Composition for the treatement of seizure or epilepsy

Abstract

The current invention relates to a pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy or seizure in a patient. Said antibody or antibody fragment is a modulator of the Wnt7 signaling pathway, preferably an agonist of the Wnt7 signaling pathway, and said antibody or antibody fragment comprises one or more Lipoprotein Receptor-related Protein (LRP) binding domains, and further one or more Gpr124 and / or Reck binding domains.

Description

[0001] COMPOSITION FOR THE TREATEMENT OF SEIZURE OR EPILEPSY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a pharmaceutical composition for use in the treatment and / or prevention of epilepsy. Said pharmaceutical composition for use comprises modulators of the Wnt7 signaling pathway, more particularly agonists of said pathway, preferably bispecific antibodies or antibody fragments that target two members of the Wnt7 receptor complex; or comprises nucleic acids encoding said antibodies or antibody fragments.

[0004] BACKGROUND

[0005] Epilepsy is a chronic neurological disorder characterized by recurrent, unprovoked seizures, resulting from abnormal electrical activity in the brain. Among the various types of epilepsy, temporal lobe epilepsy (TLE) is the most common form of focal epilepsy in adults and is frequently associated with hippocampal sclerosis and bloodbrain barrier (BBB) dysfunction. BBB disruption is also observed in other contexts such as post-traumatic brain injury (TBI), glioblastoma (GBM), and in patients experiencing seizures prior to a confirmed epilepsy diagnosis. Disruption of the BBB can exacerbate neuroinflammation and increase seizure susceptibility. Despite the availability of antiepileptic drugs (AEDs), a significant proportion of epilepsy patients, including TLE patients, fail to achieve adequate seizure control. This highlights an urgent need for novel therapeutic approaches that target the underlying neurovascular dysfunction contributing to epilepsy nd seizure disorders associated with BBB disruption..

[0006] Increasing evidence suggests that blood-brain barrier (BBB) dysfunction is a common feature across various forms of epilepsy, contributing to neuroinflammation, increased neuronal excitability, and seizure recurrence. Moreover, it is suggested that endothelial Wnt / p-catenin signaling plays a critical role in maintaining BBB integrity. Wnt / 0-catenin signaling is initiated by neural-derived Wnt7a / b ligands and regulates the blood-brain barrier (BBB). This signaling cascade is essential from the earliest stages of central nervous system (CNS) and remains crucial for maintaining BBB function throughout adulthood. Conditional deletion of 0-catenin signaling in cerebral endothelial cells (ECs) has been linked to neurovascular degenerative diseases. Conversely, enhancing Wnt / 0-catenin signaling through a constitutively active form of 0-catenin in CNS endothelium shows protective effects in models of brain cancer, stroke, and retinal vascular dysfunction.Given the central role of Wnt7a / b ligands in driving p-catenin-dependent BBB maturation, these ligands represent potential therapeutic agents for repairing dysfunctional barriers. However, natural Wnt7 ligands pose significant challenges as therapeutics due to their pleiotropic effects across tissues and organs, and the structural complexity of Wnt / Frizzled (FZD) interactions further disqualify natural Wnt ligands as safe therapeutics.

[0007] Wnt7 ligands activate Wnt signaling through two distinct receptor complexes. The first complex, composed of a Frizzled (FZD) family receptor and Lipoprotein Receptor- Related Protein 5 of 6 (LRP5 or LRP6), has broad tissue distribution and non-specific binding to Wnt7a / b, leading to systemic activation. The second complex involves GPR124 / RECK / FZD / LRP5 or LRP6 and is specifically enriched in cerebral and retinal endothelial cells where GPR124 and / or RECK are expressed.

[0008] The observation that Wnt signaling at the BBB and CNS requires a different receptor complex compared to other cell types and tissues, opens the opportunity to develop CNS-specific Wnt agonists. However, developing chemical or biologicals that bind to and stimulate a four-membered receptor complex is challenging. While W02019 / 180204 proposes several compounds, it fails to provide enabling data or experimental evidence demonstrating their efficacy in selectively modulating Wnt signaling in the CNS, let alone in epilepsy-related diseases and / or disorders.

[0009] The present invention addresses at least some of these challenges based on the unexpected observation that binding of a compound to only two specific members of the receptor complex is sufficient to activate the atypical GPR124 / RECK / FZD / LRP5 or LRP6 mediated Wnt signaling and thereby provide a treatment of epilepsy.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages. To this end, the present invention relates to a pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy or seizure in a patient according to claim 1.

[0012] In particular, said antibody or antibody fragment is a modulator of the Wnt7 signaling pathway, preferably an agonist of the Wnt7 signaling pathway, and comprises one or more Lipoprotein Receptor-related Protein (LRP) binding domains, and further one or more Gprl24 and / or Reck binding domains.Preferred embodiments of the pharmaceutical composition for use are shown in any of the claims 2 to 16.

[0013] By modulating, agonizing or stimulating the Wnt7 signaling pathway, more particularly the atypical Wnt7 signaling pathway, the present pharmaceutical composition for use is able to be used in treatment and / or prevention of epilepsy. This pathway is mediated or controlled by the four member receptor complex comprising Frizzled, LR.P5 or LR.P6, Gprl24 and Reck. The composition is effective at least against seizure in general, or epilepsy such as generalized epilepsy, focal epilepsy, temporal lobe epilepsy (TLE), and non-TLE focal epilepsy.

[0014] The specific antibodies or antibody fragments disclosed herein target and bind only two specific members of this receptor complex. Surprisingly, this selective binding activates the "on-target" atypical GPR124 / RECK / FZD / LRP5 or LRP6-mediated Wnt signaling, providing a successful treatment for epilepsy. At the same time, it does not, or only minimally, stimulate systemic canonical Wnt7 signaling, which is mediated by Fzd / LRP5 or Fzd / LRP6 alone. Avoiding this canonical pathway prevents pleiotropic, unwanted "off-target" effects across tissues and organs.

[0015] DESCRIPTION OF FIGURES

[0016] The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

[0017] Figure 1 illustrates the structure of the anti-Gprl24 / LRP6 and anti-Reck / LRP6 bispecific antibodies used in Example 1, where the LRP6 VHH is fused to the N-terminus of the VL domain of the respective anti-Gprl24 or anti-Reck IgGs.

[0018] Figure 2 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP6 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0019] Figure 3 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 1.9 nM and 1.7 nM for 01E12-05A10 and 01B01-06C03, respectively.Figure 4 shows a dose-response for bispecific antibodies 01B01-06A05 (A) and 01E12-06A05 (B) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 2.8 nM and 3.0 nM for 01B01-06A05and 01E12-06A05, respectively.

[0020] Figure 5 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP5 upon addition of the indicated bispecific antibodies (20 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0021] Figure 6 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzd5 and LRP6. The EC50 values calculated from the curves were 4.2 nM and 1.5 nM for 01E12-05A10 and 01B01-06C03, respectively.

[0022] Figure 7 shows the luminescence signal in STF cells expressing Gprl24, Fzdl and LRP6 (no Reck) upon addition of the indicated bispecific antibodies (20 nM). In the Wnt7a control, Wnt7a was delivered via transient transfection, but the natural ligand showed no activity under these conditions, i.e. in the absence of Reck. An empty vector was used as a negative control.

[0023] Figure 8 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzd4 and LRP6 (A) or Gprl24, Reck, Fzd5 and LRP6 (B) upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0024] Figure 9 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in STF cells expressing Gprl24, Reck, Fzd4 and LRP6. The EC50 values calculated from the curves were 3.7 nM and 1.3 nM for 01E12-05A10 and 01B01-06C03, respectively.

[0025] Figure 10 shows signaling activity of bispecific antibodies 01E12-05A10 and 01E12-06C03 (10 nM) in hRMEC (Human Retinal Microvascular Endothelial Cells) (A) and hBMEC (Human Brain Microvascular Endothelial Cells) (B). Recombinant Wnt3a (13.4 nM) was used as a positive control. Wnt pathway activation was assessed by monitoring the level of Axin2 mRNA by qRT-PCR.Figure 11 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hRMEC cells. Wnt pathway activation was assessed by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 66 pM and 32 pM for 01E12-05A10and 01B01-06C03, respectively.

[0026] Figure 12 shows that treatment of hRMEC cells with bispecific antibody 01B01-06C03 (10 nM) for 6 h results in a detectable increase in the mRNA expression of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and NKD1 (Naked cuticle 1). The levels of Lefl and NKD1 mRNAs were monitored by qRT-PCR.

[0027] Figure 13 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in hBMEC cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 17 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively.

[0028] Figure 14 relates to the response of bEnd.3 cells to (i) treatment with bispecific antibody 01E12-05A10 (1 nM), (ii) treatment with a control antibody obtained by combining an irrelevant VHH with the anti-Gprl24 IgG 05A10 (1 nM), (iii) treatment with a control antibody obtained by combining the anti-LRP6 VHH 01E12 with an irrelevant IgG (1 nM), or (iv) treatment with both control (1 nM each). Treatment was applied for 24 h, and Wnt pathway activation was assessed by monitoring the level of Axin2, PLVAP, Lefl and MFSD2A mRNAs by qRT-PCR. Figure 21 shows the levels of Axin2 (A), Lefl (B), PLVAP (C) and MFSD2A (D) mRNAs for treatment (i) with construct 01E12-05A10.

[0029] Figure 15 shows a dose-response for bispecific antibodies 01E12-05A10 (A) and 01B01-06C03 (B) in bEnd.3 (mouse brain endothelial) cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 25 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively. As a positive control, cells were treated with different concentrations of recombinant Wnt3a (C). The EC50 value calculated from that curve was 6.6 nM.

[0030] Figure 16 shows a dose-response for bispecific antibodies 01E12-06C03 (A) and 01F11-06C03 (B) in bEnd.3 (mouse brain endothelial) cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 18 pM for both antibodies. As a positive control,cells were treated with different concentrations of recombinant Wnt3a (C). The EC50 value calculated from that curve was 6.6 nM.

[0031] Figure 17 shows that treatment of bEnd.3 cells with bispecific antibody 01E12-05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 ("CHIR", 10 pM) for 24 h results in a detectable modulation in the level of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domaincontaining protein 2a) mRNAs, as well as in the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA, as monitored by qRT-PCR.

[0032] Figure 18 shows that treatment of bEnd.3 cells with bispecific antibody 01B01-06C03 (10 nM) for 24 h results in a detectable modulation in the level of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domaincontaining protein 2a) mRNAs, as well as in the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA, as monitored by qRT-PCR.

[0033] Figure 19 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzdl and LRP6 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0034] Figure 20 shows a dose-response for bispecific antibodies 01B01-01F11 (A), 01E12-01F11 (B), 01E12-03A04 (C) and 01B01-03A04 (D) in STF cells expressing Gprl24, Reck, Fzdl and LRP6. The EC50 values calculated from the curves were 4.2 nM, 1.3 nM, 4.3 nM, and 6.4 nM for 01B01-01F11, 01E12-01F11, 01E12-03A04 and 01B01-03A04, respectively.

[0035] Figure 21 shows the luminescence signal in STF cells expressing human Gprl24, human Reck, human Fzdl and mouse LRP5 upon addition of the indicated bispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0036] Figure 22 shows the luminescence signal in STF cells expressing Gprl24, Reck, Fzd4 and LRP6 (A) or Gprl24, Reck, Fzd5 and LRP6 (B) upon addition of the indicatedbispecific antibodies (50 nM). Wnt7a, delivered via transient transfection, was used as positive control, and an empty vector was used as a negative control.

[0037] Figure 23 shows a dose-response for bispecific antibodies 01B01-01F11 (A), 01E12-03A04 (B) and 01B01-03A04 (C) in STF cells expressing Gprl24, Reck, Fzd4 and LRP6. The EC50 values calculated from the curves were 5.5 nM, 5.9 nM, and 1.8 nM for 01B01-01F11, 01E12-03A04, and 01B01-03A04, respectively.

[0038] Figure 24 shows the modulation in bEnd.3 cells in the level of the downstream mediators of the Wnt / 0-catenin signaling pathway Axin2, Lefl, and Mfsd2a mRNAs, as well as in the level of the canonically Wnt-downregulated protein PLVAP mRNA, as monitored by qRT-PCR, upon exposure to Reck-LRP6 bispecific antibodies (10 nM) for 24 h.

[0039] Figure 25 shows a dose-response for bispecific antibodies 01B01-01A08 (A), 01E12-03A04 (B) and 01E12-01F11 (C) in bEnd.3 cells. Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR. The EC50 values calculated from the curves were 10 pM, 7 pM, and 7 pM, for 01B01-01A08, 01E12-03A04 and 01E12-01F11, respectively.

[0040] Figure 26 shows racine score evolution in KA mouse model (ip injection) in animals treated with a Gprl24 / Lrp6 bispecific antibody - according to example 2. (A) The schematic overview of the experiment. (B) Racine score evolution just after the model induction, mean + / - SD, numbers in brackets indicate 48h surviving animals / initially included animals. (C) Cumulative and (D) mean Racine score are represented. Histogram mean + / - SD, each dots represent one animal, Mann-Whithney test, **p<0.01.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention concerns novel pharmaceutical composition for use in the treatment and / or prevention of epilepsy, thereby modulating, agonizing or stimulating the Wnt7 signaling pathway, more particularly the atypical Wnt7 signaling pathway that is mediated or controlled by the four member receptor complex comprising Frizzled, LRP5 or LRP6, Gprl24 and Reck. Even more particularly, the modulators, for instance agonists, herein provided and comprised in the pharmaceutical composition are soluble antibodies or antibody fragments, or nucleic acids encoding said antibodies or antibody fragments. Said antibodies or antibody fragments are specific or selective for the Wnt7 signaling pathway that isorchestrated by the GPR124 / RECK / FZD / LRP5 / 6 complex. Said antibodies or antibody fragments, herein disclosed, do not significantly stimulate the canonical Wnt7 signaling mediated by Fzd / LRP5 or Fzd / LRP6 alone.

[0043] By specifically targeting the Wnt / B-catenin signaling pathway orchestrated by the GPR124 / RECK / FZD / LRP5 / 6 complex, the present invention provides a novel approach to restoring BBB integrity in epilepsy. Strengthening the BBB as well as reducing the permeability of the BBB through selective Wnt signaling modulation has the potential to reduce neuroinflammation, limit the spread of pathological electrical activity, and ultimately improve seizure control in TLE patients, including patients who do not respond to conventional antiepileptic drugs. This targeted strategy offers a promising new avenue for addressing the neurovascular dysfunction underlying epilepsy and represents a significant advancement in therapeutic options for patients with drug-resistant seizures.

[0044] Definitions

[0045] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0046] As used herein, the following terms have the following meanings:

[0047] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0048] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / -20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0049] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing","contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0050] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0051] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints.

[0052] The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0053] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0054] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0055] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures orcharacteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0056] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. Unless otherwise indicated, nucleotide sequences are written left to right in 5' to 3' orientation. Amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0057] The term "antibody" as used herein, refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen. "Antibodies" can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The term "immunoglobulin (Ig) domain" as used herein refers to a globular region of an antibody chain, or to a polypeptide that essentially consists of such a globular region. Immunoglobulin domains are characterized in that they retain the immunoglobulin fold (Ig fold as named herein) characteristic of antibody molecules, which consists of a two-layer sandwich of about seven to nine antiparallel P-strands arranged in two p-sheets, optionally stabilized by a conserved disulphide bond. The term "immunoglobulin (Ig) domain", includes "immunoglobulin constant domain", and "immunoglobulin variable domain" (abbreviated as "IVD"), wherein the latter means an immunoglobulin domain essentially consisting of four "framework regions" which are referred to in the art and herein below as "framework region 1" or "FR1"; as "framework region 2" or "FR2"; as "framework region 3" or "FR3"; and as "framework region 4" or "FR4", respectively; which framework regions are interrupted by three "complementarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as "complementarity determining region 2" or "CDR2"; and as "complementarity determining region 3" or "CDR3", respectively. Thus, the generalstructure or sequence of an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site.

[0058] The term "antibody fragment" refers to a portion of any antibody or antibody-like structure that by itself has a high affinity for an antigenic determinant or epitope, and contains one or more CDRs accounting for such specificity. In some particular embodiments, at least one of the targeting moieties of the antibody or antibody fragment of the application is a single-domain antibody, an immunoglobulin single variable domain, a heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody, an Affimer, a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a peptide aptamer, an alterases, a plastic antibodies, a phylomer, a stradobodies, a maxibodies, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, a peptide mimetic molecule, or a synthetic molecule, as described in US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004 / 132094, US 5,831,012, US 2004 / 023334, US 7,250,297, US 6,818,418, US 2004 / 209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004 / 146938, US 2004 / 157209, US 6,994,982, US 6,794,144, US 2010 / 239633, US 7,803,907, US 2010 / 119446, and / or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties. See also Storz 2011 MAbs 3: 310-317.

[0059] An "immunoglobulin domain" of this application also includes "immunoglobulin single variable domains" (abbreviated as "ISVD"), equivalent to the term "single variable domains", and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation. In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, aF(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen. In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH / VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs. As such, the single variable domain may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit). In one embodiment of the invention, the immunoglobulin single variable domains are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the immunoglobulin single variable domains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody. For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof. In particular, the immunoglobulin single variable domain may be a Nanobody (as defined herein) ora suitable fragment thereof. Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V. For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein, such as e.g. described in W02008 / 020079.

[0060] Immunoglobulin domains herein also include "VHH domains", also known as VHHs, VHH domains, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen-binding immunoglobulin (Ig) (variable) domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman etal (1993) Nature 363: 446-448). The term "VHH domain" has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VL domains"). For a further description of VHHs and Nanobody, reference is made to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03 / 050531 of Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 by the Institute of Antibodies; as well as WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. As described in these references, Nanobody (in particular VHH sequences and partially humanized Nanobody) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobody, including humanization and / or camelization of Nanobody, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications to increase the half-life of the Nanobody and their preparations can be found e.g. in WO 08 / 101985 and WO 08 / 142164.

[0061] "Domain antibodies", also known as "Dabs", "Domain Antibodies", and "dAbs" (the terms "Domain Antibodies" and "dAbs" being used as trademarks by the GlaxoSmithKline group of companies) have been described in e.g., EP 0368684, Ward et al. (Nature 341: 544-546, 1989), Holt et al. (Tends in Biotechnology 21: 484-490, 2003) and WO 03 / 002609 as well as for example WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. Domain antibodies essentially correspond to the VH or VL domains of non-camelid mammalians, in particular human 4-chain antibodies. In order to bind an epitope as a single antigen binding domain, i.e., without being paired with a VL or VH domain, respectively, specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences. Domain antibodieshave, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutical use in humans. It should also be noted that single variable domains can be derived from certain species of shark (for example, the so-called "IgNAR domains", see for example WO 05 / 18629).

[0062] Immunoglobulin single variable domains such as Domain antibodies and Nanobody (including VHH domains and humanized VHH domains), represent in vivo matured macromolecules upon their production, but can be further subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molecule. Affinity-matured immunoglobulin single variable domain molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et al. (Biotechnology 10:779-783, 1992), Barbas et al. (Proc. Nat. Acad. Sci, USA 91: 3809-3813, 1994), Shier et al. (Gene 169: 147-155, 1995), Yelton et al. (Immunol. 155: 1994-2004, 1995), Jackson et al. (J. Immunol. 154: 3310-9, 1995), Hawkins et al. (J. Mol. Biol. 226: 889 896, 1992), Johnson and Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). The process of designing / selecting and / or preparing a polypeptide, starting from an immunoglobulin single variable domain such as a Domain antibody or a Nanobody, is also referred to herein as "formatting" said immunoglobulin single variable domain; and an immunoglobulin single variable domain that is made part of a polypeptide is said to be "formatted" or to be "in the format of" said polypeptide. Examples of ways in which an immunoglobulin single variable domain can be formatted and examples of such formats for instance to avoid glycosylation will be clear to the skilled person based on the disclosure herein.

[0063] Immunoglobulin single variable domains such as Domain antibodies and Nanobody (including VHH domains) can be subjected to humanization, i.e. increase the degree of sequence identity with the closest human germline sequence. In particular, humanized immunoglobulin single variable domains, such as Nanobody (including VHH domains) may be immunoglobulin single variable domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, at least one framework residue) that is and / or that corresponds to a humanizing substitution (as defined herein). Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequences, afterwhich one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and / or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (the framework regions of) an immunoglobulin single variable domain, such as a Nanobody (including VHH domains) may be partially humanized or fully humanized. It should be noted that the immunoglobulin single variable domains, as well as the antigen-binding chimeric protein of the invention in their broadest sense are not limited to a specific biological source or to a specific method of preparation. For example but without the purpose of being limiting, the immunoglobulin single variable domains, in particular the antigenbinding chimeric proteins of the invention, can generally be obtained: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody, and further engineering of the sequence to obtain the antigen-binding chimeric protein; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain, in a format fused to said scaffold protein of the antigen-binding chimeric protein; (3) by "humanization" of a naturally occurring VHH domain and / or scaffold protein or by expression of a nucleic acid encoding a such humanized VHH domain and / or scaffold protein, and / or antigen-binding chimeric protein; (4) by "mutation" of a naturally occurring VHH domain to reduce binding to pre-existing antibodies or by engineering of the scaffold protein fusion sites to obtain an antigen-binding chimeric protein of the invention with reduced binding to pre-existing antibodies as compared to the natural VHH; or (5) by using synthetic or semisynthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se.

[0064] An "epitope", as used herein, refers to an antigenic determinant of a polypeptide, constituting a binding site or binding pocket on a target molecule (e.g. a protein to which an immunoglobulin or part thereof, antibody, VHH or ISVD is binding). "Binding" means any interaction, be it direct or indirect. A direct interaction implies a contact (e.g. physical or chemical) between two binding partners. An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two molecules. An interaction can be completely indirect (e.g. two molecules are part of the same complex with the help of one or more bridging molecules but don't bind in the absence of the bridging molecule(s)). An interaction may be partly direct or partly indirect: there is still a direct contact between twointeraction partners, but such contact is e.g. not stable, and is stabilized by the interaction with one or more additional molecules. The term "binding pocket", "binding domain" or "binding site" refers to a region of a molecule or molecular complex, that, as a result of its shape and charge, associates with another chemical entity, compound, protein, peptide, antibody, single domain antibody or ISVD or VHH.

[0065] An epitope could comprise 1, 2 or 3 amino acids in a spatial conformation, which is unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, 7 such amino acids, and more usually, consists of at least 8, 9, 10 such amino acids. Methods of determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and multi-dimensional nuclear magnetic resonance. A "conformational epitope", as used herein, refers to an epitope comprising amino acids in a spatial conformation that is unique to a folded 3-dimensional conformation of a polypeptide. Generally, a conformational epitope consists of amino acids that are discontinuous in the linear sequence but that come together in the folded structure of the protein. However, a conformational epitope may also consist of a linear sequence of amino acids that adopts a conformation that is unique to a folded 3-dimensional conformation of the polypeptide (and not present in a denatured state). In protein complexes, conformational epitopes consist of amino acids that are discontinuous in the linear sequences of one or more polypeptides that come together upon folding of the different folded polypeptides and their association in a unique quaternary structure. Similarly, conformational epitopes may here also consist of a linear sequence of amino acids of one or more polypeptides that come together and adopt a conformation that is unique to the quaternary structure. The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., o-helix, [3-sheet, among others), tertiary structure (e.g., the 3-dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Posttranslational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, andinteraction with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X-ray crystallography, NMR, or spin labelling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993.

[0066] A "paratope" as used herein refers to the antigen-binding site and is the part of an antibody which recognizes and binds to an antigen. The paratope of the bispecific binding agent of current disclosure thus comprises the amino acid residues of the binding agent that bind the epitope of the LR.P5 or LR.P6 protein and the amino acid residues of the binding agent that bind the epitope of the Gprl24 or Reck protein.

[0067] Antibodies or binding agents in general can bind one or more epitopes of one or more targets.

[0068] "Bispecific" as used herein in the context of current disclosure means that the binding agent can bind two different epitopes or bind two different antigens simultaneously. "Multispecific" as used herein then refers to the ability of binding two or more different binding sites or antigens simultaneously. Multispecific binding agents thus include bispecific antibodies, but it can also refer to antibodies with the ability to bind to three or more targets.

[0069] Independent of the number of targets that can be bound by the binding agent, the binding agent can comprise multiple binding sites. The number of binding sites is referred to as the valency of the antibody. A "bivalent" antibody is a typical antibody structure with two binding sites. The common structure of natural antibodies comprises two identical binding sites that bind two identical antigenic determinants or epitopes. However, a bivalent antibody can also bind two different epitopes or two different targets. A "tetravalent" antibody is an antibody that has four binding sites. It can bind to four identical or four different antigenic determinants or epitopes, or it can bind two different epitopes or two different targets that are each bound by two binding sites.

