQSDPIIVSLRHTSPGHDPVAAHWNSQALGHHGSWSLDGCQLIHSDVSISTLRCSVLSNYAVLQEIPDFPGSPSIPVEVLHP

[0506] SEQ ID NO:45 (hADAMTS10 (823-1103))

[0507] SLPPYSWHYAPWTKCSAQCAGGSQVQAVECRNQLDSSAVAPHYCSAHSKLPKRQRACNTEPCPPDWVVGNWSLCSRSCDAGVRSRSVVCQRRVSAAEEKALDDSACPQPRPPVLEACHGPTCPPEWAALDWSECTPSCGPGLRHRVVLCKSADHRATLPPAHCSPAAKPPATMRCNLRRCPPARWVAGEWGECSAQCGVGQRQRSVRCTSHTGQASHECTEALRPPTTQQCEAKCDSPTPGDGPEECKDVNKVAYCPLVLKFQFCSRAYFRQMCCKTCHGH

[0508] SEQ ID NO: 46 (hWnt7a linker (171 - 199))

[0509] VEPVRASRNKRPTFLKIKKPLSYRKPMDT

[0510] SEQ ID NO: 47 (hReck CK1 - 5 (27 - 340))

[0511] GLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVENYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTCLA

[0512] SEQ ID NO: 48 (Wnt7b linker)

[0513] VEVVRASRLRQPTFLRLKQLRSYQKPMET

[0514] SEQ ID NO: 49 (CK4 motif of Reck)

[0515] CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC

[0516] SEQ ID NO: 50 (CK5 domain RECK)

[0517] CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC

[0518] SEQ ID NO: 51 (Reck binding domain)

[0519] HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC

[0520] SEQ ID NO: 52 (RECK binding domain)

[0521] YEWRASRLRQPTFLRIKQLRSYQKPMET

[0522] SEQ ID NO: 53 (Anti-LRP5 VHH)

[0523] DVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAGSLKGRFTMSTESAKNTMYLQMNSLKPEDTAVYYCYVKLRDDDYVYRGQGTQVTVSS

[0524] SEQ ID NO: 54 (RECK binding domain)

[0525] XXXVXAXRXXXXXFLXIXXXXXYXKXXXX

[0526] SEQ ID NO: 55 (Reck binding domain)

[0527] VXAXRXXXXXFLXIXXXXXYXK

[0528] SEQ ID NO: 56 (Reck binding domain)

[0529] XXXVXAXRXXXXXFLXXXXXXXXXKXXXX

[0530] SEQ ID NO: 57 (Reck binding domain)

[0531] VXAXRXXXXXFLXXXXXXXXXK

[0532] SEQ ID NO:58 (N-terminal domain Gpr142)

[0533] APGCPLSIRSCKCSGERPKGLSGGVPGPARRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKITGLRNGSFLGLSLEKLDLRNNIISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDELPALKVVDLGTEFLTCDCHLRWLLPWAQNRSLQLSEHTL CAYPSALHAQALGSLQEAQLCCEGALHTHHLIPSLRQVVFQGDRLPFQCSASYLGNDTRIRWYHNRAPVEGDEQAGILLAESLIHDCTFITSELTLSHIGVWASGEWECTVSMAQGNASKKVEIVVLETSASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTSVPLGGGAPGTRASRR CDRAGRWEPGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKFLGYVDQIKELVEVMVDMASNLMLVDEHLLWLAQREDKACSRIVGALERIGGAALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKRRGVATPVIFAGTSGCGVGNLTEPVAVSLRHWAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELSAFPREVGGAGAGLHP

[0534] LRP6 sequence

[0535] GPR124 series

[0536] Rec sequence

[0537] SEQ ID NO: 172 (5-mer connector)

[0538] GSGGS

Claims

1. An agonist of the Wnt7 signaling pathway, wherein, The agonist is a soluble antibody or antibody fragment, and the agonist comprises one or more LRP binding domains and one or more Gpr124 or Reck binding domains.

2. The agonist of claim 1, wherein, The LRP binding domain can bind to LRP5 and / or LRP6.

3. The agonist as described in claim 1 or 2, wherein, The agonist is either divalent or tetravalent.

4. The agonist of claim 3, wherein, The agonist is tetravalent, and the agonist comprises: i) Two regions, each specifically binding to a group of one or more LRP epitopes, preferably a group of one or more LRP epitopes; and ii) Two regions, each specifically binding to one or more Reck or Gpr124 epitopes.

5. The agonist of claim 3, wherein, The agonist is divalent, and the agonist comprises: i) A region that specifically binds to a group of one or more LRP epitopes, preferably one or more LRP epitopes, such as a group of LRP5 and / or LRP6 epitopes; and ii) A region that specifically binds to one or more Reck or Gpr124 epitopes.

6. The agonist as claimed in any of the preceding claims, wherein, Each or more LRP binding domains are fused in series with one or more Reck binding domains or one or more Gpr124 binding domains, wherein the domains are optionally separated by a connector sequence.

7. The agonist as claimed in any of the preceding claims, wherein, The one or more LRP binding domains bind to an amino acid sequence having at least 75% sequence identity with a fragment of the LRP5 and / or LRP6 protein shown in SEQ ID NO: 3, 4 or 5.

8. The agonist as claimed in any of the preceding claims, wherein, The one or more Gpr124 or Reck binding domains bind to an amino acid sequence having at least 75% sequence identity with a fragment of the Gpr124 or Reck protein shown in SEQ ID NO: 6, 7 or 8.

9. The agonist as claimed in any of the preceding claims, wherein, At least one of the one or more LRP binding domains and / or the one or more Gpr124 or Reck binding domains is an antibody, an antibody fragment, an immunoglobulin single variable domain (ISVD), or a VHH.