[0070] The term "affinity" generally refers to the degree to which an antibody or other binding protein (as defined further herein) binds to a target protein so as to shift the equilibrium of target protein and binding protein toward the presence of a complex formed by their binding. Thus, for example, where an antibody and an antigen arecombined in relatively equal concentration, an antibody of high affinity will bind to the antigen so as to shift the equilibrium toward high concentration of the resulting complex. The equilibrium dissociation constant KD (or KD) is commonly used to describe the affinity between a ligand and a target protein, or an antibody and its antigen. KD is the calculated ratio of koff / kon, between the antibody and its antigen and thus measures the propensity of a complex to fall apart into its component molecules. The association constant (konor kon) is used to characterize how quickly the antibody binds to its target. The dissociation constant (kOff or koff, also referred to as kdis, Kdis, Kd or kd) is used to measure how quickly an antibody dissociates from its target and is expressed as number of units that dissociated from a target per second. Hence, the lower koff is, the higher the affinity towards the target, koff and thus also KD is inversely related to affinity. A high affinity interaction is characterized by a low KD, a fast recognizing (high kon) and a strong stability of formed complexes (low koff). It will be appreciated that within the scope of the present application, the term "affinity" is used in the context of the antibody or antibody fragment that binds an epitope of the LR.P5, LR.P6, Gprl24 and / or Reck protein, more particularly the antibody or antibody fragment is "functional" in binding its target via the CDR regions of its immunoglobulin (Ig) domain.

[0071] "Amino acids" as used herein refer to the structural units (monomers) that make up proteins. They join together to form short polymer chains called peptides or longer chains called either polypeptides or proteins. These chains are linear and unbranched, with each amino acid residue within the chain attached to two neighboring amino acids. Twenty amino acids encoded by the universal genetic code are naturally incorporated into polypeptides and are called proteinogenic or natural amino acids. Natural amino acids or naturally occurring amino acids are glycine (Gly or G), Alanine (Ala or A), Valine (Vai or V), Leucine (Leu or L), Isoleucine (He or I), Methionine (Met or M), Proline (Pro or P), Phenylalanine (Phe or F), Tryptophan (Trp or W), Serine (Ser or S), Threonine (Thr or T), Asparagine (Asn or N), Glutamine (Gin or Q), Tyrosine (Tyr or Y), Cysteine (Cys or C), Lysine (Lys or K), Arginine (Arg or R), Histidine (His or H), Aspartic Acid (Asp or D) and Glutamic Acid (Glu or E).

[0072] As used herein, the terms "nucleic acid", "nucleic acid sequence" or "nucleic acid molecule" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of nucleic acids include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular. The nucleic acid may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The nucleic acid may comprise single stranded or double stranded DNA or RNA. The nucleic acid may comprise modified bases or a modified backbone. A nucleic acid that is up to about 100 nucleotides in length, is often also referred to as an oligonucleotide. "Nucleotides" as used herein refer to the building blocks of oligonucleotides and polynucleotides, and for the purposes of present disclosure include both naturally occurring and non-naturally occurring nucleotides. In nature, nucleotides, such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucleobase moiety and one or more phosphate groups (which are absent in nucleosides). A nucleotide without a phosphate group is called a "nucleoside" and is thus a compound comprising a nucleobase moiety and a sugar moiety. As used herein, "nucleobase" means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Naturally occurring nucleobases of RNA or DNA comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0073] "Nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, the (reverse) complement DNA, and RNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occurring nucleotides with an analogue. By "nucleic acid construct" it is meant a nucleic acid sequence that has been constructed to comprise one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from lambda phage), viral genomes comprising non-native nucleic acid sequences, and the like. "Coding sequence" is a nucleotide sequence, which is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5’-terminus and a translation stop codon at the 3’-terminus. A coding sequence can include, but is not limited to mRNA, cDNA, recombinant nucleotide sequences or genomic DNA, while introns may be present as well under certain circumstances.

[0074] An "expression cassette" as used herein comprises any nucleic acid construct capable of directing the expression of a gene / coding sequence of interest, which is operably linked to a promoter of the expression cassette. Expression cassettes are generally DNA constructs preferably including (5' to 3' in the direction of transcription): a promoter region, a polynucleotide sequence, homologue, variant or fragment thereof operably linked with the transcription initiation region, and a termination sequence including a stop signal for RNA polymerase and a polyadenylation signal. It is understood that all of these regions should be capable of operating in biological cells, such as prokaryotic or eukaryotic cells, to be transformed. The promoter region comprising the transcription initiation region, which preferably includes the RNA polymerase binding site, and the polyadenylation signal may be native to the biological cell to be transformed or may be derived from an alternative source, where the region is functional in the biological cell. Such cassettes can be constructed into a "vector". The term "vector" or alternatively "vector construct", "expression vector" or "gene transfer vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, and includes any vector known to the skilled person, including any suitable type, but not limited to, for instance, plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenoviral, AAV or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments. The construction of expression vectors for use in transfecting cells is also well known in the art, and thus can be accomplished via standard techniques (see, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp.

[0075] 109-128, ed. E. J. Murray, The Humana Press Inc., Clif ton, N .), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).The terms "identical" or percent "identity" in the context of two or more nucleic acid or amino acid sequences refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues respectively that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of nucleotide or amino acid sequences.

[0076] The term "percent sequence identity" or "% sequence identity" or "percent identity" or "% identity" between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain aspects, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain aspects, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative aspects, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain aspects, the percent identity between nucleotide or amino acid sequencesis determined using the algorithm of Myers and Miller (CABIOS, 4:11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain aspects, the default parameters of the alignment software are used.

[0077] One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequencesequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is Clustal Omega, available at www.ebi.ac.uk / jdispatcher / msa / clustalo, as well as MUSCLE, which can be accessed at www.drive5.com / muscle. ClustalW2, Clustal Omega and MUSCLE are alternatively available, e.g., from the EBI (European Bioinformatics Institute).

[0078] In certain aspects, the percentage identity "X" of a first nucleotide sequence to a second nucleotide sequence is calculated as 100 x (Y / Z), where Y is the number of nucleotide residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence. Different regions within a single polynucleotide target sequence that align with a polynucleotide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0079] According to the present disclosure, the degree of identity, between a given reference nucleotide sequence and a nucleotide sequence which is a homologue of said given nucleotide sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for a nucleic acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% ofthe entire length of the reference nucleic acid sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 nucleotides, preferably contiguous nucleotides. In a particular embodiment, the degree / percentage of similarity or identity is given for the entire length of the reference nucleic acid sequence.

[0080] The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. According to the present disclosure, the degree of identity, between a given reference amino acid sequence and an amino acid sequence which is a homologue of said given amino acid sequence will preferably be at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of identity is given preferably for an amino acid region which is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of identity is given preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, or 200 amino acids, preferably contiguous amino acids. In a particular embodiment, the degree / percentage of similarity or identity is given for the entire length of the reference amino acid sequence.

[0081] "Homologue" or "homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.The term "defined by SEQ ID N°: X" or "as depicted in SEQ ID N°: X" as used herein refers to a biological sequence consisting of the sequence of amino acids or nucleotides given in the SEQ ID N°: X. For instance, a protein defined in / by SEQ ID N° : X consists of the amino acid sequence given in SEQ ID N° : X. A further example is an amino acid sequence comprising SEQ ID N°: X, which refers to an amino acid sequence longer than the amino acid sequence given in SEQ ID N°: X but entirely comprising the amino acid sequence given in SEQ ID N°: X (wherein the amino acid sequence given in SEQ ID N°: X can be located N-terminally or C-terminally in the longer amino acid sequence, or can be embedded in the longer amino acid sequence), or to an amino acid sequence consisting of the amino acid sequence given in SEQ ID N°: X.

[0082] A "modulator", as used herein, refers to a molecule (e.g., binding agent, ligand, or antibody) that interacts with a target in a signaling pathway, thereby altering the pathway's activity. Modulation can involve either activation or inhibition of the signaling pathway, depending on the nature of the interaction between the modulator and its target. In the context of the Wnt / beta-catenin signaling pathway, a modulator may either enhance or suppress the stabilization and accumulation of beta-catenin and the subsequent transcriptional activation of Wnt target genes. The modulator may exhibit a range of affinities or specificities for the target, yet still exert an effect on the pathway's activity. In the present context, the term "modulator" encompasses both activators (e.g., agonists) and inhibitors (e.g., antagonists), and includes molecules that selectively influence specific Wnt signaling components, such as the Wnt7 / beta-catenin signaling pathway.

[0083] An "agonist," as used herein, refers to a molecule (e.g., binding agent, ligand, or antibody) that, upon interaction with a receptor or another target in a signaling pathway, activates downstream signaling, thereby potentially initiating a biological response. In the context of the Wnt / beta-catenin signaling pathway, an agonist functions as an activator of this pathway, leading to the stabilization and accumulation of beta-catenin and subsequent transcriptional activation of Wnt target genes. The agonist may not necessarily exhibit strong binding affinity to the receptor but still effectively promotes pathway activation. In the present context, the term "agonist" may be used interchangeably with the term "activator", preferably of the Wnt / beta-catenin signaling pathway, more preferably of the Wnt7 / beta-catenin signaling pathway.

[0084] Conversely, an "antagonist" is a molecule that interferes with or inhibits the activation of downstream signaling pathways, thereby preventing such biological response. In the context of the Wnt / beta-catenin signaling pathway, an antagonistfunctions by blocking or inhibiting the pathway, thereby preventing the accumulation of beta-catenin and the transcriptional activation of Wnt target genes. The antagonist does not activate the pathway and may act by preventing the agonist or natural ligand from initiating the signaling cascade. In the present context, the term "antagonist" may be used interchangeably with the term "inhibitor", preferably of the Wnt / beta-catenin signaling pathway, more preferably of the Wnt7 / beta-catenin signaling pathway.

[0085] The terms "treatment" or "treating" or "treat" can be used interchangeably and are defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, injury, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders. Those in need of treatment include those already diagnosed with the disorder as well as those prone or predisposed to contract the disorder or those in whom the disorder is to be prevented. For example, in tumor (e.g. cancer) treatment, a therapeutic agent can directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents or by the subject's own immune system. For example, in treatment of epilepsy, this includes reducing the frequency, intensity, or duration of seizures, preventing seizure recurrence, improving neurological function, restoring blood-brain barrier (BBB) integrity, and / or decreasing neuroinflammation linked to epilepsy. "Prevention" refers to reducing the likelihood, delaying the onset, or entirely preventing the development of epilepsy or epilepsy-related complications. In patients at risk of epilepsy— such as those with a history of brain injury, genetic predisposition, or other risk factors— administration of the pharmaceutical composition aims to prevent or significantly reduce the incidence of seizures or neurovascular dysfunction.

[0086] As used herein, the terms "diagnosis", "prognosis" and / or "prediction" comprise diagnosing, prognosing and / or predicting a certain disease and / or disorder, thereby predicting the onset and / or presence of a certain disease and / or disorder, and / or predicting the progress and / or duration of a certain disease and / or disorder, and / or predicting the response of a patient suffering from a certain disease and / or disorder to therapy.

[0087] The term "statistically significantly" different is well known by the person skilled in the art. Statistical significance plays a pivotal role in statistical hypothesis testing. It is used to determine whether the null hypothesis should be rejected or retained. Thenull hypothesis is the default assumption that nothing happened or changed. For the null hypothesis to be rejected, an observed result has to be statistically significant, i.e. the observed p-value is less than the pre-specified significance level a. The p-value of a result, p, is the probability of obtaining a result at least as extreme, given that the null hypothesis were true. In one embodiment, a is 0.05. In a more particular embodiment, a is 0.01. In an even more particular embodiment, a is 0.001.

[0088] A peptide, polypeptide or protein can be naturally occurring, e.g., present in or isolated from nature, e.g., produced or expressed natively or endogenously by a cell or tissue and optionally isolated therefrom. A peptide, polypeptide or protein can be recombinant, i.e., produced by recombinant DNA technology, and / or can be, partly or entirely, chemically or biochemically synthesized. Without limitation, a peptide, polypeptide or protein can be produced recombinantly by a suitable host or host cell expression system and optionally isolated therefrom (e.g., a suitable bacterial, yeast, fungal, plant or animal host or host cell expression system), or produced recombinantly by cell-free translation or cell-free transcription and translation, or non-biological peptide, polypeptide or protein synthesis.

[0089] A conservative amino acid substitution is a substitution of one amino acid with another that has similar chemical and physical characteristics. Conservative amino acid substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (i.e., basic) amino acids include arginine, lysine and histidine. The negatively charged (i.e., acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non -conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.

[0090] The term "biologically active" is interchangeable with terms such as "functionally active" or "functional", denoting that the proteins disclosed herein at least partly retain the biological activity or intended functionality of the respective orcorresponding peptide, polypeptide or protein. Reference to the "activity" of a peptide, polypeptide or protein may generally encompass any one or more aspects of the biological activity of the peptide, polypeptide or protein, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and / or structural activity, e.g., within a cell, tissue, organ or an organism.

[0091] Preferably, a functionally active protein may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding protein.

[0092] In certain embodiments, a functionally active protein may even display higher biological activity or functionality compared with the corresponding peptide, polypeptide or protein, for example may display at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the intended biological activity or functionality compared with the corresponding protein. By means of an example, where the activity of a given protein can be readily measured in an assay with a quantitative output, for example an enzymatic assay or a signaling assay or a binding assay producing a quantifiable signal, a functionally active fragment or variant of the peptide, polypeptide or protein may produce a signal which is at least about 20%, or at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, or at least 60%, more preferably at least about 70%, or at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500% of the signal produced by the corresponding protein.

[0093] In the present specification, references to any peptides, polypeptides, proteins, or nucleic acids denote the respective peptides, polypeptides, proteins, or nucleic acids as commonly known under the respective designations in the art. More particularly, the references to "Wnt" and in particular to "Wnt7", to "G-protein coupled receptor 124" (GPR124), "Reversion-inducing cysteine-rich protein with Kazal motifs" (RECK), "Frizzled" (FZD), or "lipoprotein receptor-related protein" (LRP) denote the respective peptides, polypeptides, proteins or nucleic acids, as apparent from the context, as commonly known under said designations in the art."Wnt7" in the present context refers to both "Wnt7a" and / or "Wnt7b", which are part of the Wnt-family of proteins. The terms "Wnt7" and "WNT7" are used interchangeably throughout this specification. "Wnt" or "Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Inti" refers to a family of genes and proteins comprising the homologous "Wingless" or "Wg" and "Integration site" or "Int". In a particular embodiment, Wnt is Wnt7a protein with amino acid sequence as depicted in SEQ ID N°: 1. In another particular embodiment, Wnt is Wnt7b protein with amino acid sequence as depicted in SEQ ID N°: 2.

[0094] A "Gprl24 / RECK / FZD / LRP receptor complex", "Gprl24 / RECK / FZD / LRP co-receptor complex", "Gprl24 / RECK / FZD / LRP complex" or "Gprl24 / RECK / FZD / LRP protein complex" broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane-associated protein complex comprising at least one Gprl24 protein, at least one RECK protein, at least one FZD protein and at least one LRP protein. A GPR124 / RECK / FZD / LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt / 0-catenin signaling in said cell in response to extracellularly provided Wnt7 ligand.

[0095] A "FZD / LRP receptor complex", "FZD / LRP co-receptor complex", "FZD / LRP complex" or "FZD / LRP protein complex" broadly denotes a protein complex, particularly a membrane-associated protein complex, more particularly a plasma membrane associated protein complex comprising at least one FZD protein and at least one LRP protein. A FZD / LRP receptor complex, when located at the plasma membrane of a cell, is capable of activating Wnt / 0-catenin signaling in said cell in response to extracellularly provided Wnt ligand, such as, but not limited to Wnt7 ligand.

[0096] "Wnt signaling" and "Wnt / 0-catenin signaling" are used interchangeably herein.

[0097] "Frizzled", "Fz", "FZ", "Fzd" or "FZD" as used herein denotes a family of at least ten atypical G protein-coupled receptors (GPCR) that serve as receptors in the Wnt signaling pathway and other signaling pathways. When activated, Frizzled leads to activation of Dishevelled in the cytosol. In certain particularly preferred embodiments, the terms denote Fzdl, Fzd4, Fzd5 and / or Fzd8.

[0098] In a particular embodiment, Fzd is Fzdl, wherein Fzdl is the protein with amino acid sequence as depicted in SEQ ID N°: 9.

[0099] "LRP" or "lipoprotein receptor-related protein" refers to any member of the low-density lipoprotein receptor- related protein (LRP) family, which consists of single-pass transmembrane receptors involved in receptor-mediated endocytosis, signaling pathway modulation, and macromolecule trafficking. LRPs are characterized by their structural similarity to low-density lipoprotein (LDL) receptors and play key roles in lipid metabolism, neuronal signaling, and Wnt / 0-catenin pathway regulation. In certain embodiments, the term LRP denotes LRP5, LRP6, or LRP5 / 6. LRP may be LRP5 with the amino acid sequence as depicted in SEQ ID N°: 3, or LRP6 with the amino acid sequence as depicted in SEQ ID N°: 4 or 5. Both LRP5 and LRP6 may exist as multiple isoforms, which are also included. LRP5 and LRP6 function as coreceptors in the Wnt signaling pathway, regulating p-catenin-mediated transcriptional activity. The LRP family also includes other members with distinct roles. LRP1 is involved in receptor-mediated endocytosis and clearance of lipoproteins and proteases. LRP2 (Megalin) is important for renal and developmental processes, while LRP3 regulates intracellular trafficking. LRP4 is involved in neuromuscular junction formation and modulates Wnt signaling, and LRP8 (ApoER2) plays a role in neuronal signaling and synaptic plasticity. LRP10 and LRP11 are also part of this family, contributing to cellular processes like ligand uptake and intracellular signaling. For the purposes of this application, LRP encompasses any naturally occurring or modified versions of these proteins, including truncated, mutated, or chimeric forms, as long as they retain functional LRP characteristics.

[0100] The interaction between Wnt ligands, Frizzled receptors, and LRP is essential for the activation of the canonical Wnt pathway, which has wide-ranging effects on cell proliferation, differentiation, and tissue development. This binding event triggers a series of intracellular signaling events that ultimately lead to the stabilization and nuclear translocation of a protein called 0-catenin. In the nucleus, 0-catenin functions as a transcriptional co-activator, regulating the expression of target genes involved in various cellular processes.

[0101] "Reck", or "RECK" is a GPI-anchored membrane protein that stands for "Reversioninducing Cysteine-rich Protein with Kazal Motifs". In a particular embodiment, Reck is the protein with amino acid sequence as depicted in SEQ ID N°: 6.

[0102] "Gprl24" or "GPR124" is an orphan GPCR expressed in endothelial cells. Gprl24 is also named ADGRA2 or adhesion G protein-coupled receptor A2. In a particular embodiment, Gprl24 is the protein with amino acid sequence as depicted in SEQ ID N°: 7.The terms "bind", "interact", "specifically bind" or "specifically interact" as used throughout this specification mean that an agent binds to or influences one or more desired molecules or analytes substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. The terms do not necessarily require that an agent binds exclusively to its intended target(s). For example, an agent may be said to specifically bind to target(s) of interest if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or more greater, such as, e.g., at least about 1000-fold or more greater, at least about lxl04-fold or more greater, or at least about lxl05-fold or more greater, than its affinity for a nontarget molecule.

[0103] The term "gene therapy" and / or "RIMA therapy" as used herein refers to the introduction of an exogenous polynucleotide into a host cell for therapeutic or prophylactic purposes, irrespective of the method used for the introduction. Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) as described elsewhere herein. The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art. The RNA molecule may be any type of RNA molecule. For instance, the RNA molecule may be cytoplasmic RNA, a nuclear RNA, an mRNA, antisense RNA or a noncoding RNA, preferably an mRNA.

[0104] The terms "host cell" and "host organism" may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E. coli, Salmonella typhimurium, Serratia marcescens, or Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae or Pichia pastoris), (cultured) plant cells (e.g., from Arabidopsis thaliana or Nicotiana tabacum) and (cultured) animal cells (e.g., vertebrate animal cells, mammalian cells,primate cells, human cells or insect cells). Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.

[0105] Except when noted, the terms "subject" or "patient" can be used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals including rodents and primates. Said subject may be a human, a dog, cat, rodent, rabbit, primate, or any other suitable warm-blooded animal, including but not limited to livestock, companion animals, and laboratory animals. Preferred subjects are human subjects. The terms "subject" or "patient" include subjects in need of treatment, more particularly subjects that would benefit from treatment of a given condition, particularly an epilepsy. Such subjects may include, without limitation, those that have been diagnosed with said condition, those exhibiting one or more symptoms of epilepsy (e.g. seizures), those prone to develop said condition and / or those in who said condition is to be prevented. Diagnosis may be performed by any method or technique known in the art.

[0106] The term "therapeutically active / effective amount" as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a surgeon, researcher, veterinarian, medical doctor or other clinician, which may include inter alia alleviation of the symptoms of the disease or condition being treated. The term "prophylactically active / effective amount" refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician. In present context, the "therapeutically active amount" is used to refer to both the "therapeutically active amount" and "prophylactically active amount" as described above, unless the distinction is clear from the context. Methods are known in the art for determining therapeutically and / or prophylactically effective doses of a compound, a protein, a nucleic acid encoding the compound / protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein. The term "therapeutically effective dose" as used herein refers to an amount of a compound, a protein, a nucleic acid encoding the compound / protein, a nucleic acid expression cassette, or a pharmaceutical composition, as taught herein, that when administeredbrings about a positive therapeutic response with respect to treatment of a patient having a specific disease or disorder.

[0107] "Blood-brain barrier", "blood brain barrier" or "BBB" refers to highly selective semipermeable properties of endothelial cells that prevents solutes in the circulating blood from non-selectively crossing into the extracellular fluid of the central nervous system where neurons reside. The blood-brain barrier is formed by endothelial cells of the capillary wall, astrocyte end-feet ensheathing the capillary, and pericytes embedded in the capillary basement membrane. This system allows the passage of some small molecules by passive diffusion, as well as the selective and active transport of various nutrients, ions, organic anions, and macromolecules that are crucial to neural function. The blood-brain barrier, by the selectivity of tight junctions between the endothelial cells of brain capillaries, restricts the passage of pathogens, the diffusion of solutes in the blood, and large or hydrophilic molecules into the cerebrospinal fluid, while allowing the diffusion of hydrophobic molecules (O2, CO2, hormones) and small non-polar molecules. Cells of the barrier actively transport metabolic products such as glucose across the barrier using specific transport proteins. The barrier also restricts the passage of peripheral immune factors, like signalling molecules, antibodies, and immune cells, into the CNS, thus insulating the brain from damage due to peripheral immune events. The BBB is composed of endothelial cells restricting passage of substances from the blood more selectively than endothelial cells of capillaries elsewhere in the body.

[0108] In the present context, "epilepsy" refers to a chronic neurological disorder characterized by recurrent, unprovoked seizures resulting from abnormal electrical activity in the brain. "Epileptogenesis" is a process that leads to the establishment of epilepsy and which may appear latent while cellular, molecular, and morphological changes leading to pathological neuronal network reorganization occur. Epilepsy can manifest in various forms, including focal epilepsy and generalized epilepsy, with symptoms and severity depending on the brain regions affected and the underlying causes.

[0109] Epilepsy can be classified according the electroclinical syndromes following the Classification and Terminology of the International League Against Epilepsy (ILAE; Berg et al., 2010). These syndromes can be categorized by age at onset, distinctive constellations (surgical syndromes), and structural-metabolic causes, such as:

[0110] (A) age at onset:

[0111] (i) neonatal period includes benign familial neonatal epilepsy (BFNE), early myoclonic encephalopathy (EME), Ohtahara syndrome;(ii) infancy period includes epilepsy of infancy with migrating focal seizures, West syndrome, myoclonic epilepsy in infancy (MEI), benign infantile epilepsy, benign familial infantile epilepsy, Dravet syndrome, myoclonic encephalopathy in nonprogressive disorders;

[0112] (iii) childhood period includes febrile seizures plus (FS+), Panayiotopoulos syndrome, epilepsy with myoclonic atonic (previously astatic) seizures, benign epilepsy with centrotemporal spikes (BECTS), autosomal- dominant nocturnal frontal lobe epilepsy (ADNFLE), late onset childhood occipital epilepsy (Gastaut type), epilepsy with myoclonic absences, Lennox-Gastaut syndrome, epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS), Landau- Kleffner syndrome (LKS), childhood absence epilepsy (CAE);

[0113] (iv) adolescence - adult period includes juvenile absence epilepsy (JAE) juvenile myoclonic epilepsy (JME), epilepsy with generalized tonic-clonic seizures alone, progressive myoclonus epilepsies (PME), autosomal dominant epilepsy with auditory features (ADEAF), other familial temporal lobe epilepsies;

[0114] (v) variable age onset includes familial focal epilepsy with variable foci (childhood to adult), reflex epilepsies;

[0115] (B) distinctive constellations (surgical syndromes) include mesial temporal lobe epilepsy (MTLE), Rasmussen syndrome, gelastic seizures with hypothalamic hamartoma, hemiconvulsion-hemiplegia-epilepsy;

[0116] (C) epilepsies attributed to and organized by structural-metabolic causes include malformations of cortical development (hemimegalencephaly, heterotopias, etc.), neurocutaneous syndromes (tuberous sclerosis complex and Sturge- Weber), tumor, infection, trauma, angioma, perinatal insults, and stroke. The term "refractory epilepsy" refers to an epilepsy which is refractory to pharmaceutical treatment (e.g., treatment with an anti-epileptic drug); that is to say that current pharmaceutical treatment does not allow an effective treatment of a patient's disease (see, for example, Englot et al. (Journal of Neurosurgery. 118(1): 169-74. 2013)).

[0117] Non-limiting examples of anti-epileptic drugs include narrow-spectrum anti-epileptic drugs and broad-spectrum anti-epileptic drugs. Narrow-spectrum anti-epileptic drugs are primarily used to treat focal seizures and include Carbamazepine (Carbatrol, Tegretol, Epitol, Equetro), Eslicarbazepine (Aptiom) Everolimus (Afinitor, Afinitor Disperz), Gabapentin (Neurontin), Lacosamide (Vimpat), Oxcarbazepine (Trileptal, OxtellarXR), Phenobarbital, Phenytoin (Dilantin, Phenytek), Pregabalin (Lyrica), Tiagabine (Gabitril), and Vigabatrin (Sabril). Broad -spectrum anti-epilepticdrugs include Brivaracetam (Briviact), Cannabidiol (Epidiolex), Cenobamate (Xcopri), Clorazepate (Gen-Xene, Tranxene-T), Divalproex (Depakote, Depakote ER), Felbamate (Felbatol), Lamotrigine (Lamictal, Lamictal CD, Lamictal ODT, Lamictal XR), Levetiracetam (Elepsia XR, Keppra, Keppra XR, Spritam), Lorazepam (Ativan), Perampanel (Fycompa), Primidone (Mysoline), Rufinamide (Banzel), Topiramate (Topamax, Qudexy XR, Trokendi XR), Valproic acid, and Zonisamide (Zonegran).

[0118] An "epileptic seizure" refers to a transient occurrence of signs or symptoms due to abnormal, excessive, or synchronous neuronal activity in the brain. Epileptic seizures can cause sensory disturbances, involuntary movements, loss of consciousness, or behavioral changes. Seizures may be classified based on their origin in the brain and their clinical presentation.

[0119] "Generalized epilepsy" is a type of epilepsy where seizures originate simultaneously in both hemispheres of the brain, rather than from a localized region. These seizures often cause a loss of consciousness and bilateral motor involvement. Generalized epilepsy can be caused by genetic factors, metabolic conditions, or structural brain abnormalities. A "generalized seizure" refers to an epileptic seizure arising from both hemispheres of the brain simultaneously. Generalized seizures often result in a loss of consciousness and may present with bilateral motor activity. Types of generalized seizures include absence seizures, tonic-clonic seizures, myoclonic seizures, and atonic seizures, each characterized by distinct clinical manifestations.

[0120] "Focal Epilepsy" is a type of epilepsy where seizures originate in a specific, localized area of the brain. Symptoms and severity depend on the affected brain region or lobe. Focal epilepsy includes both focal aware seizures (where the person remains conscious) and focal impaired awareness seizures (where consciousness is altered or lost). Focal seizures are most common in people who have had head injuries, birth abnormalities of their brain, febrile seizures in childhood, infections of their brain (encephalitis), strokes, brain tumors, or other conditions that affect their brain . Thus, causes of focal epilepsy include brain injury, infections, and genetic factors. Focal epilepsies include idiopathic location-related epilepsies, frontal lobe epilepsy, temporal lobe epilepsy, parietal love epilepsy, and occipital lobe epilepsy. The pharmaceutical composition for use, as disclosed herein, can be used in the treatment and / or prevention of focal epilepsy. A "focal seizure" refers to an epileptic seizure originating from a specific, localized region of the brain. Focal seizures may be further categorized into focal aware seizures, where the person remainsconscious, and focal impaired awareness seizures, where consciousness is altered or lost. Symptoms depend on the affected brain region and may include motor, sensory, autonomic, or psychological changes.

[0121] "Temporal Lobe Epilepsy" (TLE) is the most common form of focal epilepsy, affecting 6 of every 10 people with focal epilepsy, and where seizures originate in one or both of the temporal lobes of the brain. The hippocampus, including the dentate gyrus (DG), has been identified as a brain region particularly susceptible to damage that leads to TLE, and, in some instances, has been associated with treatment-resistant (i.e., refractory) epilepsy (Jarero-Basulto et al., Pharmaceuticals 11 (1):17, 2018). TLE is often characterized by auras (such as feelings of deja vu or unusual sensory experiences), automatisms (e.g., repetitive movements like lip-smacking), and disturbances in memory and emotions. In some cases, seizures may progress to bilateral tonic-clonic seizures involving both brain hemispheres. Causes of TLE include mesial temporal sclerosis, brain injuries, and infections such as meningitis. The pharmaceutical composition for use, as disclosed herein, can be used in the treatment and / or prevention of TLE.

[0122] "Non-TLE Focal Epilepsy" relates to focal epilepsy where seizures originate outside the temporal lobe, such as the frontal, parietal, or occipital lobes. The symptoms vary based on the affected region. For example, frontal lobe seizures may cause sudden body movements or vocal outbursts, parietal lobe seizures may lead to sensory disturbances, and occipital lobe seizures may involve visual phenomena like flashes of light. Causes of non-TLE focal epilepsy include structural abnormalities, autoimmune conditions, and genetic mutations.

[0123] A "recurrent epileptic seizure that is refractory to treatment" refers to repeated epileptic seizures that persist despite administration of at least two appropriately selected and adequately dosed antiepileptic drugs (AEDs), whether used as monotherapy or in combination. This condition, also known as "drug-resistant epilepsy", indicates a failure to achieve sustained seizure freedom with available pharmacological treatments. Non-limiting examples of antiepileptic drugs are described above.

[0124] "Symptoms of epilepsy" refers to any observable or measurable manifestations associated with epilepsy or epileptic seizures. These symptoms may include, but are not limited to:(A) Seizure-related characteristics - frequency, duration, intensity, or severity of epileptic seizures, including focal seizures, generalized seizures, absence seizures, myoclonic seizures, tonic-clonic seizures, atonic seizures, and epileptiform discharges as measurable by electroencephalogram (EEG); (B) Neurological and cognitive impairments - sudden confusion, difficulty understanding or producing speech, cognitive dysfunction, memory impairment, and loss of consciousness or awareness;

[0125] (C) Motor dysfunction - weakness, impaired mobility, dizziness, loss of balance or coordination, paralysis, involuntary muscle contractions, or abnormal motor responses;

[0126] (D) Behavioral and emotional changes - emotional dysregulation, mood disturbances, anxiety, agitation, or other neuropsychiatric manifestations; (E) Structural or functional abnormalities - pathological sprouting of recurrent mossy fibers of dentate gyrus granule cells in the hippocampus, excitotoxicity-related neuronal cell death in the central nervous system (CNS), and disruptions in physiological excitation-inhibition balance.

[0127] Symptoms may be assessed using standard clinical and diagnostic methods, including but not limited to neurological examination, electroencephalogram (EEG), magnetoencephalogram (MEG), computed tomography (CT) scan, positron emission tomography (PET) scan, functional magnetic resonance imaging (fMRI), videography, and visual observation.

[0128] The "kainic acid (KA) model of temporal lobe epilepsy (TLE)" is a widely used experimental model to study the pathophysiology of epilepsy and to evaluate potential antiepileptic treatments. It replicates many features of human TLE, including hippocampal sclerosis, spontaneous recurrent seizures, and blood-brain barrier (BBB) dysfunction. This model employs kainic acid, a potent neurotoxin and glutamate receptor agonist (specifically targeting AMPA and kainate receptors), is administered systemically (intraperitoneally), intracerebrally (into the hippocampus), or intracerebroventricularly. Kainic acid induces excitotoxicity by overstimulating glutamate receptors, leading to prolonged neuronal depolarization, calcium influx, and eventual neuronal damage. This excitotoxic cascade mimics the hyperexcitable neural environment observed in TLE. The Kainic Acid (KA) Model of epilepsy consists of three distinct phases.

[0129] • The Acute Seizure Phase, also known as Status Epilepticus (SE), begins within minutes to hours after KA administration. This phase is characterized by continuous seizures lasting several hours, accompanied by intense neuronal firing and hippocampal excitotoxicity.• Following this, the Latent Period represents a seizure-free interval lasting from days to weeks. During this time, neuronal damage, neuroinflammation, and structural remodeling occur, including abnormal synaptic reorganization such as mossy fiber sprouting.

[0130] • Finally, the Chronic Phase is marked by the emergence of spontaneous recurrent seizures (SRS), which mimic human temporal lobe epilepsy (TLE). This phase is long-lasting and is useful for evaluating long-term therapeutic interventions.

[0131] Several pathophysiological changes occur in the KA model. One key feature is hippocampal sclerosis, which involves the loss of pyramidal neurons in the CAI and CA3 regions, along with damage to the dentate gyrus. The model also induces bloodbrain barrier (BBB) disruption, leading to increased permeability, which enhances neuroinflammation and seizure susceptibility. Neuroinflammation is another hallmark, involving the activation of microglia and astrocytes and the release of pro-inflammatory cytokines such as IL-ip and TNF-o. Additionally, synaptic reorganization occurs, with aberrant growth of mossy fibers in the dentate gyrus. Neurogenesis is also altered, contributing to circuit dysregulation.

[0132] The KA model has several applications in epilepsy research. It is widely used for drug screening, allowing the evaluation of new antiepileptic drugs (AEDs) and diseasemodifying therapies. It also facilitates molecular studies, helping to investigate pathways such as Wnt signaling and neuroinflammation. The model is particularly useful in blood-brain barrier (BBB) research, providing insights into how BBB dysfunction contributes to epilepsy progression. Furthermore, it is used in gene therapy research to explore the efficacy of gene-based treatments targeting epileptogenesis.

[0133] The model offers several advantages. It closely mimics human TLE by replicating features such as spontaneous seizures and hippocampal damage. It is also suitable for studying epileptogenesis and chronic epilepsy, with flexible dosing and administration routes.

[0134] The "Racine scoring system" is a widely used classification method to assess seizure severity in animal models of epilepsy, particularly in rodents.

[0135] While variations exist depending on the laboratory or experimental setup, the version applied here is defined as follows:

[0136] • Score 0: no change in behaviour;

[0137] • Score 1: immobility;

[0138] • Score 2: head nodding;

[0139] • Score 3: forelimb clonus;• Score 4: forelimb clonus with rearing and falling;

[0140] • Score 5: wild running and jumping, often followed by generalized tonic-clonic activity and loss of postural tone;

[0141] • Score 6: death.

[0142] This scoring system provides a structured way to evaluate seizure progression and severity in experimental epilepsy models, offering valuable insights into the effects of epileptogenic stimuli and potential therapeutic interventions.

[0143] Detailed description

[0144] The inventors demonstrate herein a pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy or seizure in a patient. Said antibody or antibody fragment is a modulator of the Wnt7 signaling pathway, preferably an agonist of the Wnt7 signaling pathway. Said antibody or antibody fragment comprises one or more Lipoprotein Receptor-related Protein (LRP) binding domains, and further one or more Gprl24 and / or Reck binding domains.

[0145] Said antibody or antibody fragment is a modulator, preferably agonist, of the Wnt7 signaling pathway, and is capable of activating Wnt7 signaling selectively in cells expressing RECK and / or GPR124; for instance cells expressing GPR124, or cells expressing GPR124 and RECK i.e. for instance in cerebral endothelial cells. Hence, the invention allows providing inter alia modulators which are agonists of the Wnt7 signaling pathway, capable of stimulating Wnt / 0-catenin signaling in cerebral endothelial cells (also referred to as "on-target activity") with only low crossreactivity with other Frizzled pathways (i.e. "unwanted" or "off-target activity"), and thereby effective for use in the treatment and / or prevention of epilepsy or seizure in a patient.

[0146] The inventors surprisingly discovered that the direct recruitment or binding of the antibody or antibody fragment disclosed herein to LRP, such as LRP5 or LRP6 or any of the further LRP molecules as described above, preferably LRP 5 or LRP6, and at least Gprl24 or RECK is sufficient for stimulating Wnt7 signaling specifically through the Gprl24 / RECK / FZD / LRP receptor complex. This results in stimulating Wnt7 signaling in cerebral endothelial cells with impaired BBB, thereby restoring said BBB and ameliorating associated disease effects, in particular thereby treating and / or preventing epilepsy or seizure in a patient.In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0147] Accordingly, in a first aspect, the invention provides a pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy or seizure in a patient, wherein said antibody or antibody fragment is a modulator of the Wnt7 signaling pathway, preferably an agonist of the Wnt7 signaling pathway, and wherein said antibody or antibody fragment comprises one or more Lipoprotein Receptor-related Protein (LRP) binding domains, and further one or more Gprl24 and / or Reck binding domains. Said Wnt7 signaling pathway is mediated by the Gprl24 / RECK / Frizzled / LRP receptor complex, even more particularly of the Wnt7 signaling pathway mediated by the Gprl24 / RECK / Frizzled / LRP (LRP5 or LRP6) receptor complex.

[0148] It will be clear to a person skilled in the art that, in aspects, the invention thus also provides a method for treating and / or preventing epilepsy or seizure in a patient comprising administering the pharmaceutical composition to said patient or to a patient in need thereof, as well as a use in the preparation of a medicament for the treatment and / or prevention of epilepsy or seizure in a patient; and that all these aspects are connected. All features as described in one of the aspects, as described above as well as below, can relate to any of these aspects, even if they are described in conjunction with a specific aspect.

[0149] In one embodiment, said antibody or antibody fragment is a selective or specific modulator, preferably agonist, of the Gprl24- and Reck-dependent Wnt / p-catenin pathway. The term "selective agonist of the Gprl24- and Reck-dependent Wnt / p-catenin pathway" or "specific agonist of the Gprl24- and Reck-dependent Wnt / p-catenin pathway" is to be understood as an agonist of said pathway that is able to activate said pathway with high selectivity and specificity (on-target activity), with no, no statistically significantly or only minimal activation of other Wnt signaling pathways (off-target activity). In embodiments, the antibody or antibody fragment disclosed herein is an agonist of Gprl24- and Reck-dependent Wnt / 0-catenin pathway that shows less than 20% off-target activity, more particularly less than15%, 10%, 5% or 1% off-target activity, as measured in cell culture Super TOP-Flash assays (as known it the art and discussed in the example section below).

[0150] In embodiments, the pharmaceutical composition for use, as disclosed herein, comprises an antibody or antibody fragment, or nucleic acid encoding said antibody or antibody fragment. Said antibody or antibody fragment is preferably an agonist of the GPR124 / RECK / Frizzled / LRP complex, and is capable of binding to LRP such as LRP5 or LRP6 and the GPR124 protein, or to LRP such as LRP5 or LRP6 and the RECK protein. In embodiments, said antibody or antibody fragment is not able to or has an impaired capacity to bind to or activate Frizzled (Fzd), and / or does not comprise Fzd binding domains. Said pharmaceutical composition is used in the treatment and / or prevention of epilepsy.

[0151] Epilepsy, as also defined above, refers to one or more neurological disorders that clinically present with, generally recurrent, epileptic seizures, and with symptoms and severity depending on the brain regions affected and the underlying causes. Such epileptic seizure refers to a transient occurrence of signs or symptoms due to abnormal, excessive, or synchronous neuronal activity in the brain. Epileptic seizures can cause sensory disturbances, involuntary movements, loss of consciousness, or behavioral changes. Seizures may be classified based on their origin in the brain and their clinical presentation.

[0152] In embodiments of said pharmaceutical composition for use in the treatment and / or prevention of epilepsy or seizure in a patient, said epilepsy is generalized epilepsy. In generalized epilepsy, seizures originate simultaneously in both hemispheres of the brain, rather than from a localized region. These seizures often cause a loss of consciousness and bilateral motor involvement. Generalized epilepsy can be caused by genetic factors, metabolic conditions, or structural brain abnormalities.

[0153] In embodiments of said pharmaceutical composition for use in the treatment and / or prevention of epilepsy or seizure in a patient, said epilepsy is focal epilepsy. In focal epilepsy, seizures originate in a specific, localized area of the brain. Symptoms and severity depend on the affected brain region or lobe. Focal epilepsy includes both focal aware seizures (where the person remains conscious) and focal impaired awareness seizures (where consciousness is altered or lost).

[0154] In embodiments, where said epilepsy is focal epilepsy, said epilepsy may be temporal lobe epilepsy (TLE) or non-TLE focal epilepsy. As defined above, in TLE, seizuresoriginate in one or both of the temporal lobes of the brain. TLE is often characterized by auras, automatisms, and disturbances in memory and emotions. In some cases, seizures may progress to bilateral tonic-clonic seizures involving both brain hemispheres. Non-TLE focal epilepsy relates to focal epilepsy where seizures originate outside the temporal lobe, such as the frontal, parietal, or occipital lobes. The symptoms vary based on the affected region.

[0155] In embodiments of the pharmaceutical composition for use in the treatment and / or prevention of epilepsy in a patient, said patient exhibits one or more symptoms of epilepsy, including epileptic seizure.

[0156] Epileptic seizure is a transient occurrence of signs or symptoms due to abnormal, excessive, or synchronous neuronal activity in the brain. Epileptic seizures can cause sensory disturbances, involuntary movements, loss of consciousness, or behavioral changes. Seizures may be classified based on their origin in the brain and their clinical presentation.

[0157] "Symptoms of epilepsy" refer to any observable or measurable manifestations associated with epilepsy or epileptic seizures. These symptoms may include, but are not limited to:

[0158] (A) Seizure-related symptoms - frequency, duration, intensity, or severity of epileptic seizures, including focal seizures, generalized seizures, absence seizures, myoclonic seizures, tonic-clonic seizures, atonic seizures, and epileptiform discharges as measurable by electroencephalogram (EEG), or combinations thereof;

[0159] (B) Symptoms related to neurological and cognitive impairment - sudden confusion, difficulty understanding or producing speech, cognitive dysfunction, memory impairment, and loss of consciousness or awareness, or combinations thereof;

[0160] (C) Symptoms related to motor dysfunction - weakness, impaired mobility, dizziness, loss of balance or coordination, paralysis, involuntary muscle contractions, or abnormal motor responses, or combinations thereof;

[0161] (D) Symptoms related to behavioral and emotional changes - emotional dysregulation, mood disturbances, anxiety, agitation, or other neuropsychiatric manifestations, or combinations thereof;

[0162] (E) Symptoms related to structural or functional abnormalities - pathological sprouting of recurrent mossy fibers of dentate gyrus granule cells in the hippocampus, excitotoxicity- related neuronal cell death in the central nervoussystem (CNS), and disruptions in physiological excitation-inhibition balance, or combinations thereof;

[0163] or combinations of one or more of said symptoms.

[0164] Symptoms may be assessed using standard clinical and diagnostic methods, including but not limited to neurological examination, electroencephalogram (EEG), magnetoencephalogram (MEG), computed tomography (CT) scan, positron emission tomography (PET) scan, functional magnetic resonance imaging (fMRI), videography, and visual observation, as known in the art.

[0165] The pharmaceutical composition for use of the present invention is able to treat and / or prevent epilepsy, thereby reducing at least one or more of the above mentioned symptoms in the patient. Preferably, at least one or more seizure-related symptoms are reduced by said treatment.

[0166] As used herein, "treatment" refers to any therapeutic intervention that mitigates, alleviates, delays the onset, inhibits the progression, or reduces the severity of epilepsy or its associated symptoms in a patient. Treatment may be curative, palliative, prophylactic, or preventive. This includes reducing the frequency, intensity, or duration of seizures, preventing seizure recurrence, improving neurological function, restoring blood-brain barrier (BBB) integrity, or decreasing neuroinflammation linked to epilepsy. "Prevention" refers to reducing the likelihood, delaying the onset, or entirely preventing the development of epilepsy or epilepsy-related complications. In patients at risk of epilepsy— such as those with a history of brain injury, genetic predisposition, or other risk factors— administration of the pharmaceutical composition aims to prevent or significantly reduce the incidence of seizures or neurovascular dysfunction.

[0167] As used herein, "reduction" means a measurable decrease in at least one seizure-related symptom, which may be evaluated through clinical assessments, electrophysiological recordings (e.g., EEG), patient-reported outcomes, or any standard clinical or diagnostic method as known in the art. Reduction may include, but is not limited to:

[0168] (i) A decrease in seizure frequency by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% relative to baseline or an untreated control.

[0169] (ii) A reduction in seizure severity, including a decrease in the duration of seizures, the intensity of motor symptoms, or other measurable clinical manifestations, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% relative to baseline or an untreated control.

[0170] (iii)An extension of seizure-free periods by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% relative to baseline or an untreated control.

[0171] (iv)Improvements in cognitive or memory deficits associated with epilepsy, quantified by standardized neurocognitive tests by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% improvement relative to baseline or an untreated control, in performance scores.

[0172] (v) Restoration or maintenance of blood-brain barrier integrity as determined by biomarkers or imaging techniques, with at least a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more reduction in BBB permeability markers (e.g., albumin extravasation or other suitable indicators).

[0173] (vi)A decrease in neuroinflammation or gliosis as confirmed by biochemical or histological analysis, with at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50%, or more reduction in inflammatory markers (e.g., cytokine levels, activated glial cell counts, or c-fos immunolabelling with EEG readout).

[0174] The degree of reduction may vary depending on patient characteristics, the severity of epilepsy, and the administration regimen but preferably involves a statistically significant improvement compared to untreated patients or baseline conditions. The pharmaceutical composition of the present invention may be administered as a monotherapy or in combination with existing antiepileptic drugs (AEDs) to enhance therapeutic outcomes.

[0175] In an embodiment where said patient exhibits one or more symptoms of epilepsy, including epileptic seizure, said epileptic seizures may be recurrent epileptic seizure. Optionally, said recurrent epileptic seizure is refractory to treatment, as defined above, and / or said epilepsy is refractory epilepsy, as defined above.

[0176] In embodiments where the epilepsy is generalized epilepsy, the patient exhibits one or more symptoms of epilepsy as described above, including generalized epileptic seizure.

[0177] In embodiments, where the epilepsy is focal epilepsy, the patient exhibits one or more symptoms of epilepsy as defined above, including focal epileptic seizure.As mentioned above, said pharmaceutical composition for use in the treatment and / or prevention of epilepsy or seizure in a patient of the invention, comprises a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment.

[0178] In an embodiment, the antibodies or antibody fragments as comprised in the pharmaceutical composition for use, or as encoded by the nucleic acid comprised in the pharmaceutical composition for use as disclosed herein may be engineered.

[0179] In an embodiment, said antibody or antibody fragment as comprised in the pharmaceutical composition for use, or as encoded by the nucleic acid comprised in the pharmaceutical composition for use as disclosed herein, comprises one or more LRP binding domains, and further one or more Gprl24 and / or Reck binding domains.

[0180] In embodiments, said LRP binding domains are able to bind LRP5 and / or LRP6.

[0181] In another or further embodiment, the antibody or antibody fragment as comprised in the pharmaceutical composition for use or as encoded by the nucleic acid comprised in the pharmaceutical composition for use described herein is water soluble. By "water soluble", or short "soluble" it is meant a composition that is soluble in aqueous buffers in the absence of detergent, usually soluble at a concentration that provides a biologically effective dose of the polypeptide. The antibody or antibody fragment disclosed herein typically forms a substantially homogeneous aqueous solution at concentrations of at least 2.5 pM, and higher, e.g., at least 2.5 pM, 3 pM, 4 pM, 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 40 pM, or 50 pM, and in some embodiments at least 60 pM, 70 pM, 80 pM, or 90 pM, sometimes as much as 100 pM, 120 pM, or 150 pM. In other words, the antibody or antibody fragment disclosed herein typically forms a substantially homogeneous aqueous solution at concentrations of about 2.5 mg / ml, about 0.5 mg / ml, about 1.0 mg / ml, about 1.5 mg / ml, about 2.0 mg / ml, 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.