10. The agonist as claimed in any of the preceding claims, wherein, The agonist is a bispecific antibody, a bispecific antibody fragment, a bispecific ISVD, or a VHH.

11. The agonist as claimed in any of the preceding claims, wherein, The one or more LRP binding domains are LRP5 and / or LRP6 binding domains, and wherein the LRP6 binding domain comprises the CDR3 region shown in SEQ ID NO: 85, 86, 87 or 88 or consists of an amino acid sequence having at most two different amino acids from SEQ ID NO: 85, 86, 87 or 88.

12. The agonist as claimed in any of the preceding claims, wherein, The one or more LRP binding domains are LRP5 and / or LRP6 binding domains, and wherein the LRP6 binding domain includes the CDR2 region shown in SEQ ID NO: 81, 82, 83 or 84 and / or the CDR1 region shown in SEQ ID NO: 77, 78, 79 or 80.

13. The agonist as claimed in any of the preceding claims, wherein, The agonist comprises an anti-LRP VHH containing the sequence according to SEQ ID No: 59, 60, 61 or 62.

14. The agonist of claim 13, wherein, The agonist comprises IgG containing a VH sequence according to SEQ ID NO: 63, 65 or 67 and a VL sequence according to SEQ ID NO: 64, 66 or 68, optionally separated by a linker sequence; preferably, the VH sequence according to SEQ ID NO: 63 and the VL sequence according to SEQ ID NO: 64, the VH sequence according to SEQ ID NO: 65 and the VL sequence according to SEQ ID NO: 66, or the VH sequence according to SEQ ID NO: 67 and the VL sequence according to SEQ ID NO:

68.

15. The agonist according to claim 13, wherein, The agonist comprises IgG, which comprises a VH sequence according to SEQ ID NO: 69, 71, 73 or 75, and a VL sequence according to SEQ ID NO: 70, 72, 74 or 76, optionally separated by a linker sequence; preferably, it comprises a VH sequence according to SEQ ID NO: 69 and a VL sequence according to SEQ ID NO: 70, a VH sequence according to SEQ ID NO: 71 and a VL sequence according to SEQ ID NO: 72, a VH sequence according to SEQ ID NO: 73 and a VL sequence according to SEQ ID NO: 74, or a VH sequence according to SEQ ID NO: 75 and a VL sequence according to SEQ ID NO:

76.

16. The agonist as claimed in any of the preceding claims, wherein, The agonist does not contain an Fzd binding domain.

17. The agonist as claimed in any of the preceding claims, wherein, The agonist cannot bind to Fzd.

18. The agonist as claimed in any of the preceding claims, wherein, The agonist does not contain any further binding domains beyond the one or more LRP binding domains and one or more Gpr124 or Reck binding domains.

19. The agonist as claimed in any of the preceding claims, wherein, The binding domain of the agonist is selected from one or more LRP binding domains and one or more Gpr124 or Reck binding domains.

20. The agonist of claim 19, wherein, The binding domain of the agonist is selected from one or more LRP binding domains and one or more Gpr124 binding domains.

21. The agonist of claim 20, wherein, The agonist is a bispecific antibody having two binding domains, including an LRP binding domain and a Gpr124 binding domain.

22. The agonist of claim 19, wherein, The binding domain of the agonist is selected from one or more LRP binding domains and one or more Reck binding domains.

23. The agonist of claim 22, wherein, The agonist is a bispecific antibody having two binding domains, including an LRP binding domain and a Reck binding domain.

24. A nucleic acid encoding the agonist of any one of claims 1-23, or a vector comprising said nucleic acid.

25. A pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent or carrier, and an agonist as described in any one of claims 1-23 or a nucleic acid or carrier as described in claim 24.

26. The agonist of any one of claims 1-23, the nucleic acid or carrier of claim 24, for use as a drug.

27. The agonist of any one of claims 1-23, the nucleic acid or vector of claim 24, for use in blood-brain barrier endothelial cell-guided gene therapy, or for use in the treatment of conditions related to blood-brain barrier integrity, or for the treatment of blood-brain barrier integrity.

28. The agonist of any one of claims 1-23, the nucleic acid or vector of claim 24, for the treatment of neurological disorders, wherein the neurological disorders are preferably selected from: retinopathy, ischemic stroke, hemorrhagic stroke, ischemia / reperfusion injury, cerebral aneurysm, arteriovenous malformation (AVM), cavernous malformation, vasculitis, cerebral hemorrhage, subarachnoid hemorrhage, spinal vascular malformation, carotid artery stenosis, moyamoya disease, intracranial atherosclerosis and combinations thereof, or multiple sclerosis, brain cancer, glioblastoma, human monogenic neurological disorders, epilepsy, neurodegenerative diseases, dementia, vascular dementia, HIV-1 related dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Charcot-Marie-Tue disease, dystonia, infectious brain disease, traumatic brain injury, migraine, neuroinflammation, COVID-19 and chronic traumatic encephalopathy and combinations thereof.

29. The agonist of any one of claims 1-23, the nucleic acid or vector of claim 24, for use in blood-retinal barrier endothelial cell-guided gene therapy, or for use in the treatment of conditions related to blood-retinal barrier integrity, or for the treatment of blood-retinal barrier integrity.

30. The agonist of any one of claims 1-23, the nucleic acid or carrier of claim 24, for the treatment of an ophthalmic disease or disorder, preferably selected from the group consisting of: retinopathy, retinal vascular disorders such as Norrie's disease, diabetic retinopathy, macular degeneration, familial exudative vitreoretinopathy, osteoporosis-pseudoglioma syndrome, retinal vein occlusion, and retinopathy of prematurity.

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