[0182] In an embodiment, the antibody or antibody fragment as disclosed herein is capable of simultaneously binding to LRP proteins, more particularly LRP5 or LRP6, in addition to the GPR124 or the RECK proteins.In an embodiment, the antibody or antibody fragment, which is a modulator of the Wnt7 signaling pathway, comprises one or more LRP binding domains and one or more Gprl24 or Reck binding domains. In a further embodiment, said agonist comprises one, two, three, four, or five LRP binding domains, and one, two, three, four, or five Gprl24 or Reck binding domains. In a preferred embodiment, agonist comprises one or two LRP binding domains, and one or two Gprl24 or Reck binding domains, more preferably two LRP binding domains, and two Gprl24 or Reck binding domains.

[0183] In an embodiment, the LRP binding domains or binding regions are LRP5 and / or LRP6 binding domains or binding regions, and / or said LRP binding domains or binding regions are able to bind to LRP5 and / or LRP6.

[0184] In an embodiment, the antibody or antibody fragment as described herein may be tetravalent or bivalent.

[0185] In an embodiment, the antibody or antibody fragment as described herein is tetravalent.

[0186] In a particular embodiment, said antibody or antibody fragment comprises:

[0187] i) two regions that each specifically bind to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and / or LRP6 epitopes; and

[0188] ii) two regions that each specifically bind to a set of one or more Reck or Gprl24 epitopes.

[0189] In an embodiment, each one or more LRP binding domain such as LRP5 / 6 binding domain is fused in tandem to either one or more Reck binding domains and / or one or more Gprl24 binding domains, and wherein said domains are optionally separated by a linker sequence.

[0190] Said linkers may be any linker known in the art, or described in this text. As a nonlimiting example, said linker is a 5-mer linker such as GSGGS (SEQ ID N°: 172).

[0191] In another embodiment, the antibody or antibody fragment as described herein is bivalent.

[0192] In a particular embodiment, said antibody or antibody fragment comprises:

[0193] i) one region that specifically binds to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and / or LRP6 epitopes; andii) one region that specifically binds to a set of one or more Reck or Gprl24 epitopes.

[0194] In an embodiment, the LRP binding domain, such as LRP5 / 6 binding domain, is fused in tandem to a Reck or Gprl24 binding domain, and wherein said domains are optionally separated by a linker sequence.

[0195] Said linkers may be any linker known in the art, or described in this text. As a nonlimiting example, said linker is a 5-mer linker such as GSGGS (SEQ ID N°: 172).

[0196] In a particular embodiment, said antibody or antibody fragment does not comprise a Fzd binding domain. According to particular embodiments, the antibody or antibody fragment disclosed herein is not able to bind FZD.

[0197] In other or further embodiments, the antibody or antibody fragment as disclosed herein cannot stimulate the Wnt7 signaling pathway in cells which are GPR124 / RECK negative. In further embodiments, GPR124 / RECK negative means having no or low expression of GPR124 and / or RECK proteins, no or low expression of GPR124 and / or RECK in a cell, no or low presence of GPR124 and / or RECK proteins in a cell or no or low presence of GPR124 and / or RECK proteins at a cell surface.

[0198] In other embodiments, GPR124 / RECK positive then refers to the expression of GPR124 and / or RECK in a cell, presence of GPR124 and / or RECK proteins in a cell and / or presence of GPR124 and / or RECK proteins at a cell surface.

[0199] In other or further embodiments, FZD / LRP positive can mean the expression of FZD and / or LRP in a cell, presence of FZD and / or LRP protein in a cell, and / or presence of FZD and / or LRP proteins at a cell surface.

[0200] In particular embodiments, cells expressing GPR124, RECK, FZD and LRP proteins, more particularly LRP5 or LRP6, at its plasma membrane, are cells naturally expressing all GPR124, RECK, FZD and LRP proteins at the cell surface, such as a cerebral endothelial cell.

[0201] In some embodiments "the presence of RECK and GPR124" may refer to the presence of the RECK and GPR124 proteins at or close to the cell membrane, preferably in close proximity with the Frizzled and LRP proteins, more particularly LRP5 or LRP6. The close mutual proximity can facilitate the formation of the GPR124 / RECK / Frizzled / LRP receptor complex under conditions conducive thereto,such as when the agonist as taught herein is externally supplied to a cell. In other or further embodiments, when used in gene or RIMA therapy, the agonist as taught herein could be expressed from the target cell population. On the other hand, the phrase "the absence of RECK and / or GPR124" may refer to the lack or nonoccurrence of the RECK and / or GPR124 proteins at the cell membrane. The absence of RECK and / or GPR124 at or close to the cell membrane may occur when a cell does not express, translate, or correctly translocate RECK and / or GPR124. The absence of RECK and / or GPR124 need not denote the complete absence of the RECK and / or GPR124 protein at the cell membrane, but may for example refer to an amount of RECK and / or GPR124 protein which is not detectable by, or falls below the sensitivity range of, conventional protein detection or quantification assays known by the person skilled in the art, such as for example immunoblotting, immunocytochemistry or immunofluorescence.

[0202] In other or further embodiments, said antibody or antibody fragment herein disclosed is capable of activating the GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling, wherein said antibody or antibody fragment does not or not substantially activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124.

[0203] In particular embodiments, the capability of the antibody or antibody fragment herein disclosed to activate GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling, but not or not substantially activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of the antibody or antibody fragment to activate Wnt7 signaling in cells positive for GPR124, RECK, FZD and LRP, more particularly LRP5 or LRP6, but not in cells positive for FZD and LRP and negative for GPR124 and / or RECK, wherein the cells positive for GPR124, RECK, FZD and LRP, more particularly LRP5 or LRP6, and the cells positive for FZD and LRP and negative for GPR124 and / or RECK are otherwise substantially identical.

[0204] In particular embodiments, the capability of the antibody or antibody fragment to activate GPR124 / RECK / Frizzled / LRP-mediated Wnt7 signaling, but not activate the canonical Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124, denotes the capability of said antibody or antibody fragment to activate the atypical Wnt signaling in the presence of RECK and GPR124, but not in the absence of RECK and / or GPR124.The capability of activating Wnt7 signaling refers to the ability of the antibody or antibody fragment as disclosed herein to mimic, reproduce or approximate the signal transduction effect and / or activity of a natural Wnt7 ligand binding to a Gprl24 / RECK / FZD / LRP complex.

[0205] Activation of Wnt7 signaling may be suitably determined and / or quantitated by measuring the expression of one or more Wnt7 target genes, TCF reporter gene expression, beta-catenin stabilization, LRP phosphorylation, and / or translocation of Axin from cytoplasm to cell membrane as known in the art. For instance, activation of Wnt signaling may be suitably determined and / or quantitated by measuring the expression of TCF gene (e.g., by RT-PCR or any other transcript detection method), a primary output of Wnt signaling. For example, a TCF reporter assay (also known as TOP / FOP or TOP flash) may be used to assess changes in the transcription of TCF / LEF controlled genes. The TCF reporter assay may be a luciferase reporter assay. Further for example, activation of Wnt signaling may be suitably determined and / or quantitated by measuring the expression of c-myc, n-myc, LEF1, or c-jun. Alternatively, activation of Wnt signaling may be determined by measuring the location, level and / or phosphorylation status of 0-catenin. A non-limiting example of such an assay is the "0-Catenin Redistribution Assay” (Thermo Scientific) which provides recombinant U20S cells stably expressing human p-catenin fused to the C-terminus of enhanced green fluorescent protein (EGFP). The assay allows visualization and monitoring of the translocation of a GFP-p-catenin fusion protein from the membrane to the nucleus. Another way of determining activation of Wnt signaling is the visualization of Axin translocation, for example with a GFP-Axin fusion protein.

[0206] In particular embodiments, the antibody or antibody fragment as disclosed herein may be considered capable of activating the central nervous system (CNS) Wnt7 signaling or the Gprl24 / RECK / FZD / LRP5 or LRP6 dependent Wnt7 signaling, if the antibody or antibody fragment enhances Wnt / 0-catenin signaling at least 10-fold more, at least 20-fold more, at least 30-fold more, at least 40-fold more, at least 50-fold more, at least 100-fold more, at least 250-fold more, at least 500-fold more, at least 750-fold more, at least 1000-fold more, at least lxl04-fold more, or at least lxl05-fold more compared to Wnt / 0-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.In particular embodiments, the antibody or antibody fragment as disclosed herein may be considered to not activate or not substantially activate (canonical) Wnt signaling if the compound enhances Wnt / 0-catenin signaling less than 10-fold more, such as particularly at most 5-fold more or at most 2.5-fold more, or if the protein does not enhance or even reduces (e.g., 2-fold less or 5-fold less or 10-fold less) Wnt / 0-catenin signaling compared to Wnt / 0-catenin signaling baseline or background induced by a neutral substance or negative control, for example as measured in an assay as described elsewhere herein.

[0207] In particular embodiments, the antibody or antibody fragment as disclosed herein may be considered to activate the GPR124 / RECK / Frizzled / LRP5 or LRP6-mediated Wnt7 signaling (denoted "on-target activity"), but not or not substantially activate Frizzled / LRP-mediated Wnt signaling in the absence of RECK and / or GPR124 (denoted "off-target activity"), if the on-target signaling activity is induced at least 30% and off-target activity is induced less than 20% by said antibody or antibody fragment. In embodiments, said antibody or antibody fragment can activate GPR124 / RECK / Frizzled / LRP5 or LRP6-mediated Wnt7 signaling more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein, preferably more than 70% compared to a control situation, for example in the absence of said antibody or antibody fragment.

[0208] In embodiments, said antibody or antibody fragment can activate Frizzled / LRP-mediated Wnt in the absence of RECK and / or GPR124 less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein, preferable less than 10% compared to a control situation, for example in the presence of Wnt7. Before comparing the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity (denoted "on-target activity") and the Frizzled / LRP-mediated Wnt signaling activity in the absence of RECK and / or GPR124 (denoted "off -target activity" in this paragraph), on-target activity and off-target activity induced by the antibody or antibody fragment may be normalized to on-target activity induced by wild-type Wnt7a or b, the latter for example set to represent 100% activity.

[0209] In one embodiment, the antibody or antibody fragment acts by Wnt7-specific RECK / GPR124 / Frizzled / LRP5 or LRP6-mediated signaling, in which said antibody or antibody fragment binds specifically to LRP5 or LRP6 and RECK in a FZD- and Gprl24-independent manner. The antibody or antibody fragment thereby recruitsGprl24 via RECK to the FZD / LRP complex, thereby assembling the Wnt7-ligand specific RECK / GPR124 / FZD / LRP signalosomes and activating the canonical Wnt7 signaling. According to further embodiments, the antibody or antibody fragment of the disclosed invention thus induces the heteromerization of RECK and LRP5 or LRP6 proteins in the presence of Frizzled and GPR124.

[0210] In another embodiment, the antibody or antibody fragment of the present invention acts by Wnt7-specific RECK / GPR124 / Frizzled / LRP-mediated signaling, in which said antibody or antibody fragment binds specifically to LRP5 or LRP6 and Gprl24 in a RECK- and FZD-independent manner. The antibody or antibody fragment thereby recruits RECK via Gprl24 to the FZD / LRP complex, thereby assembling the Wnt7-ligand specific RECK / GPR124 / FZD / LRP signalosomes and activating the canonical Wnt7 signaling. According to further embodiments, the antibody or antibody fragment of the disclosed invention thus induces the heteromerization of Gprl24 and LRP5 or LRP6 proteins in the presence of Frizzled and RECK.

[0211] Accordingly, in particular embodiments, the antibody or antibody fragment as disclosed herein is able to bind to LRP5 and / or LRP6 proteins and a protein selected from Gprl24 and RECK, preferably at a cell membrane. In particular embodiments, the binding of the antibody or antibody fragment to LRP5 and / or LRP6 and to RECK and / or Gprl24 occurs via epitopes that are located in the extracellular domains of these receptor proteins, thereby mediating extracellular engagement of the target molecules at the cell surface. The LRP5 and / or LRP6 binding domains of the antibody or antibody fragment thus recognize and bind amino acid sequences within the extracellular domain of LRP5 and / or LRP6, including, for example, the DKKl-binding site and / or the Wnt-binding site and / or one or more of the 0-propeller-EGF-like modules P1E1, P2E2, P3E3 and / or P4E4 as described herein. Likewise, the RECK-binding and / or Gprl24-binding domains of the antibody or antibody fragment recognize and bind epitopes within the extracellular regions of RECK and / or Gprl24, such as the CK4 and / or CK5 motifs of RECK and / or the extracellular segments of Gprl24 as defined herein. Accordingly, the antibody or antibody fragment of the present disclosure is explicitly intended to bind its LRP5 and / or LRP6 and RECK and / or Gprl24 targets in an extracellular manner, without requiring intracellular access to the cytoplasmic or transmembrane portions of these proteins.

[0212] The skilled person shall further appreciate that a GPR124 / RECK / FZD / LRP receptor complex, as envisaged herein, may include further component(s), which may or need not functionally modulate the complex. For example, the complex may includeDishevelled (Dvl), forming intracellular scaffolds capable of bridging GPR124 and Frizzled.

[0213] In particular embodiments, the antibody or antibody fragment as disclosed herein induces or has a Wnt signaling activity of at least 35%, preferably at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, preferably 100%, of the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In preferred embodiments, the antibody or antibody fragment as disclosed herein has at least 70% of the GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling activity of the full-length wild-type Wnt7 protein. In particular embodiments, the antibody or antibody fragment of the invention as disclosed herein may activate GPR124 / RECK / Frizzled / LRP-mediated Wnt signaling in the presence of all four coreceptors more than 30% of the activation by full-length wild-type Wnt7 protein. For example, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 99% and all ranges and subranges therein.

[0214] In particular embodiments, the antibody or antibody fragment as disclosed herein may activate Wnt signaling in the absence of one of the four co-receptors (i.e. RECK, Fzd, LRP or GPR124) less than 20% of the activation by full-length wild-type Wnt7 protein. For example, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, less than 1% and all ranges and subranges therein.

[0215] In one embodiment, the antibody or antibody fragment disclosed herein is a binding agent comprising one or more LRP binding domains such as LRP5 or LRP6 binding domains and one or more Gprl24 or Reck binding domains. In a further embodiment, the binding agent is a fusion protein comprising a first polypeptide that can bind at least one LRP protein such as LRP5 or LRP6 protein and a second polypeptide that can bind at least one Gprl24 or Reck protein.

[0216] In an embodiment, the binding domains or polypeptides may be separated from each other via a linker or spacer sequence, such as the flexible GS or (Gly)n linker. Other suitable linker sequences will be known to the person skilled in the art.

[0217] In embodiments, the antibody or antibody fragment disclosed herein can bind to the LRP protein, particularly to LRP5 or LRP6, for example to the extracellular domain ofthe LRP protein, to the DKK-binding site and / or the Wnt-binding site of the LRP protein. In more particular embodiments, the antibody or antibody fragment disclosed herein is capable of binding to the DKKl-binding site of the LRP5 and / or LRP6 protein. In other particular embodiments the antibody or antibody fragment as disclosed herein is capable of binding to 0-propeller-EGF-like domains 1 and 2 (P1E1P2E2) and / or p-propeller-EGF-like domains 3 and 4(P3E3P4E4) of the LRP protein, more particularly of the LRP5 and / or LRP6 protein.

[0218] In particular embodiments, the antibody or antibody fragment as disclosed herein may be selective for the LRP5 or LRP6 proteins, for example having a specificity for LRP5 and / or LRP6 of at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least lxl04-fold, or at least lxl05-fold, compared to other non-preferred LRP proteins.

[0219] In an embodiment, the one or more LRP binding domains, such as one or more LRP5 and / or one or more LRP6 binding domains, bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the LRP5 or LRP6 proteins as depicted in SEQ ID N°: 3, 4 and 5. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids.

[0220] In an embodiment, said fragment of the LRP5 protein is the amino acid sequence between position 32 and 614 or between position 644 and 1254 of SEQ ID N°: 3. In an embodiment, said fragment of the LRP6 protein is the amino acid sequence between position 20 and 628 or between position 631 and 1244 of SEQ ID N°: 4.

[0221] In some embodiments, the one or more LRP5 and / or LRP6 binding domains are derived from one or more antigen-binding fragments of an antibody. In some embodiments, the one or more antigen-binding fragments are selected from the group consisting of: IgG, scFv, Fab, and VHH or sdAbs. In some embodiments, the one or more antigen-binding fragments are humanized.

[0222] In an embodiment, the antibody or antibody fragment herein disclosed can bind to RECK proteins involved in Wnt signaling. RECK is composed of five N-terminal cysteine-knot (CK) motifs or regions (i.e. CK1, CK2, CK3, CK4 and CK5), a cysteine-rich domain (CRD) and three Kazal motifs preceding a Glycosylphosphatidylinositol (GPI)-anchor site. The CK motifs, the CRD and the Kazal motifs are locatedextracellularly. Accordingly, in embodiments, the agonist herein disclosed capable of binding to the RECK protein may bind the CK1 motif, CK2 motif, CK3 motif, CK4 motif, CK5 motif, CRD, and / or one or more of the Kazal motifs of the RECK protein, preferably said agonist binds to the CK4 and / or CK5 regions of the RECK protein.

[0223] Accordingly, in particular embodiments, the antibody or antibody fragment as disclosed herein is capable of binding to the CK4 and / or CK5 regions of RECK polypeptide. The CK4 motif spans from amino acid C216 to C263, and the CK5 motif spans from amino acid C292 to C338 of the amino acid sequence of the human RECK protein annotated under NCBI Genbank accession number NP 066934.1 as disclosed elsewhere herein. Accordingly, the CK4 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC (SEQ ID N°: 49) and the CK5 motif of human RECK comprises, consists essentially of or consists of the amino acid sequence CCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVSMLTC (SEQ ID N°: 50). In particular embodiments, the antibody or antibody fragment as disclosed herein is capable of binding to the amino acid sequence of SEQ ID N°: 49 and / or the amino acid sequence of SEQ ID N°: 50.

[0224] In another or further particular embodiment, the one or more Reck binding domains bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the Reck protein as depicted in SEQ ID N° : 6. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In another further particular embodiment, said fragment of the Reck protein is the amino acid sequence between position 23 and 789 or between position 37 and 338 or between position 627 and 673 or between position 698 and 752 or between position 753 and 789 of SEQ ID N°: 6.

[0225] In an embodiment, said RECK-binding domain comprises a RECK specific antibody or comprises the RECK-binding region, preferably the variable region sequence or CDRs, of one or more RECK specific antibody or antibodies. Preferably, the RECK specific antibody is directed to the extracellular portion of the RECK polypeptide, such as directed to the CK4 and / or CK5 region(s) of the RECK polypeptide. Nonlimiting examples of RECK antibodies include antibodies which are known in the art and commercially available, such as from Abeam (e.g. ab88249 and ab899l5 specific for human full-length RECK), Cell Signaling Technology (e.g. #3433 specific forhuman, mouse, rat, monkey RECK (D8C7)), Santa Cruz (e.g. sc-373929 specific for C-terminus of human RECK).

[0226] In another or further embodiment, the antibody or antibody fragment described herein comprises a full-length multimeric protein that includes two heavy chains and two light chains. Each heavy chain includes one variable region (e.g. VH) and at least three constant regions (e.g. CHI, CH2 and CH3), and each light chain includes one variable region (VL) and one constant region (CL). As described above in the definitions section, the variable regions determine the specificity of the antibody and comprise three hypervariable regions also known as complementarity-determining regions (CDRs) that contribute to the antibody binding specificity.

[0227] In particular embodiments, the RECK-binding domain comprises at least one, at least two, at least three, at least four, at least five or at least six CDR(s), each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of a RECK specific antibody.

[0228] In particular embodiments, the RECK-binding domain is or comprises a RECK specific IgG or scFv, such as an IgG or scFv comprising one or more (preferably all 6) CDR(s) of a RECK specific antibody.

[0229] In particular embodiments, the RECK-binding domain is or comprises a RECK specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a RECK specific heavy chain antibody.

[0230] In particular embodiments, the RECK-binding domain of the antibody or antibody fragment as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different RECK specific antibodies.

[0231] In another or further embodiment, the antibody or antibody fragment described herein comprises one or more (preferably all 6) CDR(s) of a RECK specific antibody. Said CDRs may be CDRs as described in the table below, and although the CDRs shown below are linked to specific VH and VL sequences, they may be combined as desired.In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRH1 according to SEQ ID N°: 118, 119, 120, or 121, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 118, 119, 120, or 121.

[0232] In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRH2 according to SEQ ID N°: 122, 123, 124, or 125, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 122, 123, 124, or 125.

[0233] In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRH3 according to SEQ ID N°: 126, 127, 128, or 129, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 126, 127, 128, or 129.

[0234] In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRL1 according to SEQ ID N°: 146, 147, 148, or 149, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 146, 147, 148, or 149.

[0235] In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRL2 according to SEQ ID N°: 150, 151, 152, or 153, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 150, 151, 152, or 153.

[0236] In an embodiment, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRL3 according to SEQ ID N°: 154, 155, 156, or 157, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 154, 155, 156, or 157.

[0237] In preferred embodiments, the antibody or antibody fragment comprises one or more Reck binding domains comprising a CDRH1, CDRH2, CDRH3; and / or a CDRL1, CDRL2, CDRL3 as combined in the table below.

[0238] In an embodiment, the antibody or antibody fragment described herein comprises an anti-Reck IgG or scFv comprising:

[0239] a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and

[0240] a VL sequence according to SEQ ID N° 70, 72, 74, or 76,- the VH and VL optionally separated by a linker sequence.

[0241] In embodiments, the VH and / or VL comprises framework regions (FRs; FRH1, FRH2, FRH3, FRH4 for VH (SEQ ID N° : 130 to 145) and FRL1, FRL2, FRL3, FRL4 for VL (SEQ ID N°: 158 to 171), respectively) as described in the table below, or having maximally 1, 2, 3, 4, or 5 amino acid difference to any one of said sequences, and although said FRs shown below are linked to specific VH and VL sequences, they may be combined as desired.

[0242] Preferably, the agonist described herein comprises an anti-Reck IgG or scFv comprising:

[0243] - a VH sequence according to SEQ ID N° 69 and a VL sequence according to SEQ ID N° 70,

[0244] - a VH sequence according to SEQ ID N° 71 and a VL sequence according to SEQ ID 72,

[0245] - a VH sequence according to SEQ ID N° 73 and a VL sequence according to SEQ ID N° 74, or

[0246] - a VH sequence according to SEQ ID N° 75 and a VL sequence according to SEQ ID N° 76,

[0247] optionally having 1, 2, 3, 4, or 5 amino acid difference to any one of the FRs in said VH and / or VL sequences.

[0248] Table: Anti-Reck IgG or scFv sequences

[0249] CD CD CD FR FR FR FR CD CD CD FRL FRL FRL FRL ID VH RH RH RH VL

[0250] Hl H2 H3 H4 RL1 RL2 RL3 1 2 3 4 1 2 3

[0251] 01A08 RLT GV QL MS PS QL VD RFS QLQLQESG QE TSK DIVMTQS GS PGRVKPSE SG NQ PSSLSAS GS TLSLTCKVS PG FSL VGDRVTI DIV GT GFSISRGYY RV KM TCRASQS MT DFT WAWVRQA KPS WV RS ISRYLNW QS WY LTI PGKGLEWI NIH AFY ETL RQ VTA YQQKPG PSS QQ SSL GNIHHSGS HS GS SLT AP AD WG KAPKLLM RA LSA KP QP TYYSPSLKS GS GN CK GK TAV QG YAASSLQ SQ QQ SV GK ED FG RLTMSVDT RG TYY YYV VS GL YF TTV SGVPSRF SIS AA SYS GD AP FAT GG SKNQFSLK YY SPS TAL GF EWI CA IVS SGSGSGT RYL SSL TPL RVT KLL YY TKV MRSVTAAD WA LKS DV SIS G R S DFTLTISS N QS T ITC MY C EIK TAVYFCARA (SE (SE (SE (SE (SE (SE (SE LQPEDFA (SE (SE (SE (SE (SE (SE (SE FYGSGNYY Q Q Q Q Q Q Q TYYCQQS Q Q Q Q Q Q Q VTALDVWG ID ID ID ID ID ID ID YSTPLTF ID ID ID ID ID ID ID QGTTVIVSS N°: N°: N°: N°: N°: N°: N°: GGGTKVE N°: N°: N°: N°: N°: N°: N°: (SEQ ID N0: 118 122 126 130 134 138 142 IK (SEQ 146 150 154 158 162 165 168

[0252]

[0253] 69) ) ) ) ) ) ) ) ID N°: 70) ) ) ) ) ) ) )01F11 RFT GV EV ISR PS QL DN RFS EVQLVESG VES SN DIQLTQS GS GALVQPGG GG NR PSSLSAS GS SLRLSCAAS ALV LYL VGDRVTI DIQ GT GFTVSSNY AEL QP QM TCRASQ LTQ DFT MSWVRQA VIY LPF GG WV NS GISSYLA SPS WY LTI PGKGLEWV SG RV SLR RQ LRA WYQQKP SLS QQ SG SVIYSGGST GS GV LS AP EDT WG GKAPKLLI RA AS KP LQ YYAGSVKG TYY VIS CA GK GV QG YAASTLQ SQ QQ VG GK AE FG RFTISRDNS SN AG KYY AS GL YY TTV SGVPSRF GIS AA SYS DR AP DF QG NNRLYLQM YM SVK GL GFT EW CA TVS SGSGSGT SYL STL SPI VTI KLL ATY TRL NSLRAEDT S G DV VS VS R S DFTLTISG A QS T TC IY YC DIK GVYYCARA (SE (SE (SE (SE (SE (SE (SE LQAEDFA (SE (SE (SE (SE (SE (SE (SE ELLPFRVGV Q Q Q Q Q Q Q TYYCQQS Q Q Q Q Q Q Q VISKYYGLD ID ID ID ID ID ID ID YSSPITFG ID ID ID ID ID ID ID VWGQGTTV N°: N°: N°: N°: N°: N°: N°: QGTRLDI N°: N°: N°: N°: N°: N°: N°: TVSS (SEQ 119 123 127 131 135 139 143 K (SEQ ID 147 151 155 159 163 166 169 ID N°: 71) ) ) ) ) ) ) ) N°: 72) ) ) ) ) ) ) ) O3AO4 RFT GV EV ISR PS EVQLVESG QL DN RFS GGLVQPG VES SN DIQMTQS GS GSLRLACTT GG DTL PSSLSAS GS SGFTFSNY NS GL YL AGDRVTI DIQ GT AMSWVRQ GR VQ QM TCRASQS MT DFT TPGGRLEW SIIA TIF PG WV HS INTYLNW QS WY LTI VSSIIASDV SD GV GS RQ LR YQQKPG PSS QQ SSL STYYADSVR VST VMI LRL TP DE WG KAPKLLIY RA LSA KP QP GRFTISRDN YYA GE AC GG DT QG AASNLQS SQ AA SA GK ED FG SNDTLYLQ NY DS YG TTS RLE AVY TLV GVPSRFS SIN SN HQ GD AP FAT QG MHSLRDED AM VR FD GFT WV FC TVS GSGSGT TYL LQ TYT RVT KLL YY TKL TAVYFCAK S G S FS S AK S DFTLTISS N S THT ITC IY C EIK NSGRTIFGV (SE (SE (SE (SE (SE (SE (SE LQPEDFA (SE (SE (SE (SE (SE (SE (SE VMIGEYGF Q Q Q Q Q Q Q TYYCHQT Q Q Q Q Q Q Q DSWGQGT ID ID ID ID ID ID ID YTTHTFG ID ID ID ID ID ID ID LVTVSS N°: N°: N°: N°: N°: N°: N°: QGTKLEI N°: N°: N°: N°: N°: N°: N°: (SEQ ID N0: 120 124 128 132 136 140 144 K (SEQ ID 148 152 156 160 163 165 170 73) ) ) ) ) ) ) ) N°: 74) ) ) ) ) ) ) ) 07B05 RVT GIP QV ISV QSVLTQP DR QVQLVESG QL DM PSVSAAP FTG PGLVKPSQ VES SK GQHVTIS QS SKS TLSLTCTVS GP NQ CSGSSS VLT GT GGSISSGD GL FSL NVGKNF QP SAT YYWSWIRQ YIY VKP Wl KLS VSWYQQ PSV WY LGI HPGKGLE YS SQ RQ SVT LPGTVPK SG SA QQ TGL WIGYIYYSG GS ERL TLS HP AA WG LLIYDNN SSS GT AP LP QT STYHNPSL SG TYH KG LTC GK DT QG RRPSGIP NV DN WD GQ GT GD FG KSRVTISVD DY NP LD TVS GL AVY TM DRFTGSK GK NR TSL HV VPK EA GG MSKNQFSL YW SLK AF GG EWI YC VTV SGTSATL NF RP SA TIS LLI DY TRL KLSSVTAAD S S DI SIS G AR SS GITGLQT VS S W C Y YC TVL TAVYYCARE (SE (SE (SE (SE (SE (SE (SE GDEADYY (SE (SE (SE (SE (SE (SE (SE RLKGLDAF Q Q Q Q Q Q Q CGTWDT Q Q Q Q Q Q Q DIWGQGT ID ID ID ID ID ID ID SLSAWF ID ID ID ID ID ID ID MVTVSS N°: N°: N°: N°: N°: N°: N°: GGGTRLT N°: N°: N°: N°: N°: N°: N°: (SEQ ID N0: 121 125 129 133 137 141 145 VL (SEQ 149 153 157 161 164 167 171

[0254]

[0255] 75) ) ) ) ) ) ) ) ID N°: 76) ) ) ) ) ) ) )In another or further particular embodiment, the one or more Gprl24 binding domains bind an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a fragment of the Gprl24 protein as depicted in SEQ ID N°: 7. In a further particular embodiment, said amino acid sequence has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or 30 amino acids. In another further particular embodiment, said fragment of the Gprl24 protein is the amino acid sequence between position 34 and 344 or between position 82 and 106 or between position 107 and 130 or between position 131 and 154 or between position 156 and 178 or between position 190 and 240 or between position 247 and 344 of SEQ ID N°: 7.

[0256] In particular embodiments, the Gprl24-binding domain is or comprises a Gprl24 specific IgG or scFv, such as an IgG or scFv comprising one or more (preferably all 6) CDR(s) of a Gprl24 specific antibody.

[0257] In particular embodiments, the Gprl24-binding domain is or comprises a Gprl24 specific VHH, such as VHH comprising one or more (preferably all 3) CDR(s) of a Gprl24 specific heavy chain antibody.

[0258] In particular embodiments, the Gprl24-binding domain of the antibody or antibody fragment as disclosed herein may comprise two or more CDR(s) each independently having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, preferably 100%, sequence identity with the respective CDR(s) of two or more different Gprl24 specific antibodies.

[0259] In another or further embodiment, the antibody or antibody fragment described herein comprises one or more (preferably all 6) CDR(s) of a Gprl24 specific antibody. Said CDRs may be CDRs as described in the table below, and although the CDRs shown below are linked to specific VH and VL sequences, they may be combined as desired.

[0260] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRH1 according to SEQ ID N°: 89 or 90, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 89 or 90.

[0261] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRH2 according to SEQ ID N°: 91 or 92, orhaving maximally 1 amino acid difference to a sequence according to SEQ ID N°: 91 or 92.

[0262] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRH3 according to SEQ ID N°: 93 or 94, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 93 or 94.

[0263] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRL1 according to SEQ ID N°: 103 or 104, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 103 or 104.

[0264] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRL2 according to SEQ ID N°: 105 or 106, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 105 or 106.

[0265] In an embodiment, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRL3 according to SEQ ID N°: 107, 108, or 109, or having maximally 1 amino acid difference to a sequence according to SEQ ID N°: 107, 108, or 109.

[0266] In preferred embodiments, the antibody or antibody fragment comprises one or more Gprl24 binding domains comprising a CDRH1, CDRH2, CDRH3; and / or a CDRL1, CDRL2, CDRL3 as combined in the table below.

[0267] In another or further embodiment, the antibody or antibody fragment described herein comprises an anti-Gprl24 IgG or scFv comprising:

[0268] - a VH sequence according to SEQ ID N° 63, 65, or 67, and

[0269] - a VL sequence according to SEQ ID N° 64, 66, or 68,

[0270] - the VH and VL optionally separated by a linker sequence.

[0271] In embodiments, the VH and / or VL comprises framework regions (FRs; FRH1, FRH2, FRH3, FRH4 for VH (SEQ ID N° : 95 to 102) and FRL1, FRL2, FRL3, FRL4 for VL (SEQ ID N°: 110 to 117), respectively) as described in the table below, or having maximally 1, 2, 3, 4, or 5 amino acid difference to any one of said sequences, andalthough said FRs shown below are linked to specific VH and VL sequences, they may be combined as desired.

[0272] Preferably, the antibody or antibody fragment described herein comprises an anti-Gprl24 IgG or scFv comprising:

[0273] - a VH sequence according to SEQ ID N° 63 and a VL sequence according to SEQ ID N° 64,

[0274] - a VH sequence according to SEQ ID N°65 and a VL sequence according to SEQ ID N° 66, or

[0275] - a VH sequence according to SEQ ID N° 67 and a VL sequence according to SEQ ID N° 68,

[0276] optionally having 1, 2, 3, 4, or 5 amino acid difference to any one of the FRs in said VH and / or VL sequences.

[0277] Table: Anti-Gprl24 IgG or scFv sequences

[0278] CD CD CD FR FR FR FR CD CD CD FRL FRL FRL FRL ID VH RH RH RH VL

[0279] Hl H2 H3 H4 RL1 RL2 RL3 1 2 3 4 1 2 3

[0280] 05A10 GV PD RLT QAVLTQP RFS QV ISV PSASGTP GS QVQLQESG QL DTS GQRVTIS KS PGLVKPSET QE RS CSGSSS QA GT LSLTCTVSG SG QF NIGSNYV VLT SAS GSISGSSYY PG SLK YWYQQL QP WY LAI WSWIRQPP NIY LVK Wl LSS PGTAPKL AA PSA QQ SG GRGLEWIG YS PSE RQ VTA WG LIYRNNQ SG WD SG LP LRS NIYYSGSTY GS LLV TLS PP AD QG RPSGVPD SSS RN DS TP GT ED FG YNPSLKSRL TYY QG LTC GR TAV TM RFSGSKS NIG NQ LS GQ AP EA GG TISVDTSRS GS NP VD TVS GL YY VTV GTSASLAI SN RP GV RVT KLL DY TKL QFSLKLSSV SYY SLK AF GG EWI CA SS SGLRSED YVY S V ISC IY YC TVL TAADTAVYY WS S DI SIS G R (SE EADYYCA (SE (SE (SE (SE (SE (SE (SE CARLLVQG (SE (SE (SE (SE (SE (SE Q AWDDSL Q Q Q Q Q Q Q VDAFDIWG Q Q Q Q Q Q ID SGWFG ID ID ID ID ID ID ID QGTMVTVS ID ID ID ID ID ID N°: GGTKLTV N°: N°: N°: N°: N°: N°: N°: S (SEQ ID N°: N°: N°: N°: N°: N°: 102 L (SEQ ID 103 105 107 110 113 115 117 N°: 63) 89) 91) 93) 95) 97) 99) ) N°: 64) ) ) ) ) ) ) ) 06C03 QVQLQESG QV RLT SYVLTQP SYV WY GV PGLVKPSET NIY QL Wl ISV PSASGTP AA LTQ QQ PD LSLTCTVSG YS QE RQ DT WG GQRVTIS SG WD PPS LP RFS GSISGSSYY GS LLV SG PP SR QG CSGSSS SSS RN DS AS GT GS FG WSWIRQPP TYY QG PG GK NQ TM NIGSNYV NIG NQ LN GT AP KS GG GKGLEWIG GS NP VD LVK GL FSL VTV YWYQQL SN RP GV PG KLL GS TKL NIYYSGSTY SYY SLK AF PSE EWI KLS SS PGTAPKL YVY S V QR IY SAS TVL YNPSLKSRL WS S DI TLS G SVT (SE LIYRNNQ (SE (SE (SE VTI (SE LAI (SE TISVDTSRN (SE (SE (SE LTC (SE AA Q RPSGVPD Q Q Q SC Q SG Q QFSLKLSSV Q Q Q TVS Q DT ID RFSGSKS ID ID ID (SE ID LQ ID TAADTAVYY ID ID ID GG ID AVY N°: GSSASLA N°: N°: N°: Q N°: SE N°: CARLLVQG N°: N°: N°: SIS N°: YC 102 ISGLQSE 103 105 108 ID 113 DE 117

[0281]

[0282] VDAFDIWG 89) 91) 93) (SE 98) AR ) DEADYYC ) ) ) N°: ) AD )QGTMVTVS Q (SE AAWDDS 111 YY S (SEQ ID ID Q LNGWFG ) C N°: 65) N°: ID GGTKLTV (SE 95) N°: L (SEQ ID Q 100 N°: 66) ID ) N°:

[0283] 116 ) 06A05 GV RVT PD QV MS SYELTQP RFS QL VD PSASGTP GS QVQLQESG QE TSK GQRVTIS KS PGLVKPSET SG NQ CSGSSS SYE GT LSLSCTVSG PG FSL NIGTNYV LTQ SAS DSISSRSHY LVK NL YWYQQL PPS WY LAI WGWIRQP TIY PSE Wl SSV PGMAPKL AT AS QQ SG PGKGLEWI YS TLS RQ TAA WG LIYSDDQ SG WD GT LP LRS GTIYYSGNT GN LR LS pp DT QG RPSGVPD SSS SD DS PG GM ED FG YYNPSLKSR SR TYY GS CT GK AVY TM RFSGSKS NIG DQ LS QR AP EA GG VTMSVDTS SH NP VD VS GL FC VTV GTSASLAI TNY RP GV VTI KLL DY TKL KNQFSLNL YW SLK AF GD EWI Al SS SGLRSED VY S V SC IY YC TVL SSVTAADTA G S DI SIS G (SE (SE EADYYCA (SE (SE (SE (SE (SE (SE (SE VYFCAILRG (SE (SE (SE (SE (SE Q Q TWDDSL Q Q Q Q Q Q Q SVDAFDIW Q Q Q Q Q ID ID SGWFG ID ID ID ID ID ID ID GQGTMVTV ID ID ID ID ID N°: N°: GGTKLTV N°: N°: N°: N°: N°: N°: N°: SS (SEQ ID N°: N°: N°: N°: N°: 101 102 L (SEQ ID 104 106 109 112 114 115 117

[0284]

[0285] N°: 67) 90) 92) 94) 96) 98) ) ) N°: 68) ) ) ) ) ) ) )

[0286] In particular embodiments, at least one of the binding domains of the antibody or antibody fragment described herein is or is derived of a single-domain antibody, such as a VHH. The VHH may be derived from, for example, an organism that produces VHH antibodies such as a camelid, a shark, or the VHH may be a designed VHH. VHHs are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy-chain antibodies (see definition section above). In some embodiments, the single domain antibody as described herein is an immunoglobulin single variable domain or ISVD. In most particular embodiments, at least one of the binding domains of the antibody or antibody fragment is or is derived of a VHH. In further most particular embodiments, said VHH comprises a single amino acid chain having four "framework regions" and three "complementary determining regions" or CDRs. As used herein, "framework region" refers to a region in the variable domain which is located between the CDRs. As used herein, "complementary determining region" or "CDR" refers to variable regions in VHHs that contains the amino acid sequences capable of specifically binding to antigenic targets. In various embodiments, the antibody or antibody fragment described herein comprises a VHH or ISVD targeting LRP5 or LRP6 having a variable domain comprising at least one CDR1, CDR2, and / or CDR3 sequence. The CDR3 sequence represents an essential feature of a family of ISVDs, moreparticularly VHHs. An ISVD family is defined herein as a group of ISVD amino acid sequences with high similarity, or even identical, in the CDR.3 sequence. By default, ISVDs belong to the same family when binding to the same target epitope. Variations in an ISVD family may be interesting if expression / stability / affinity / crystallization of a representative of that family is poor, as small deviations like single amino acid mutations occurring within one family may improve these properties.

[0287] In a specific embodiment, the antibody or antibody fragment comprises an ISVD or VHH targeting LR.P5 comprises a CDR.3 region selected from the list consisting of SEQ ID N°: 38-41, SEQ ID N°: 85-88, or having an amino acid sequence with maximally two amino acids different to SEQ ID N°: 38-41 or SEQ ID N°: 85-88, or with maximally one amino acid different to SEQ ID N°: 38-41 or SEQ ID N°: 85-88.

[0288] In a specific embodiment, said ISVD or VHH comprises a CDR2 region as depicted in SEQ ID N°: 42, 81, 82, 83, or 84, and / or a CDR1 region as depicted in SEQ ID N°: 43, 77, 78, 79, or 80, or with maximally 1 or 2 amino acid different to said SEQ ID N°: 42, 81, 82, 83, or 84, or SEQ ID N°: 43, 77, 78, 79, or 80, respectively.

[0289] In a most specific embodiment, said anti-LRP5 ISVD or VHH comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to SEQ ID N°: 34, 35, 36, 37, 53, 59, 60, 61, or 62. In particular embodiments, said differences in amino acid sequence between said homologues and SEQ ID N°: 34, 35, 36, 37, 53, 59, 60, 61, or 62 are found in the framework regions, i.e. regions between said CDRs in said VHH sequence.

[0290] In another specific embodiment, the antibody or antibody fragment comprises an ISVD or VHH targeting LRP6 and comprises a CDR3 as depicted in SEQ ID N°: 15, 24, 25, or 26, or SEQ ID N°: 85-88, or having an amino acid sequence with maximally two amino acids different to SEQ ID N°: 15, 24, 25, or 26, or SEQ ID N°: 85-88, or with maximally one amino acid different to SEQ ID N°: 15, 24, 25, or 26, or SEQ ID N°: 85-88,.

[0291] In a further specific embodiment, said ISVD or VHH comprises a CDR2 region as depicted in SEQ ID N°: 16, 27, 28, 29, 81, 82, 83, or 84, and / or a CDR1 region as depicted in SEQ ID N°: 17, 30, 31, 32, 33, 77, 78, 79, or 80; or with maximally 1 or 2 amino acid different to said SEQ ID N°: 16, 27, 28, 29, 81, 82, 83, or 84, or SEQ ID N°: 17, 30, 31, 32, 33, 77, 78, 79, or 80, respectively.In a most specific embodiment, said anti-LRP6 ISVD or VHH comprises an amino acid sequence of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology to any of SEQ ID N°: 14, 18-23, 59, 60, 61, or 62.

[0292] In particular embodiments, said differences in amino acid sequence between said homologues and any of SEQ ID N°: 14, 18-23, 59, 60, 61, or 62 are found in the framework regions , i.e. regions between said CDRs in said VHH sequence.

[0293] Preferably, the antibody or antibody fragment described herein comprises an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62.

[0294] Anti-LRP (Anti-LRP5 or Anti-LRP6) VHH sequences

[0295] ID VHH CDRH1 CDRH2 CDRH3

[0296] 01B01 QVQLQESGGGLVQPGGSLRLSCAASGF DYDMS TISWNGAGTYYAES YAGNTIATSKSTYDY TFDDYDMSWVRQAPGKGLEWLSTISWN (SEQ ID N°: VKG (SEQ ID N°: (SEQ ID N°: 85) GAGTYYAESVKGRFTISRDNTKNTLYLQM 77) 81)

[0297] NSLKPDDTAVYYCAAYAGNTIATSKSTYD YWGQGTQVTVSS (SEQ ID N°: 59)

[0298] 01E12 QVQ LVESGGG LVQAGGSLSLSCVASG R DFGMA SINWSGSSTYYED RHSNRVKEYDD (SEQ NFDDFGMAWFRQAPGKDREFVASINW (SEQ ID N°: SVKG (SEQ ID N°: ID N°: 86) SGSSTYYEDSVKGRFTISRDNAKNTVYLQ 78) 82)

[0299] MNSLKSEDTAVYYCVRRHSNRVKEYDD WGQGTQVTVSS (SEQ ID N°: 60)

[0300] 01F11 QVQLVESGGGLVQPGGSLRLSCTASGF NHAIG (SEQ SIGNSDGSTYYADS QSMMINRNY (SEQ ID SLDNHAIGWFRQAPGKEREWVSSIGNS ID N°: 79) VKG (SEQ ID N°: N°: 87) DGSTYYADSVKGRFTISRDNAKNALYLQ 83)

[0301] MNSLKPDDTAVYYCNAQSMMINRNYW GQGTQVTVSS (SEQ ID N°: 61)

[0302] 02G02 QLQLVESGGGLVQPGGSLRVSCVASGR GNNMG EITYRGNTNYADSV RSRNLIRNY (SEQ ID PFSGNNMGWYRQAPGKQREWIAEITYR (SEQ ID N°: KG (SEQ ID N°: 84) N°: 88) GNTNYADSVKGRFIISRDNAKNTVYLQM 80)

[0303] NSLKPEDTAVYYCNARSRNLIRNYWGQ

[0304]

[0305] GTQVTISS (SEQ ID N°: 62)

[0306] In a preferred embodiment, the antibody or antibody fragment described herein comprises:

[0307] an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62, or comprising CDRs as described in the table above in any combination,

[0308] and

[0309] either: an anti-Gprl24 IgG or scFv comprising:

[0310] - a VH sequence according to SEQ ID N° 63, 65, or 67, and

[0311] - a VL sequence according to SEQ ID N° 64, 66, or 68,

[0312] - the VH and VL optionally separated by a linker sequence,optionally having 1, 2, 3, 4, or 5 amino acid difference to any one of the FRs in said VH and / or VL sequences.

[0313] Or: an anti-Reck IgG or scFv comprising:

[0314] a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and

[0315] a VL sequence according to SEQ ID N° 70, 72, 74, or 76,

[0316] - the VH and VL optionally separated by a linker sequence,

[0317] optionally having 1, 2, 3, 4, or 5 amino acid difference to any one of the FRs in said VH and / or VL sequences.

[0318] Based on the above, any combination of VHH and IgG or scFv listed above is deemed disclosed herein. Most preferred combinations are shown in the example section.

[0319] In a preferred embodiment, the VHH is fused to the N-terminus of the VL of the IgG, preferably using a linker such as a 5-mer linker, preferably having the sequence GSGGS (SEQ ID N°: 172). Preferably, there is no linker between the VL of the IgG and VH of the IgG.

[0320] As already described above, in an embodiment, antibody or antibody fragment disclosed herein does not comprise a Fzd binding domain. In another or further embodiment, said antibody or antibody fragment is not able to bind or not capable of binding to Fzd. This is particularly remarkable as it is not obvious based on what was previously known that such molecule, not capable of binding to Fzd and / or without Fzd binding domain and / or thus without crosslinking said molecule with Fzd, can show agonist activity, i.e. can activate the Wnt / beta-catenin signaling pathway and thus can act as an activator of said signaling pathway. The inventors, however, were able to show that the antibody or antibody fragment of the present invention, without Fzd binding domain and / or without the capability of binding Fzd, was able to activate said signaling pathway, and thereby treat and / or prevent epilepsy, or any subtype of epilepsy as described above.

[0321] In an embodiment, the antibody or antibody fragment does not comprise further binding domains over said one or more LRP binding domains and one or more Gprl24 or Reck binding domains.

[0322] In an embodiment, the binding domains of said antibody or antibody fragment are selected from one or more LRP binding domains and one or more Gprl24 or Reck binding domains.In a preferred embodiment, the binding domains of said antibody or antibody fragment are selected from one or more LRP binding domains and one or more Gprl24 binding domains. In another preferred embodiment, the binding domains of said antibody or antibody fragment are selected from one or more LRP binding domains and one or more Reck binding domains.

[0323] In an embodiment, said antibody or antibody fragment is a bispecific antibody with two binding domains. In a preferred embodiment, said antibody or antibody fragment is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Gprl24 binding domain. In another preferred embodiment, said antibody or antibody fragment is a bispecific antibody with two binding domains, said binding domains including one LRP binding domain and one Reck binding domain.

[0324] Similarly as described above in relation to said antibody or antibody fragment not having a Fzd binding domain and / or not being able of binding Fzd, it could not be expected based on what was previously known that such molecule, having as binding domains only one or more LRP binding domains and one or more Gprl24 or Reck binding domains, or having only two binding domains chosen from an LRP binding domain and either a Reck binding domain or a Gprl24 binding domain, can show agonist activity, i.e. can activate the Wnt / beta-catenin signaling pathway and thus can act as an activator of said signaling pathway, and thereby treat and / or prevent epilepsy, or any subtype of epilepsy as described above.

[0325] In another or further embodiment, the antibody or antibody fragment described herein is capable of activating the Wnt / beta-catenin signaling pathway and thus can act as an activator of said signaling pathway in the absence of Reck.

[0326] In certain embodiments, one or more and preferably all of Wnt7, GPR124, RECK, FZD and LRP as employed herein is or are of animal origin, preferably warm-blooded animal origin, more preferably vertebrate origin, yet more preferably mammalian origin, including human origin and non-human mammalian origin, still more preferably human origin.

[0327] A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be precursors of the respective peptides, polypeptides, proteins, or nucleic acids and may include parts that are processed away from mature molecules; or may represent sequences that are ofmature molecules while also the precursor sequences are considered part of the present concept.

[0328] The terms encompass the peptides, polypeptides, proteins, or nucleic acids when forming a part of a living organism, organ, tissue, or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass the peptides, polypeptides, proteins, or nucleic acids when produced by recombinant or synthetic means.

[0329] Unless otherwise apparent from the context, reference herein to any peptide, polypeptide, protein, or nucleic acid also encompasses modified forms of said peptide, polypeptide, protein, or nucleic acid, such as forms bearing post-expression modifications including, for example, phosphorylation, glycosylation, palmitoylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, ubiquitination, oxidation of methionine to methionine sulphoxide or methionine sulphone, signal peptide removal, N-terminal Met removal, conversion of proenzymes or pre-hormones into active forms, and the like. A broader definition is given above.

[0330] Humanization

[0331] In one embodiment, the binding agent of current application comprises one or more immunoglobulin single variable domains or VHHs and / or further binding domains, for example targeting LRP such as LR.P5 or LR.P6, that has been "humanized", i.e. one or more amino acid residues in the amino acid sequence of the VHH obtained by immunization is replaced by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being to increase the degree of sequence identity with the closest human germline sequence. Potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring VHH sequence with the corresponding framework sequence of one or more closely related human VH sequence(s), after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said VHH sequence (in any manner known per se, as further described herein) and the resulting humanized VHH sequences can be tested for affinity for the target, for stability, for ease and level of expression, and / or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person. Also, based on what is described before, (theframework regions of) an immunoglobulin single variable domain, such as a VHH domain may be partially humanized or fully humanized.

[0332] Therefore, in various embodiments, one or more binding domains of said antibody or antibody fragment comprise an amino acid sequence having one or more amino acid mutations with respect to SEQ ID N°: 12, 13, 14, 18, 19, 20, 21, 22, 23, 34, 35, 36, 53, 37, 59, 60, 61, and / or 62. In various embodiments, the binding domains of said antibody or antibody fragment comprise an amino acid sequence having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to SEQ ID N°: 12, 13, 14, 18, 19, 20, 21, 22, 23, 34, 35, 36, 53, 37, 59, 60, 61, and / or 62. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and / or non-conservative substitutions. In particular embodiments, the one or more amino acid mutations may be in the CDRs of the binding domains (e.g., the CDR1, CDR2 or CDR3 regions). In other particular embodiments, the one or more amino acid mutations may be in the framework regions of the binding domains (e.g., the FR1, FR2, FR3, or FR4 regions). In most particular embodiments, said one or more amino acid mutations are only present in the framework regions of said FRo binding agents. The same applies to humanization of the binding domains for Gprl24 and / or Reck, which may take place in either of the above-mentioned sequences, either in the CDRs, FRs, or a combination thereof, and either having one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty amino acid mutations with respect to the binding domain sequences as described above. "Conservative substitutions" may be made, for instance, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed within the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu retains one negative charge in the so modified polypeptide. In addition, glycine and proline may be substituted for one another based on their ability to disrupt o-helices.As used herein, "non-conservative substitutions" are defined as exchanges of an amino acid by another amino acid listed in a different group of the six standard amino acid groups (1) to (6) shown above.

[0333] In various embodiments, the substitutions may also include non-classical amino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionine 0-alanine, GABA and 6-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4-diaminobutyric acid, o-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, 0-alanine, fluoro-amino acids, designer amino acids such as [3 methyl amino acids, C o-methyl amino acids, N o-methyl amino acids, and amino acid analogs in general).

[0334] Humanization can be performed using humanization techniques known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions may be determined by methods known in the art, for example without the purpose of being limiting, by a comparison between the sequence of a VHH and / or further binding domains, and the sequence of a naturally occurring human VH domain. In some embodiments, the humanizing substitutions are chosen such that the resulting humanized VHHs, and / or further binding domains, still retain advantageous functional properties. Generally, as a result of humanization, the VHHs and / or further binding domains of the application may become more "human-like", while still retaining favourable properties such as a reduced immunogenicity, compared to the corresponding naturally occurring VHH domains. In various embodiments, the humanized VHHs and / or further binding domains of the application can be obtained in any suitable manner known in the art and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH and / or further binding domain as a starting material. Indeed, modification of the amino acid sequences may be achieved using any known technique in the art e.g., site-directed mutagenesis or PCR based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., 1989.

[0335] In various embodiments, the mutations that were introduced for example to humanize the binding agent do not substantially reduce the present antibody or antibody fragment's capability to specifically bind to the human LR.P5 or LR.P6 andGprl24 or Reck, to agonize the Gprl24 / Reck / Fzd / LRP5 or LRP6-mediated Wnt7 signaling, and to treat and / or prevent epilepsy or any subtype of epilepsy as described above.

[0336] Methods of production

[0337] The antibodies or antibody fragments of the application, more particularly comprising the IgGs, scFvs, ISVDs or VHHs described herein are not limited to a specific biological source or to a specific method of preparation. Methods for producing the antibodies or antibody fragments of the application are described herein. For example, DNA sequences encoding the antibodies or antibody fragments of the application can be easily prepared by the art-known techniques such as cloning, hybridization screening and Polymerase Chain Reaction (PCR). Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989), Maniatis et al. (1982), Wu (ed.) (1993) and Ausubel et al. (1992). Alternatively, DNA sequences encoding the antibodies or antibody fragments of the application can be chemically synthesized using methods known in the art. Synthetic DNA sequences can be ligated to other appropriate nucleotide sequences, including for example expression control sequences, to produce gene expression constructs encoding the desired antibodies or antibody fragments.

[0338] Accordingly, in various embodiments, the present application provides for a pharmaceutical composition for use as described before, comprising a nucleic acid comprising a nucleotide sequence encoding the antibodies or antibody fragments described in current application for example, encoding the proteins disclosed herein and formulated for vectorized gene therapy delivery for in vivo or ex vivo expression, and, where the coding sequence exceeds a single-vector packaging capacity (e.g., AAV), employing dual-vector strategies such as dual AAV delivery.

[0339] Dual AAV," as used herein, refers to a two-vector adeno-associated virus delivery strategy in which complementary portions of a coding sequence and / or expression cassette are packaged separately into two AAV genomes and co-administered such that, following infection of the same target cell, the full-length expression unit is reconstituted (e.g., by homologous overlap / concatemerization, 5' / 3' trans-splicing, and / or split-intein-mediated protein reconstitution), thereby enabling expression of transgenes exceeding the packaging capacity of a single AAV vector. One embodiment further discloses an expression cassette comprising said nucleic acidmolecule. More specific embodiments disclose the expression cassette wherein elements for cell- or tissue-specific expression are present e.g., promoters and / or enhancers conferring brain or blood-brain barrier endothelial cell specificity. Further embodiments relate to a vector comprising said expression cassette or said nucleic acid molecule. More particular, said vector may be a viral vector or lentiviral vector, preferably a viral vector specifically directed towards the central and / or peripheral nervous system (e.g., a brain-specific viral vector). In further particular embodiments, the viral vector is a central nervous system (CNS) neuron-specific adeno-associated virus serotype 9 (AAV9) mutant. In preferred embodiments, the viral vector is a blood brain barrier endothelial cell-specific viral vector. In further preferred embodiments, the viral vector is a blood brain barrier endothelial cellspecific capsid adeno-associated virus serotype 2 (AAV2) mutant. This may be configured as a single- or dual-AAV system for delivery of larger transgenes; in certain embodiments, reconstitution of the full-length coding sequence in target cells is achieved using overlapping genomes, 5' / 3' trans-splicing, and / or split-intein— mediated protein reconstitution.

[0340] Factors of importance in selecting a particular vector include inter alia: choice of recipient cell, ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in particular recipient cells; whether it is desired for the vector to integrate into the chromosome or to remain extra-chromosomal in the recipient cells; and whether it is desirable to be able to "shuttle" the vector between recipient cells of different species, as well as the packaging capacity of the vector and whether single- or multi-vector (e.g., dual AAV) delivery is indicated by transgene size.

[0341] Expression vectors can be autonomous or integrative. A nucleic acid can be introduced into a cell in the form of an expression vector such as a plasmid, phage, transposon, cosmid or virus particle. The recombinant nucleic acid can be maintained extra-chromosomally or it can be integrated into the cell chromosomal DNA. Expression vectors can contain selection marker genes encoding proteins required for cell viability under selected conditions (e.g., URA3, which encodes an enzyme necessary for uracil biosynthesis, or LEU2, which encodes an enzyme required for leucine biosynthesis, or TRP1, which encodes an enzyme required for tryptophan biosynthesis) to permit detection and / or selection of those cells transformed with the desired nucleic acids. Expression vectors can also include an autonomous replication sequence (ARS). The ARS may comprise a centromere (CEN) and an origin of replication (ORI). For example, the ARS may be ARS18 or ARS68.Integrative vectors generally include a serially arranged sequence of at least a first insertable DNA fragment, a selectable marker gene, and a second insertable DNA fragment. The first and second insertable DNA fragments are each about 200 (e.g., about 250, about 300, about 350, about 400, about 450, about 500, or about 1000 or more) nucleotides in length and have nucleotide sequences which are homologous to portions of the genomic DNA of the cell species to be transformed. A nucleotide sequence containing a nucleic acid of interest for expression is inserted in this vector between the first and second insertable DNA fragments, whether before or after the marker gene. Integrative vectors can be linearized prior to transformation to facilitate the integration of the nucleotide sequence of interest into the cell genome. Prior to introducing the vectors into a cell of interest, the vectors can be grown (e.g., amplified) in bacterial cells such as Escherichia coli (E. coli). The vector DNA can be isolated from bacterial cells by any of the methods known in the art, which result in the purification of vector DNA from the bacterial milieu. The purified vector DNA can be extracted extensively with phenol, chloroform, and ether, to ensure that no E. coli proteins are present in the plasmid DNA preparation, since these proteins can be toxic to mammalian cells, and, when used for vectorized gene therapy delivery, the resulting vector preparations can be formulated for single- or dual-AAV administration as described herein.

[0342] In order to produce the antibodies or antibody fragments of current application, expression vectors comprising a nucleic acid sequence encoding said antibodies or antibody fragments can then be introduced into host cells through transfection, transformation, or transduction techniques. Hence, in various embodiments, the present application provides for a host cell comprising a nucleic acid encoding one of the antibodies or antibody fragments of the present application. For example, nucleic acids encoding the antibody or antibody fragment of the application can be introduced into host cells by retroviral transduction. Illustrative host cells are E.coli cells, Chinese hamster ovary (CHO) cells, yeast cells such as Pichia spp., human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the antibody or antibody fragment of the application.

[0343] Following expression, the antibodies or antibody fragments can be harvested and purified using techniques well known in the art, e.g. affinity tags such as glutathione-S-transferase (GST) and histidine (His) tags or by chromatography. Specific expression and purification conditions will vary depending upon the expressionsystem employed. For example, if a gene is to be expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g. Trp or Tac, and a prokaryotic signal sequence. In another example, if the engineered gene is to be expressed in eukaryotic host cells, e.g. CHO cells, it is first inserted into an expression vector containing for example, a suitable eukaryotic promoter, a secretion signal, enhancers, and various introns. In an embodiment, the antibody or antibody fragment of the application comprises a His tag, a FLAG-tag and / or a Myc tag. In an embodiment, the antibody or antibody fragment of the application comprises a His tag and a proteolytic site to allow cleavage of the His tag.

[0344] Current application thus also provides a host cell comprising one of the antibodies or antibody fragments described herein. Host cells comprising one of the nucleic acid molecules or the expression cassettes or the vectors of the application are provided herein as well. Host cells can be either prokaryotic or eukaryotic. Representative host cells that may be used with the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells. Bacterial host cells suitable for use with the invention include Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Yeast host cells suitable for use with the invention include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are the most commonly used yeast hosts and are convenient fungal hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (most particularly derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems (e.g. review Jarvis 2003 Virology 310:1-7). Nonlimiting examples of plant cells include tobacco cells, Arabidopsis cells, tomato cells, maize cells, algae cells, among others. The host cells may be provided in suspension or flask cultures, tissue cultures, organ cultures and the like. Alternatively, the host cells may also be transgenic animals.

[0345] Animal or mammalian host cells suitable for harboring, expressing, and producing one of the FRo binding agents of the application include Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al 1986 Som Cell Mol Genet 12:555-556; Kolkekar et al 1997 Biochemistry 36:10901-10909), CHO-K1 Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire,UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-Kl / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), dihydrofolate reductase negative CHO cells (CHO / -DHFR, Urlaub & Chasin 1980 PNAS 77:4216), and dpl2.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293T cells, or 293 cells subcloned for growth in suspension culture, Graham et al 1977 J Gen Virol 36:59, or GnTI KO HEK293S cells, Reeves et al 2002 PNAS 99:13419); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1, ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4, Mather 1980 Biol Reprod 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad. Sci., 383:44-68); MCR 5 cells; FS4 cells. According to a particular embodiment, the cells are mammalian cells selected from Hek293 cells or COS cells.

[0346] The host cells described above can be transiently or stably transfected. Such transfection of DNA, such as nucleic acid molecules, expression cassettes or expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. For all standard techniques see, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2* Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.). The host cell may also be a recombinant host cell, which involves a cell which has been genetically modified to contain an isolated DNA molecule, nucleic acid molecule or expression construct or vector of the invention. The DNA can be introduced by any means known to the art which are appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or viral mediated transduction.

[0347] Furthermore, in alternative embodiments, the use of the herein described nucleic acid molecules, expression cassettes, or vectors encoding the antibodies or antibody fragments of the application, particularly comprising IgGs, scFvs, or VHHs areprovided for the production of said antibodies or antibody fragments. In a particular embodiment, said use is provided for production of an intrabody. An intracellular antibody or "intrabody" is an antibody or a fragment of an antibody that is heterologously expressed within a designated intracellular compartment, a process which is made possible through the in-frame incorporation of intracellular trafficking signals. An intrabody can be expressed in any shape or form such as an intact IgG molecule or a Fab fragment, more particularly as genetically engineered antibody fragment for example as single domain intrabodies or VHHs. For a review see Zhu, and Marasco, 2008 (Therapeutic Antibodies. Handbook of Experimental Pharmacology 181. Ed. Springer-Verlag Berlin Heidelberg).

[0348] Medical formulations and applications

[0349] As described above, the invention provides a pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy in or seizure a patient; a method for treating and / or preventing epilepsy or seizure in a patient comprising administering the pharmaceutical composition to said patient or to a patient in need thereof; as well as a use in the preparation of a medicament for the treatment and / or prevention of epilepsy or seizure in a patient.

[0350] Said antibody or antibody fragment may be as described above in any one of the embodiments.

[0351] As described above, said epilepsy may be generalized epilepsy or focal epilepsy. Said focal epilepsy may be temporal lobe epilepsy (TLE) or non-TLE focal epilepsy. In addition, said patient may exhibit one or more symptoms of epilepsy, including epileptic seizure, such as generalized epileptic seizure or focal epileptic seizure. Said epileptic seizure may be recurrent epileptic seizures or non-recurrent epileptic seizures, and said epileptic seizures may further be recurrent epileptic seizure is refractory to treatment and / or said epilepsy may be refractory epilepsy.

[0352] In an embodiment, said pharmaceutical composition for use is administered to a patient in need thereof, preferably a patient exhibiting or at risk of epilepsy, and / or exhibiting one or more symptoms of epilepsy, as described above.In embodiments, the pharmaceutical composition for use here described are particularly suitable to be administered to a patient prone to develop or suffering from epilepsy.

[0353] In embodiments, pharmaceutical compositions for use as intended herein may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), parenteral administration (such as, e.g., subcutaneous, intravenous (I.V., intraocular), intramuscular, intraperitoneal, intrathecal or intracisternal injection or infusion), transdermal or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration, rectal, vaginal or intra-tracheal instillation, and the like. In this way, the therapeutic effects attainable by the compositions can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application.

[0354] In preferred embodiments, the pharmaceutical composition comprising the antibody or antibody fragment or nucleic acid encoding the antibody or antibody fragment, as taught herein is administered parenterally, .g., intravenously, intraperitoneally, subcutaneously, or intraocularly. More preferably, the pharmaceutical composition as taught herein is administered intravenously, for example by infusion, or intrathecally.

[0355] In particular embodiments, the pharmaceutical composition for use as taught herein is administered to the subject by the injection (e.g., intravenously or intraocularly) or transplantation of allogeneic cells transformed with the vector comprising the nucleic acid or the nucleic acid expression cassette as taught herein. When administered, the injected or transplanted allogenic cells will transcribe and translate the nucleic acid encoding the antibody or antibody fragment as taught herein in vivo.

[0356] For example, for parenteral administration, pharmaceutical compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc. Suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned. The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol,1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. The compound and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations. For example, one illustrative example of a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI). Other illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion. Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.

[0357] Where aqueous formulations are preferred, such may comprise one or more surfactants. For example, the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant. Illustrative examples of phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (DPMA), dipahnitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA); and diacyl phosphatidyl ethanolamines such as dimyristoyl phosphatidyl ethanolamine (DPME), dipalmitoyl phosphatidyl ethanolamine (DPPE) and distearoyl phosphatidyl ethanolamine (DSPE). Typically, a surfactant:active substance molar ratio in an aqueous formulation will be from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.

[0358] In particular embodiments, the antibody or antibody fragment, or nucleic acid encoding said antibody or antibody fragment, as part of the pharmaceuticalcomposition for use, is provided in carriers, such as liposomes, lipid nanoparticles, nanostructured lipid carriers, nanoemulsions, polymer nanoparticles, polymer micelle or dendrimers. In a preferred embodiment, said carriers are liposomes or lipid nanoparticles (LNPs). In an embodiment, said carriers are lipid-based carriers. Said lipid-based carriers comprise one or more lipids. The one or more lipids can be in solid and / or liquid form. Said lipid-based carriers may be LNPs, lipoplexes, liposomes, phospholipid micelles, solid lipid nanoparticles, nanostructured lipid carriers or nano-emulsions. Lipid-based carriers useful according to the invention include, for example, cationic lipids, liposomes, in particular cationic liposomes, and micelles, and nanoparticles. Cationic lipids may form complexes with negatively charged nucleic acids. Any cationic lipid may be used according to the invention. Liposomes are phospholipid and cholesterol self-assembled bilayer membranes that enclose an aqueous core, where hydrophilic molecules can be incorporated. Hydrophobic compounds can also be incorporated in the lipid bilayer. Liposomes can be classified in (i) small unilamellar vesicles (SUVs); (ii) large unilamellar vesicles (LUVs) and (iii) multilamellar vesicles (MLVs), according to their size and lamellarity. Solid lipid nanoparticles (SLNs) have a spherical shape with an average diameter of 10-1000 nm. They are used as a colloidal NP drug delivery system in which lipid drug carrier solidifies at room temperature as well as at body temperature. Different solid lipids can be exploited to produce SLNs, such as, tripalmitin, cetyl alcohol, cetyl palmitate, glyceryl monostearate, trimyristin, tristearin, stearic acid, etc. SLNs comprise of solid lipid, such as triglycerides, fatty acids, waxes, partial glycerides, and polyethylene glycosylated lipid; emulsifiers, such as polysorbates, poloxamer and lecithin; and water. Nanostructured lipid carriers (NLC), comprise a blend of solid and liquid lipids which results in a partially crystallized lipid system and many have advantages such as enhanced drug loading capacity, drug release modulation flexibility and improved stability. In an embodiment, said lipid-based carrier is a lipid nanoparticle. Solid lipid nanoparticles (SLNs, sLNPs), or lipid nanoparticles (LNPs), are nanoparticles composed of lipids that are suited to be used as a drug delivery vehicle for drug compounds, especially polynucleotides such as RNA or DNA.

[0359] One skilled in this art will recognize that the above description is illustrative rather than exhaustive. Indeed, many additional formulations techniques and pharmaceutically-acceptable excipients and carrier solutions are well-known to those skilled in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.In an embodiment, said pharmaceutical composition for use is administered as part of a gene therapy, preferably to treat epilepsy via treating BBB integrity and / or as part of BBB endothelial cell-directed gene therapy. This preferably comprises introducing in the patient, in particular in the central and / or peripheral nervous system of the patient, the pharmaceutical composition comprising the nucleic acid, preferably as part of a nucleic acid expression cassette or a vector as described herein, and expressing a therapeutically effective amount of the soluble antibody or antibody fragment encoded by the nucleic acid as taught herein in the patient, in particular in the central and / or peripheral nervous system of the patient.

[0360] In an embodiment, said pharmaceutical composition for use is administered as part of a mRNA therapy, preferably to treat epilepsy via treating BBB integrity and / or as part of BBB endothelial cell-directed mRNA therapy. This preferably comprises introducing in the patient, in particular in the central and / or peripheral nervous system of the patient, the pharmaceutical composition comprising the nucleic acid, preferably as part of a nucleic acid expression cassette or a vector as described herein, and expressing a therapeutically effective amount of the soluble antibody or antibody fragment encoded by the nucleic acid as taught herein in the patient, in particular in the central and / or peripheral nervous system of the patient.

[0361] An advantage of the use of RNAs therapy, it is generally believed that RNAs do not integrate into the genome and therefore do not have the risk of insertional mutagenesis.

[0362] The dosage or amount of the pharmaceutical composition for use as taught herein, optionally in combination with one or more other active compounds to be administered such as antiepileptic drugs (AEDs) as described above, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, the unit dose and regimen depend on the nature and the severity of the type of epilepsy to be treated, and also on factors such as the species of the subject, the sex, age, body weight, general health, diet, mode and time of administration, immune status, and individual responsiveness of the human or animal to be treated, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the pharmaceutical composition for use described in any of the embodiments above or below. In order to optimize therapeutic efficacy, the pharmaceutical composition for use as taught herein can be first administered at different dosing regimens. Typically, levels of thepharmaceutical composition for use, and then in particular of the antibody or antibody fragment as described herein, in a tissue can be monitored using appropriate screening assays as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. The frequency of dosing is within the skills and clinical judgement of medical practitioners (e.g., doctors, veterinarians or nurses). Typically, the administration regime is established by clinical trials which may establish optimal administration parameters. However, the practitioner may vary such administration regimes according to the one or more of the aforementioned factors, e.g., subject's age, health, weight, sex and medical status. The frequency of dosing can be varied depending on whether the treatment is prophylactic or therapeutic.

[0363] Said pharmaceutical composition for use in the treatment and / or prevention of epilepsy or seizure in a patient of the invention, comprises a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment.

[0364] And although the dosage depends on multiple factors as described above, without limitation, a typical dosage (e.g., a typical daily dosage or a typical intermittent dosage, e.g., a typical dosage for every two days, every three days, every four days, every five days, every six days, every week, every 1.5 weeks, every two weeks, every three weeks, every month, or other) of the antibody or antibody fragment as taught herein may range from about 0.1 mg / kg to about 20 mg / kg body weight of the subject, per dose, depending on the factors mentioned above, e.g., such as about 0.2 mg / kg to 20 mg / kg body weight, or about 0.1 mg / kg to 18 mg / kg body, or about 0.1 mg / kg to 15 mg / kg body, or about 0.1 mg / kg to 13 mg / kg body, or about 0.1 mg / kg to 12 mg / kg body weight, or about 0.2 mg / kg to 12 mg / kg body weight, or about 0.5 mg / kg to 12 mg / kg body weight, or about 0.5 mg / kg to 10 mg / kg body, or about 1 mg / kg to 10 mg / kg body weight, or about 2 mg / kg to 8 mg / kg body weight, or about 4 mg / kg to 6 mg / kg body weight, or about 4.5 mg / kg to 5.5 mg / kg body weight, 5 mg / kg body weight of the subject.

[0365] By means of example and without limitation, the antibody or antibody fragment as taught herein may be administered at about 0.1 mg / kg, about 0.2 mg / kg, about 0.3 mg / kg, about 0.4 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 12 mg / kg, or about 15 mg / kg per dose, or about 18 mg / kg per dose, or about 20 mg / kg per dose.Thus, in an embodiment, the pharmaceutical composition for use is administered to a patient in need thereof, in order to provide a dose as described above in any of the typical dosages. Said dose may be a single dose or may be repeated once, twice, or 3, 4, 5 or more times, with intervals of 1, 2, 3, 4, 5 days, 1, 2, 3, 4 weeks, 1, 2, 3, 4, 5, 6 months.

[0366] Toxicity and therapeutic efficacy of the pharmaceutical composition for use as described in any of the embodiments, comprising the antibody or antibody fragment or nucleic acid encoding said antibody or antibody fragment can be determined by known pharmaceutical procedures in, for example, cell cultures or experimental animals. These procedures can be used, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit high therapeutic indices are preferred. While pharmaceutical compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to normal cells (e.g., nontarget cells) and, thereby, reduce side effects.

[0367] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in appropriate subjects. The dosage of such pharmaceutical compositions lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For a pharmaceutical composition used as described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the pharmaceutical composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0368] In particular embodiments, the pharmaceutical composition for use as taught herein is administered using a sustained delivery system, such as a (partly) implanted sustained delivery system. Skilled person will understand that such a sustaineddelivery system may comprise a reservoir for holding the pharmaceutical composition for use as taught herein, a pump and infusion means (e.g., a tubing system). For example, the sustained delivery system may be a mini-osmotic pump system implanted in the brain.

[0369] In an embodiment, administration of a therapeutically active amount of the pharmaceutical composition for use decreases one or more of (e.g., 2 or more, 3 or more, 4 or more of):

[0370] (a) seizure recurrence;

[0371] (b) excitotoxicity and associated neuronal cell death in the CNS;

[0372] (c) one or more symptoms of a epilepsy (e.g., frequency, duration, or intensity of epileptic seizures, weakness, absence, sudden confusion, trouble understanding or producing speech, cognitive impairment, impaired mobility, dizziness, or loss of balance or coordination, paralysis, and emotional dysregulation), and

[0373] (d) pathological sprouting of recurrent mossy fibers of dentate gyrus granule cells in the hippocampus

[0374] by 10% or more (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more);

[0375] (e) BBB leakage, as assessed by MRI.

[0376] In an embodiment, administration of a therapeutically active amount of the pharmaceutical composition for use also restores a physiological excitation-inhibition balance in the affected region of the CNS. In particular, administration of the composition:

[0377] (a) reduces the number of seizures per day and / or reduces epileptiform discharges in the subject, for example, as measured by an electroencephalogram and standardized to seizure frequency per 30 days,

[0378] (b) improves the subject's measurements on routine laboratory parameters, such as hematology, biochemistry, coagulation, and urinalysis parameters, within at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 1 year after the composition is administered,

[0379] (c) reduces frequency of interictal discharges, as measured by an electroencephalogram,

[0380] (d) reduces aberrant neurological behavior by the subject, and / or

[0381] (e) produces no adverse effects after 4 weeks.The above-listed symptoms of epilepsy may be assessed using standard methods, such as neurological examination, electroencephalogram, magnetoencephalogram, CT scan, PET scan, fMRI scan, videography, and visual observation. Measures of epilepsy symptoms from before and after administration of the composition can be compared to evaluate the efficacy of the treatment.

[0382] A finding of a reduction in the symptoms of epilepsy described above indicates that the pharmaceutical composition has been successfully in its use in the treatment of the epilepsy or seizure in the patient.

[0383] In other or further embodiments, administration of the pharmaceutical composition for use to said patient results in a reduced seizure severity, as measured by reduction in Racine stage, preferably by at least one or two stages. This preferably applies in epilepsy in general, and / or one or more of generalized epilepsy, focal epilepsy, TLE, and non-TLE focal epilepsy. Amounts of the antibody or antibody fragment, doses, and optionally repeated dosings are preferably as described above in any one of the embodiments.

[0384] The "Racine scoring system" is a widely used classification method to assess seizure severity in animal models of epilepsy, particularly in rodents.

[0385] While variations exist depending on the laboratory or experimental setup, the version applied here is defined as follows:

[0386] • Score 0: no change in behaviour;

[0387] • Score 1: immobility;

[0388] • Score 2: head nodding;

[0389] • Score 3: forelimb clonus;

[0390] • Score 4: forelimb clonus with rearing and falling;

[0391] • Score 5: wild running and jumping, often followed by generalized tonic-clonic activity and loss of postural tone;

[0392] • Score 6: death.

[0393] This scoring system provides a structured way to evaluate seizure progression and severity in experimental epilepsy models, offering valuable insights into the effects of epileptogenic stimuli and potential therapeutic interventions.

[0394] In specific embodiments, the efficacy of the pharmaceutical composition for use may be shown using the kainic acid model in rodents, where preferably administration of the pharmaceutical composition for use results in a reduced seizure severity, as measured by reduction in Racine score (stage), preferably by at least one or twostages. It is to be noted that there are a variety of other models that could be used as alternative to the KA model as well.

[0395] The "kainic acid (KA) model of focal epilepsy", such as temporal lobe epilepsy (TLE), is a widely used experimental model that replicates key features of human TLE, including hippocampal sclerosis, spontaneous recurrent seizures, and blood-brain barrier (BBB) dysfunction. Kainic acid, a neurotoxin and glutamate receptor agonist, is administered systemically or directly into the brain to induce excitotoxicity. This leads to excessive neuronal firing, calcium influx, and eventual neuronal damage, mimicking the hyperexcitable state observed in epilepsy. The model progresses through three phases: an acute seizure phase (status epilepticus), a latent period (seizure-free interval with neuronal remodeling), and a chronic phase characterized by spontaneous recurrent seizures (SRS), making it suitable for studying epileptogenesis and long-term therapeutic interventions.

[0396] The KA model is valuable for epilepsy research and drug screening, providing insights into neuroinflammation, synaptic reorganization, and BBB dysfunction. It closely mimics human TLE and allows for flexible dosing and administration routes. It therefore is a crucial tool for testing new antiepileptic drugs (AEDs), investigating molecular pathways, and exploring gene therapy approaches targeting epilepsy.

[0397] Therefore, in embodiments of the pharmaceutical composition for use, said epilepsy is focal epilepsy, preferably temporal lobe epilepsy, preferably induced using the kainic acid model, and said pharmaceutical composition is administered to a rodent patient.

[0398] In another or further embodiment, said administration comprises a single dose of between 1 and 15 mg of antibody or antibody fragment in said pharmaceutical composition / kg body weight of the rodent patient, preferably between 2 and 12 mg / kg, more preferably between 2 and 10 mg / kg, more preferably between 3 and 8 mg / kg, more preferably between 4 and 6 mg / kg, most preferably about 5mg / kg body weight of the rodent patient.

[0399] In another or further embodiment, administration of the pharmaceutical composition reduces seizure severity, as measured by reduction in Racine score (stage), preferably by at least one stage, more preferably by at least two stages.In a specific embodiment of the pharmaceutical composition for use, wherein said epilepsy is focal epilepsy, preferably temporal lobe epilepsy, preferably induced using the kainic acid model, wherein a dose of said pharmaceutical composition is administered to a patient, wherein said dose comprises between 4 and 6 mg of antibody or antibody fragment / kg body weight of the patient, wherein said administration reduces seizure severity, as measured by reduction in Racine score (stage), preferably by at least one stage. Optionally, said patient is a rodent patient.

[0400] In particular embodiment, the antibody or antibody fragment, or the nucleic acid encoding said antibody or antibody fragment as disclosed herein is the main or only active ingredient of the pharmaceutical composition for use.

[0401] In another embodiment, the antibody or antibody fragment, or the nucleic acid encoding said antibody or antibody fragment as described in any of the embodiments is combined with a second therapy.

[0402] In another or further embodiment, the pharmaceutical composition for use as described in any of the embodiments is combined with a second therapy, preferably chosen from antiepileptic drugs (AEDs), surgery, chemotherapy, radiotherapy or immunotherapy. Examples of said AEDs are described above.

[0403] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0404] EXAMPLES AND / OR DESCRIPTION OF FIGURES

[0405] Example 1: Generation of antibody-based Wnt7a mimics

[0406] Material and methods

[0407] Preparation of recombinant Gprl24, Reck, and LRP6 proteins

[0408] - Human and mouse Gprl24 extracellular domains (residues 34-344) were produced in a mammalian expression system either as Fc-fusion proteins or with a C-terminal 6-His extension, and purified by conventional chromatography techniques.- Human and mouse Reck extracellular domains (residues 27-340) were produced in a mammalian expression system with a C-terminal 6-His extension, and purified by conventional chromatography techniques.

[0409] - Purified human LRP6 extracellular domain (residues 20-630) was purchased from Sino Biological (cat. 17052-H08H).

[0410] - Mouse LRP6 extracellular domain (residues 20-628) was produced in a mammalian expression system with a C-terminal 6-His extension, and purified by conventional chromatography techniques.

[0411] Generation of anti-Gprl24, anti -Reck and anti-LRP6 antibodies

[0412] - Anti-Gprl24 and anti-Reck antibodies (IgGs) we isolated from naive Fab-on- phage, fully human libraries by performing multiple (up to four) rounds of selection on the corresponding antigens. Human and mouse antigens were alternated during selection to favor the identification of human / mouse cross- reactive antibodies. HEK293 cells transiently expressing the corresponding, full-length human antigens were also use at specific stages of phage libraries selection, typically round 4.

[0413] - Anti-LRP6 nanobodies (VHHs) we isolated from naive VHH-on-phage libraries by performing multiple (up to four) rounds of selection on recombinant LRP6. Human and mouse antigens were alternated during selection to favor the identification of human / mouse cross-reactive VHHs. HEK293 cells transiently expressing the corresponding, full-length human LRP6 protein were also use at specific stages of phage libraries selection, typically round 4.

[0414] Generation of anti-Gprl24 / LRP6 and anti- Reck / LRP6 bispecific antibodies

[0415] - Anti-LRP6 VHHs were fused to the N-terminus of the VL (variable light) domain of either anti-Gprl24 or anti-Reck IgGs (human IgGl with Fc- silencing L234A, L235A, P329G "LA - LA - PG" mutations - Lo et al., J. Biol. Chem. 292, 3900-3908, 2017) via a 5-mer linker (GSGGS - SEQ ID N°: 172). Constructs displaying agonist activity were identified by screening in STF (Super Top Flash) cells transiently expressing human Gprl24, Reck, Frizzled- 1 (Fzdl), and LRP6.

[0416] Constructs with agonist activity could be identified by combining the following anti-Gprl24 or anti-Reck IgGs with some or any of the below-listed LRP6 VHHs:

[0417] o Gprl24 IgGs: 05A10 (SEQ ID N° 63 and 64), 06C03 (SEQ ID N° 65 and 66), 06A05 (SEQ ID N° 67 and 68)O Reck IgGs: 01A08 (SEQ ID N° 69 and 70), 01F11 (SEQ ID N° 71 and 72), 03A04 (SEQ ID N° 73 and 74), 07B05 (SEQ ID N° 75 and 76) o LRP6 VHH: 01F11 (SEQ ID N° 61), 01B01 (SEQ ID N° 59), 01E12 (SEQ ID N° 60), 02G02 (SEQ ID N° 62)

[0418] Figure 1 illustrates the structure of the anti-Gprl24 / LRP6 and anti-Reck / LRP6 bispecific antibodies, where the LRP6 VHH is fused to the N-terminus of the VL domain of the respective anti-Gprl24 or anti-Reck IgGs.

[0419] Each construct is tetravalent, meaning it has two binding sites for Gprl24 or Reck and two binding sites for LRP6, unless explicitly mentioned otherwise.

[0420] 5.1: Gprl24 / LRP6 or Gprl24 / LRP5 bispecific antibodies

[0421] Gprl24 / LRP6

[0422] Bispecific antibodies nomenclature

[0423] Name VHH IgG

[0424] 01E12-05A10 01E12 05A10

[0425] 01F11-05A10 01F11 05A10

[0426] 02G02-05A10 02G02 05A10

[0427] 01B01-06C03 01B01 06C03

[0428] 01E12-06C03 01E12 06C03

[0429] 01F11-06C03 01F11 06C03

[0430] 02G02-06C03 02G02 06C03

[0431]

[0432] Identification of bispecific constructs with agonist activity

[0433] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

[0434] - Cells transfected with empty vector were used as a negative control ("Control")

[0435] Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal.

[0436] Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

[0437] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0438] Results: all tested constructs were capable of activating the Wnt / p-catenin pathway to a level that exceeded the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 2.STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

[0439] Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the indicated concentrations

[0440] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0441] Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 1.9 nM and 1.7 nM for 01E12-05A10 and 01B01-06C03, respectively), as shown in Figure 3.

[0442] Two additional bispecific antibodies were tested subsequently:

[0443] Name VHH IgG

[0444] 01B01-06A05 01B01 06A05

[0445] 01E12-06A05 01E12 06A05

[0446]

[0447] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

[0448] Bispecific antibodies 01B01-06A05 (A) or 01E12-06A05 (B) were added to the cells at the indicated concentrations

[0449] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0450] Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 2.8 nM and 3.0 nM for 01B01-06A05and 01E12-06A05, respectively), as shown in Figure 4.

[0451] Gorl24 / LRP5

[0452] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP5

[0453] - Cells transfected with empty vector were used as a negative control ("Control")

[0454] Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP5 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal Bispecific antibodies were added to the cells as indicated at a concentration of 20 nMLuminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0455] Results: bispecific antibodies 01E12-05A10, 01B01-06C03 & 01E12-06C03 were capable of activating the Wnt / 0-catenin pathway in STF cells expressing hLRP5 instead of hLRP6, as shown in Figure 5. This indicates (1) that VHHs 01B01 & 01E12 exhibit cross-reactivity with hLRP5 and (2) that agonist activity can be achieved by engaging either Gprl24 & LRP6 or Gprl24 & LRP5.

[0456] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP5

[0457] Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the indicated concentrations

[0458] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0459] Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 4.2 nM and 1.5 nM for 01E12-05A10 and 01B01-06C03, respectively), as shown in Figure 6.

[0460] Reck independency

[0461] STF cells were transfected with vectors encoding human Gprl24, Fzdl & LRP6 - Cells transfected with empty vector were used as a negative control ("Control")

[0462] Cells transfected with vectors encoding human Gprl24, Fzdl, LRP6 & Wnt7a were used as an additional control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

[0463] Bispecific antibodies were added to the cells as indicated at a concentration of 20 nM

[0464] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0465] Results: all tested bispecific antibodies were capable of activating the Wnt / p-catenin pathway in a Reck-independent manner, i.e. in the absence of Reck, as shown in Figure 7. By contrast, and as predicted, the natural ligand Wnt7a showed no activity in the absence of Reck.

[0466] Fzd receptor dependency

[0467] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6 (A), or Gprl24, Reck, Fzd5 & LRP6 (B)- Cells transfected with empty vector were used as a negative control ("Control")

[0468] Cells transfected with vectors encoding human Gprl24, Reck, Fzd4, LRP6 & Wnt7a (A), or Gprl24, Reck, Fzd5, LRP6 & Wnt7a (B) were used as additional controls ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

[0469] Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

[0470] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0471] Results: all tested constructs were capable of activating the Wnt / p-catenin pathway in the presence of Gprl24, Reck, LRP6, and either Fzd4 or Fzd5 (also Fzdl, see above), to a level that exceeded the level of activation achieved with the natural ligand under these experimental conditions, as shown in Figure 8. Based on the data, the bispecific antibodies described here can use any of the 10 Fzd receptors present in human for activity.

[0472] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6

[0473] Bispecific antibodies 01E12-05A10 (A) or 01B01-06C03 (B) were added to the cells at the indicated concentrations

[0474] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0475] Results: both tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 3.7 nM and 1.3 nM for 01E12-05A10 and 01B01-06C03, respectively), as shown in Figure 9.

[0476] Bispecific antibodies display agonist activity in hRMEC and hBMEC

[0477] hRMEC (Human Retinal Microvascular Endothelial Cells) (A) and hBMEC (Human Brain Microvascular Endothelial Cells) (B) were treated with either recombinant Wnt3a (13.4nM) or the indicated antibodies (10 nM)

[0478] Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

[0479] Results: both tested bispecific antibodies were capable of activating the Wnt pathway to an extent that was comparable to the level of pathway activation achieved with the natural ligand Wnt3a, as shown in Figure 10.

[0480] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly potent agonists in hRMEChRMEC (Human Retinal Microvascular Endothelial Cells) were treated with either 01E12-05A10 (A) or 01B01-06C03 (B) at the indicated concentrations Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

[0481] Results: both tested bispecific antibodies were capable of activating the Wnt pathway with low EC50 values (EC50 = 66 pM and 32 pM for 01E12-05A10 and 01B01-06C03, respectively), as shown in Figure 11.

[0482] 01B01-06C03 regulates other mediators of the Wnt / (3-catenin pathway in hRMEC cells

[0483] hRMEC cells were treated with 01B01-06C03 (10 nM) for 6 h

[0484] - The level of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and NKD1 (Naked cuticle 1) mRNAs were monitored by qRT-PCR.

[0485] Results: treatment of hRMEC cells with 01B01-06C03 resulted in a moderate but detectable increase in the expression of Lefl and NKD1, as shown in Figure 12.

[0486] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly potent agonists in hBMEC

[0487] hBMEC (Human Brain Microvascular Endothelial Cells) were treated with either 01E12-05A10 (A) or 01B01-06C03 (B) at the indicated concentrations Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

[0488] Results: both tested bispecific antibodies were capable of activating the Wnt pathway with low EC50 values (EC50 = 17 pM and 61 pM for 01E12-05A10and 01B01-06C03, respectively), as shown in Figure 13.

[0489] Agonist activity requires engagement of both Gprl24 and LRP6

[0490] bEnd.3 cells (mouse brain endothelial cells) were treated with 01E12-05A10, with an irrelevant VHH linked to 05A10, or 01E12 linked to an irrelevant IgG (1 nM), or with both 'irrelevant VHH'-05A10and 01E12- 'irrelevant IgG' (1 nM each), for 24 h

[0491] o 01E12-05A10

[0492] o 'irrelevant VHH'-05A10

[0493] o 01E12- 'irrelevant IgG'

[0494] - The levels of the Axin2, Lefl, Mfsd2a, and PLVAP mRNAs were monitored by qRT-PCRResults: As shown in Figure 14, activity was detected only with the construct capable of engaging both Gprl24 and LRP6 (i.e. 01E12-05A10).

[0495] Bispecific antibodies 01E12-05A10 & 01B01-06C03 are highly potent agonists in bEnd.3 cells

[0496] bEnd.3 cells (mouse brain endothelial cells) were treated with either 01E12- 05A10 (A), 01B01-06C03 (B), or recombinant Wnt3a (C) at the indicated concentrations

[0497] Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

[0498] Results: both tested bispecific antibodies were capable of activating the Wnt pathway in this particular cell line with EC50 values markedly lower than the one of the natural ligand Wnt3a (EC50 = 25 pM and 61 pM for 01E12-05A10 and 01B01-06C03, respectively, vs. 6.56 nM for Wnt3a). Maximum signal amplitude was also larger for the bispecific antibodies compared to Wnt3a, as shown in Figure 15.

[0499] Bispecific antibodies 01E12-06C03 & 01F11-06C03 are highly potent agonists in bEnd.3 cells

[0500] bEnd.3 cells (mouse brain endothelial cells) were treated with either 01E12- 06C03 (A), 01F11-06C03 (B), or recombinant Wnt3a (C) at the indicated concentrations

[0501] Wnt pathway activation was assess by monitoring the level of Axin2 mRNA by qRT-PCR

[0502] Results: both tested bispecific antibodies were capable of activating the Wnt pathway in this particular cell line with EC50 values markedly lower than the one of the natural ligand Wnt3a (EC50 = 18 pM for both 01E12-06C03and 01F11-06C03, vs. 6.1 nM for Wnt3a). Maximum signal amplitude was also larger for the bispecific antibodies compared to Wnt3a, as shown in Figure 16.

[0503] 01E12-05A10 regulates other mediators of the Wnt / 3-catenin pathway in bEnd.3 cells

[0504] bEnd.3 cells were treated either with 01E12-05A10 (1 nM) or the glycogen synthase kinase 3 (GSK-3) inhibitor CHIR 99021 ("CHIR", 10 pM) for 24 h - The level of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domain-containing protein 2a) mRNAs, as well as the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA were monitored by qRT-PCR.Results: treatment of bEnd.3 cells with both CHIR 99021 and 01E12-05A10resulted in an increased expression of Lefl and Mfsd2a, and a decreased expression of PLVAP, as shown in Figure 17.

[0505] 01B01-06C03 regulates other mediators of the Wnt / ^-catenin pathway in bEnd.3 cells

[0506] bEnd.3 cells were treated with 01B01-06C03 (10 nM) for 24 h

[0507] - The level of the downstream mediators of the Wnt / 0-catenin signaling pathway Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domain-containing protein 2a) mRNAs, as well as the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA were monitored by qRT-PCR.

[0508] Results: treatment of bEnd.3 cells with 01B01-06C03 resulted in an increased expression of Lefl and Mfsd2a, and a decreased expression of PLVAP, as shown in Figure 18.

[0509] Agonist activity can be achieved with bivalent molecules

[0510] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6 - Cells transfected with empty vector were used as a negative control ("Control")

[0511] - Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

[0512] - The following antibodies were added to the cells at a concentration of 20 nM (see also Figure 34B)

[0513] o 01F11-06C03, 01B01-06C03: see elsewhere

[0514] o 01E12-06C03 (VHH-Fab): VHH-Fab fragment derived from 01F11- 06C03

[0515] o 01E12-05A10 (VHH-Fab): VHH-Fab fragment derived from 01B01- 06C03

[0516] - Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0517] Results: All constructs, whether tetravalent (VHH-tethered IgG) or bivalent (VHH-Fab) showed a similar activity, as shown in Figure 34A.

[0518] 5.2: Reck / LRP6 bisoecific antibodiesBispecific antibodies nomenclature

[0519] Name VHH IgG

[0520] 01B01-01A08 01B01 01A08

[0521] 01E12-01A08 01E12 01A08

[0522] 01B01-01F11 01B01 01F11

[0523] 01E12-01F11 01E12 01F11

[0524] 01B01-03A04 01B01 03A04

[0525] 01E12-03A04 01E12 03A04

[0526] 01B01-07B05 01B01 07B05

[0527] 01E12-07B05 01E12 07B05

[0528]

[0529] Identification of bispecific constructs with agonist activity

[0530] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

[0531] - Cells transfected with empty vector were used as a negative control ("Control")

[0532] Cells transfected with vectors encoding human Gprl24, Reck, Fzdl, LRP6 & Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

[0533] Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0534] Results: all tested constructs were capable of activating the Wnt / p-catenin pathway to a level that, for some constructs at least, was close to the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 19.

[0535] STF cells were transfected with vectors encoding human Gprl24, Reck, Fzdl & LRP6

[0536] - Bispecific antibodies 01B01-01F11 (A), 01E12-01F11 (B), 01E12-03A04 (C) or 01B01-03A04 (D) were added to the cells at the indicated concentrations Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0537] Results: All tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 4.2 nM, 3.4 nM, 1.3 nM, and 6.4 nM for 01B01-01F11, 01E12-03A04, 01E12-01Flland 01B01-03A04, respectively), as shown in Figure 20.- STF cells were transfected with vectors encoding human Gprl24, human Reck, human Fzdl & mouse LRP5

[0538] - Cells transfected with empty vector were used as a negative control ("Control")

[0539] - Cells transfected with vectors encoding human Gprl24, human Reck, human Fzdl, mouse LRP5 & human Wnt7a were used as a positive control ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

[0540] - Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

[0541] - Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0542] Results: All tested antibodies were capable of activating the Wnt / p-catenin pathway in STF cells expressing mouse LRP5, as shown in Figure 21, indicating (1) that VHHs 01B01 & 01E12 exhibit at least some degree of cross -reactivity with mouse LRP5 and (2) that agonist activity can be achieved by engaging Reck and either LRP6 or LRP5.

[0543] Fzd receptor dependency (1)

[0544] - STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6 (Fig 22A), or Gprl24, Reck, Fzd5 & LRP6 (Fig 22B)

[0545] - Cells transfected with empty vector were used as a negative control ("Control")

[0546] - Cells transfected with vectors encoding human Gprl24, Reck, Fzd4, LRP6 & Wnt7a (A), or Gprl24, Reck, Fzd5, LRP6 & Wnt7a (B) were used as additional controls ("Wnt7a"). Of note, the amount of Wnt7a vector used was chosen to give the highest possible signal

[0547] - Bispecific antibodies were added to the cells as indicated at a concentration of 50 nM

[0548] - Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0549] Results: All tested constructs were capable of activating the Wnt / p-catenin pathway in the presence of Gprl24, Reck, LRP6, and either Fzd4 or Fzd5 (also Fzdl, see above), to a level that equaled or exceeded the level of activation achieved with the natural ligand Wnt7a, as shown in Figure 22.

[0550] Based on the data, the bispecific antibodies described here can use any of the 10 Fzd receptors present in human for activity.Fzd receptor dependency (2)

[0551] - STF cells were transfected with vectors encoding human Gprl24, Reck, Fzd4 & LRP6

[0552] - Bispecific antibodies 01B01-01F11 (A), 01E12-03A04 (B), or 01B01-03A04 (C) were added to the cells at the indicated concentrations

[0553] - Luminescence measurements were used to assess activation of the Wnt / 0- catenin pathway

[0554] Results: As shown in Figure 23, all tested bispecific antibodies showed agonist activity with low EC50 values (EC50 = 5.5 nM, 5.9 nM, and 1.8 nM for 01B01-01F11, 01E12-03A04, and 01B01-03A04, respectively).

[0555] Reck / LRP6 Abs regulate several Wnt / ^-catenin target genes in bEnd.3 cells

[0556] - bEnd.3 cells were treated with the indicated Abs (10 nM) for 24 h

[0557] - The level of the downstream mediators of the Wnt / 0-catenin signaling pathway Axin2 (axis inhibition protein 2), Lefl (Lymphoid enhancer-binding factor 1) and Mfsd2a (major facilitator superfamily domain-containing protein 2a) mRNAs, as well as the level of the canonically Wnt-downregulated protein PLVAP (Plasmalemma vesicle- associated protein) mRNA were monitored by qRT-PCR

[0558] Results: as shown in Figure 24, treatment of bEnd.3 cells with Reck / LRP6 bispecific Abs resulted in an increased expression of Axin2 (A and B), Lefl (C) and Mfsd2a (D), and a decreased expression of PLVAP (E and F).

[0559] Bispecific antibodies 01B01-01A08, 01E12-03A04 and 01E12-01F11 are highly potent agonists in bEnd.3 cells

[0560] - bEnd.3 cells were treated with 01B01-01A08 (A), 01E12-03A04 (B) or 01E12- 01F11 (C) at the indicated concentrations for 24 h

[0561] - Wnt pathway activation was assessed by monitoring the mRNA level of Axin2 by qRT-PCR

[0562] Results: as shown in Figure 25, 01B01-01A08, 01E12-03A048<. 01E12-OlFllactivated the Wnt pathway in this particular cell line with EC50 value of 10 pM, 7 pM, and 7 pM, respectively.

[0563] Example 2: 01B01-06C03 (Gprl24 / LRP6 bispecific) reduces disease severity in the kainic acid model of temporal lobe epilepsy.Adult mice received intraperitoneal injection of IgG control or Gprl24 / Lrp6 bispecific antibody 01B01-06C03 at a dose of 5 mg / kg right before the induction of the disease model.

[0564] The KA model was achieved by 4 intraperitoneal injection of kainic acid, 5 mg / kg each every 15 min (total 20 mg / kg in Ih). The behavior of the mice was followed for the next 2h according to the Racine Scale to assess apparition of the status epilepticus (SE). The schematic overview of the experiment is shown in Figure 26A. While the animals treated with the control IgG have a progressive increase of the Racine Scale, the mice treated with Gprl24 / Lrp6 bispecific antibody have a stable and lower score (Fig 26B). This is further exemplified with significantly reduced cumulative and mean Racine score (Figs 26 C and D, respectively). Of note, no death was observed in the Gprl24 / Lrp6 treated group while 2 animals out of 5 died in the control group (numbers in brackets Fig 26B).

[0565] The animals were placed back in their cage and euthanized 48h later to collect the brain and further assess neuronal tissue modifications and BBB leakage.

[0566] • Score 0: no change in behaviour;

[0567] • Score 1: immobility;

[0568] • Score 2: head nodding;

[0569] • Score 3: forelimb clonus;

[0570] • Score 4: forelimb clonus with rearing and falling;

[0571] • Score 5: wild running and jumping, often followed by generalized tonic-clonic activity and loss of postural tone;

[0572] • Score 6: death.

[0573] This example is repeated with all other antibodies described above and similar results are obtained.

[0574] The kainic acid (KA) model of temporal lobe epilepsy (TLE) is a widely used experimental model to study the pathophysiology of epilepsy and to evaluate potential antiepileptic treatments. It replicates many features of human TLE, including hippocampal sclerosis, spontaneous recurrent seizures, and blood-brain barrier (BBB) dysfunction.

[0575] This model employs kainic acid, a potent neurotoxin and glutamate receptor agonist (specifically targeting AMPA and kainate receptors), is administered systemically (intraperitoneally), intracerebrally (into the hippocampus), orintracerebroventricularly. Kainic acid induces excitotoxicity by overstimulating glutamate receptors, leading to prolonged neuronal depolarization, calcium influx, and eventual neuronal damage. This excitotoxic cascade mimics the hyperexcitable neural environment observed in TLE.

[0576] The Kainic Acid (KA) Model of epilepsy consists of three distinct phases. The Acute Seizure Phase, also known as Status Epilepticus (SE), begins within minutes to hours after KA administration. This phase is characterized by continuous seizures lasting several hours, accompanied by intense neuronal firing and hippocampal excitotoxicity. Following this, the Latent Period represents a seizure-free interval lasting from days to weeks. During this time, neuronal damage, neuroinflammation, and structural remodeling occur, including abnormal synaptic reorganization such as mossy fiber sprouting. Finally, the Chronic Phase is marked by the emergence of spontaneous recurrent seizures (SRS), which mimic human temporal lobe epilepsy (TLE). This phase is long-lasting and is useful for evaluating long-term therapeutic interventions.

[0577] Several pathophysiological changes occur in the KA model. One key feature is hippocampal sclerosis, which involves the loss of pyramidal neurons in the CAI and CA3 regions, along with damage to the dentate gyrus. The model also induces bloodbrain barrier (BBB) disruption, leading to increased permeability, which enhances neuroinflammation and seizure susceptibility. Neuroinflammation is another hallmark, involving the activation of microglia and astrocytes and the release of pro-inflammatory cytokines such as IL-ip and TNF-o. Additionally, synaptic reorganization occurs, with aberrant growth of mossy fibers in the dentate gyrus. Neurogenesis is also altered, contributing to circuit dysregulation.

[0578] The KA model has several applications in epilepsy research. It is widely used for drug screening, allowing the evaluation of new antiepileptic drugs (AEDs) and diseasemodifying therapies. It also facilitates molecular studies, helping to investigate pathways such as Wnt signaling and neuroinflammation. The model is particularly useful in blood-brain barrier (BBB) research, providing insights into how BBB dysfunction contributes to epilepsy progression. Furthermore, it is used in gene therapy research to explore the efficacy of gene-based treatments targeting epileptogenesis.

[0579] The model offers several advantages. It closely mimics human TLE by replicating features such as spontaneous seizures and hippocampal damage. It is also suitablefor studying epileptogenesis and chronic epilepsy, with flexible dosing and administration routes. However, there are limitations. High doses of KA can result in high mortality, and there is variable seizure onset and severity across different animals. Additionally, species-specific differences can limit the direct translation of findings to humans.

[0580] Scoring may be performed according to a modified Racine scoring system, which is a widely used classification method to assess seizure severity in animal models of epilepsy, particularly in rodents. It is based on observable behavioral and motor manifestations of seizures, progressing through defined stages of increasing severity. In Score 0, no behavioral change is observed. Score 1 is characterized by immobility, followed by head nodding in Score 2. Forelimb clonus appears in Score 3. In Score 4, the animal exhibits forelimb clonus combined with rearing and falling. Score 5 involves wild running and jumping, often followed by generalized tonic-clonic activity and loss of postural tone. In some cases, seizures may progress to Score 6, which is marked by death. This scoring system provides a structured way to evaluate seizure progression and severity in experimental epilepsy models, offering valuable insights into the effects of epileptogenic stimuli and potential therapeutic interventions.

[0581] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.Overview of sequences

[0582] SEQ ID N°:l (mature human Wnt7a) LGASIICNKIPGLAPRQRAICQSRPDAIIVIGEGSQMGLDECQFQFRNGRWNCSALGERTVFG KELKVGSREAAFTYAIIAAGVAHAITAACTQGNLSDCGCDKEKQGQYHRDEGWKWGGCSA DIRYGIGFAKVFVDAREIKQNARTLMNLHNNEAGRKILEENMKLECKCHGVSGSCTTKTCWT TLPQFRELGYVLKDKYNEAVHVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYCEE DPVTGSVGTQGRACNKTAPQASGCDLMCCGRGYNTHQYARVWQCNCKFHWCCYVKCNTC SERTEMYTCK

[0583] SEQ ID N°:2 (mature human Wnt7b) LGANIICNKIPGLAPRQRAICQSRPDAIIVIGEGAQMGINECQYQFRFGRWNCSALGEKTVFG QELRVGSREAAFTYAITAAGVAHAVTAACSQGNLSNCGCDREKQGYYNQAEGWKWGGCSA DVRYGIDFSRRFVDAREIKKNARRLMNLHNNEAGRKVLEDRMQLECKCHGVSGSCTTKTCW TTLPKFREVGHLLKEKYNAAVQVEVVRASRLRQPTFLRIKQLRSYQKPMETDLVYIEKSPNYCE EDAATGSVGTQGRLCNRTSPGADGCDTMCCGRGYNTHQYTKVWQCNCKFHWCCFVKCNT

[0584] SEQ ID N°:3 (human LRP5) MEAAPPGPPWPLLLLLLLLLALCGCPAPAAASPLLLFANRRDVRLVDAGGVKLESTIVVSGLED AAAVDFQFSKGAVYWTDVSEEAIKQTYLNQTGAAVQNVVISGLVSPDGLACDWVGKKLYWT DSETNRIEVANLNGTSRKVLFWQDLDQPRAIALDPAHGYMYWTDWGETPRIERAGMDGSTR KIIVDSDIYWPNGLTIDLEEQKLYWADAKLSFIHRANLDGSFRQKVVEGSLTHPFALTLSGDT LYWTDWQTRSIHACNKRTGGKRKEILSALYSPMDIQVLSQERQPFFHTRCEEDNGGCSHLCL LSPSEPFYTCACPTGVQLQDNGRTCKAGAEEVLLLARRTDLRRISLDTPDFTDIVLQVDDIRHA IAIDYDPLEGYVYWTDDEVRAIRRAYLDGSGAQTLVNTEINDPDGIAVDWVARNLYWTDTGT DRIEVTRLNGTSRKILVSEDLDEPRAIALHPVMGLMYWTDWGENPKIECANLDGQERRVLVN ASLGWPNGLALDLQEGKLYWGDAKTDKIEVINVDGTKRRTLLEDKLPHIFGFTLLGDFIYWTD WQRRSIERVHKVKASRDVIIDQLPDLMGLKAVNVAKVVGTNPCADRNGGCSHLCFFTPHAT RCGCPIGLELLSDMKTCIVPEAFLVFTSRAAIHRISLETNNNDVAIPLTGVKEASALDFDVSNN HIYWTDVSLKTISRAFMNGSSVEHVVEFGLDYPEGMAVDWMGKNLYWADTGTNRIEVARLD GQFRQVLVWRDLDNPRSLALDPTKGYIYWTEWGGKPRIVRAFMDGTNCMTLVDKVGRAND LTIDYADQRLYWTDLDTNMIESSNMLGQERVVIADDLPHPFGLTQYSDYIYWTDWNLHSIER ADKTSGRNRTLIQGHLDFVMDILVFHSSRQDGLNDCMHNNGQCGQLCLAIPGGHRCGCAS HYTLDPSSRNCSPPTTFLLFSQKSAISRMIPDDQHSPDLILPLHGLRNVKAIDYDPLDKFIYWV DGRQNIKRAKDDGTQPFVLTSLSQGQNPDRQPHDLSIDIYSRTLFWTCEATNTINVHRLSGE AMGVVLRGDRDKPRAIVVNAERGYLYFTNMQDRAAKIERAALDGTEREVLFTTGLIRPVALVV DNTLGKLFWVDADLKRIESCDLSGANRLTLEDANIVQPLGLTILGKHLYWIDRQQQMIERVE KTTGDKRTRIQGRVAHLTGIHAVEEVSLEEFSAHPCARDNGGCSHICIAKGDGTPRCSCPVH LVLLQNLLTCGEPPTCSPDQFACATGEIDCIPGAWRCDGFPECDDQSDEEGCPVCSAAQFPC ARGQCVDLRLRCDGEADCQDRSDEADCDAICLPNQFRCASGQCVLIKQQCDSFPDCIDGSD ELMCEITKPPSDDSPAHSSAIGPVIGIILSLFVMGGVYFVCQRVVCQRYAGANGPFPHEYVSG TPHVPLNFIAPGGSQHGPFTGIACGKSMMSSVSLMGGRGGVPLYDRNHVTGASSSSSSSTK ATLYPPILNPPPSPATDPSLYNMDMFYSSNIPATARPYRPYIIRGMAPPTTPCSTDVCDSDYSA SRWKASKYYLDLNSDSDPYPPPPTPHSQYLSAEDSCPPSPATERSYFHLFPPPPSPCTDSSSEQ ID N°:4 (human LRP6) MGAVLRSLLACSFCVLLRAAPLLLYANRRDLRLVDATNGKENATIVVGGLEDAAAVDFVFSHG LIYWSDVSEEAIKRTEFNKTESVQNVVVSGLLSPDGLACDWLGEKLYWTDSETNRIEVSNLD GSLRKVLFWQELDQPRAIALDPSSGFMYWTDWGEVPKIERAGMDGSSRFIIINSEIYWPNGL TLDYEEQKLYWADAKLN FIH KSN LDGTN RQAVVKGSLPH PFALTLFEDI LYWTD WSTH SI LAC NKYTGEGLREIHSDIFSPMDIHAFSQQRQPNATNPCGIDNGGCSHLCLMSPVKPFYQCACPT GVKLLENGKTCKDGATELLLLARRTDLRRISLDTPDFTDIVLQLEDIRHAIAIDYDPVEGYIYWT DDEVRAIRRSFIDGSGSQFVVTAQIAHPDGIAVDWVARNLYWTDTGTDRIEVTRLNGTMRKI LISEDLEEPRAIVLDPMVGYMYWTDWGEIPKIERAALDGSDRVVLVNTSLGWPNGLALDYDE GKIYWGDAKTDKIEVMNTDGTGRRVLVEDKIPHIFGFTLLGDYVYWTDWQRRSIERVHKRS AEREVIIDQLPDLMGLKATNVHRVIGSNPCAEENGGCSHLCLYRPQGLRCACPIGFELISDMK TCIVPEAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDFDVTDNRIYWTDISLKTISRAF MNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNRIEVSKLDGQHRQVLVWKDLDSP RALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTLVPNVGRANGLTIDYAKRRLYWTDLD TNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIYWTDWSRRSIERANKTSGQNRTIIQGHL DYVMDILVFHSSRQSGWNECASSNGHCSHLCLAVPVGGFVCGCPAHYSLNADNRTCSAPTT FLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVRAIDYDPLDKQLYWIDSRQNMIRKAQEDGS QGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYWTCEATNVINVTRLDGRSVGVVLKGEQDRPR AVVVNPEKGYMYFTNLQERSPKIERAALDGTEREVLFFSGLSKPIALALDSRLGKLFWADSDL RRIESSDLSGANRIVLEDSNILQPVGLTVFENWLYWIDKQQQMIEKIDMTGREGRTKVQARI AQLSDIHAVKELNLQEYRQHPCAQDNGGCSHICLVKGDGTTRCSCPMHLVLLQDELSCGES QFQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGKHKKCDHNVDC SDKSDELDCYPTEEPAPQATNTVGSVIGVIVTIFVSGTVYFICQRMLCPRMKGDGETMTNDYV VHGPASVPLGYVPHPSSLSGSLPGMSRGKSMISSLSIMGGSSGPPYDRAHVTGASSSSSSST KGTYFPAILNPPPSPATERSHYTMEFGYSSNSPSTHRSYSYRPYSYRHFAPPTTPCSTDVCDSD YAPSRRMTSVATAKGYTSDLNYDSEPVPPPPTPRSQYLSAEENYESCPPSPYTERSYSHHLYPP PPSPCTDSS

[0585] SEQ ID N°:5 (human LRP6) MGAVLRSLLACSFCVLLRAAPLLLYANRRDLRLVDATNGKENATIVVGGLEDAAAVDFVFSHG LIYWSDVSEEAIKRTEFNKTESVQNVVVSGLLSPDGLACDWLGEKLYWTDSETNRIEVSNLD GSLRKVLFWQELDQPRAIALDPSSGFMYWTDWGEVPKIERAGMDGSSRFIIINSEIYWPNGL TLDYEEQKLYWADAKLN FIH KSN LDGTN RQAVVKGSLPH PFALTLFEDI LYWTD WSTH SI LAC NKYTGEGLREIHSDIFSPMDIHAFSQQRQPNATNPCGIDNGGCSHLCLMSPVKPFYQCACPT GVKLLENGKTCKDGATELLLLARRTDLRRISLDTPDFTDIVLQLEDIRHAIAIDYDPVEGYIYWT DDEVRAIRRSFIDGSGSQFVVTAQIAHPDGIAVDWVARNLYWTDTGTDRIEVTRLNGTMRKI LISEDLEEPRAIVLDPMVGYMYWTDWGEIPKIERAALDGSDRVVLVNTSLGWPNGLALDYDE GKIYWGDAKTDKIEVMNTDGTGRRVLVEDKIPHIFGFTLLGDYVYWTDWQRRSIERVHKRS AEREVIIDQLPDLMGLKATNVHRVIGSNPCAEENGGCSHLCLYRPQGLRCACPIGFELISDMK TCIVPEAFLLFSRRADIRRISLETNNNNVAIPLTGVKEASALDFDVTDNRIYWTDISLKTISRAF MNGSALEHVVEFGLDYPEGMAVDWLGKNLYWADTGTNRIEVSKLDGQHRQVLVWKDLDSP RALALDPAEGFMYWTEWGGKPKIDRAAMDGSERTTLVPNVGRANGLTIDYAKRRLYWTDLD TNLIESSNMLGLNREVIADDLPHPFGLTQYQDYIYWTDWSRRSIERANKTSGQNRTIIQGHL DYVMDILVFHSSRQSGWNECASSNGHCSHLCLAVPVGGFVCGCPAHYSLNADNRTCSAPTT FLLFSQKSAINRMVIDEQQSPDIILPIHSLRNVRAIDYDPLDKQLYWIDSRQNMIRKAQEDGS QGFTVVVSSVPSQNLEIQPYDLSIDIYSRYIYWTCEATNVINVTRLDGRSVGVVLKGEQDRPR AIVVNPEKGYMYFTNLQERSPKIERAALDGTEREVLFFSGLSKPIALALDSRLGKLFWADSDLR RIESSDLSGANRIVLEDSNILQPVGLTVFENWLYWIDKQQQMIEKIDMTGREGRTKVQARIAQLSDIHAVKELNLQEYRQHPCAQDNGGCSHICLVKGDGTTRCSCPMHLVLLQDELSCGESQ FQCASGQCIDGALRCNGDANCQDKSDEKNCEVLCLIDQFRCANGQCIGKHKKCDHNVDCS DKSDELDCYPTEEPAPQATNTVGSVIGVIVTIFVSGTVYFICQRMLCPRMKGDGETMTNDYVV HGPASVPLGYVPHPSSLSGSLPGMSRGKSMISSLSIMGGSSGPPYDRAHVTGASSSSSSSTK GTYFPAILNPPPSPATERSHYTMEFGYSSNSPSTHRSYSYRPYSYRHFAPPTTPCSTDVCDSDY APSRRMTSVATAKGYTSDLNYDSEPVPPPPTPRSQYLSAEENYESCPPSPYTERSYSHHLYPPP PSPCTDSS

[0586] SEQ ID N°:6 (human Reck) MATVRASLRGALLLLLAVAGVAEVAGGLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSES RLKHLLQRAPDYCPETMVEIWNCMNSSLPGVFKKSDGWVGLGCCELAIALECRQACKQASS KNDISKVCRKEYENALFSCISRNEMGSVCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVE NYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLI EGCKTQPLPQDPLWQCFLESSQSVHPGVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLY SMSWGNTQSWQEFDRFCEYNPVEVSMLTCLADVREPCQLGCRNLTYCTNFNNRPTELFRSC NAQSDQGAMNDMKLWEKGSIKMPFINIPVLDIKKCQPEMWKAIACSLQIKPCHSKSRGSIIC KSDCVEILKKCGDQNKFPEDHTAESICELLSPTDDLKNCIPLDTYLRPSTLGNIVEEVTHPCNP NPCPANELCEVNRKGCPSGDPCLPYFCVQGCKLGEASDFIVRQGTLIQVPSSAGEVGCYKICS CGQSGLLENCMEMHCIDLQKSCIVGGKRKSHGTSFSIDCNVCSCFAGNLVCSTRLCLSEHSS EDDRRTFTGLPCNCADQFVPVCGQNGRTYPSACIARCVGLQDHQFEFGSCMSKDPCNPNPC QKNQRCIPKPQVCLTTFDKFGCSQYECVPRQLACDQVQDPVCDTDHMEHNNLCTLYQRGKS LSYKGPCQPFCRATEPVCGHNGETYSSVCAAYSDRVAVDYYGDCQAVGVLSEHSSVAECAS VKCPSLLAAGCKPIIPPGACCPLCAGMLRVLFDKEKLDTIAKVTNKKPITVLEILQKIRMHVSVP QCDVFGYFSIESEIVILIIPVDHYPKALQIEACNKEAEKIESLINSDSPTLASHVPLSALIISQVQ VSSSVPSAGVRARPSCHSLLLPLSLGLALHLLWTYN

[0587] SEQ ID N°:7 (human Gprl24) / Adhesion G protein-coupled receptor A2 (ADGRA2) MGAGGRRMRGAPARLLLPLLPWLLLLLAPEARGAPGCPLSIRSCKCSGERPKGLSGGVPGPA RRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKITGLRNGSFLGLSLLEKLDLRNNIISTVQPG AFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRLNISGNIFSSLQPGVFDELPALKVVDLGTEF LTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSALHAQALGSLQEAQLCCEGALELHTHHLIPSL RQVVFQGDRLPFQCSASYLGNDTRIRWYHNRAPVEGDEQAGILLAESLIHDCTFITSELTLSH IGVWASGEWECTVSMAQGNASKKVEIVVLETSASYCPAERVANNRGDFRWPRTLAGITAYQ SCLQYPFTSVPLGGGAPGTRASRRCDRAGRWEPGDYSHCLYTNDITRVLYTFVLMPINASNA LTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKFLGYVDQIKELVEVMVDMASNLMLVDEHLL WLAQREDKACSRIVGALERIGGAALSPHAQHISVNARNVALEAYLIKPHSYVGLTCTAFQRRE GGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTTGRPNVSLSSFHIKNSVALASIQLPPSLFSS LPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSRPGAAGPGKRRGVATPVIFAGTSGCGVGNL TEPVAVSLRHWAEGAEPVAAWWSQEGPGEAGGWTSEGCQLRSSQPNVSALHCQHLGNVA VLMELSAFPREVGGAGAGLHPVVYPCTALLLLCLFATIITYILNHSSIRVSRKGWHMLLNLCFHI AMTSAVFAGGITLTNYQMVCQAVGITLHYSSLSTLLWMGVKARVLHKELTWRAPPPQEGDPA LPTPSPMLRFYLIAGGIPLIICGITAAVNIHNYRDHSPYCWLVWRPSLGAFYIPVALILLITWIYF LCAGLRLRGPLAQNPKAGNSRASLEAGEELRGSTRLRGSGPLLSDSGSLLATGSARVGTPGP PEDGDSLYSPGVQLGALVTTHFLYLAMWACGALAVSQRWLPRVVCSCLYGVAASALGLFVFT HHCARRRDVRASWRACCPPASPAAPHAPPRALPAAAEDGSPVFGEGPPSLKSSPSGSSGHPL ALGPCKLTNLQLAQSQVCEAGAAAGGEGEPEPAGTRGNLAHRHPNNVHHGRRAHKSRAKGHRAGEACGKNRLKALRGGAAGALELLSSESGSLHNSPTDSYLGSSRNSPGAGLQLEGEPMLT PSEGSDTSAAPLSEAGRAGQRRSASRDSLKGGGALEKESHRRSYPLNAASLNGAPKGGKYD DVTLMGAEVASGGCMKTGLWKSETTV

[0588] SEQ ID N°:9 (human Fzdl) MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWL LEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIP LCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALP PCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEF WTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYG LMYFGPEELRFSRTWIGIWSVLCCASTLFTVLTYLVDMRRFSYPERPIIFLSGCYTAVAVAYIAG FLLEDRVVCNDKFAEDGARTVAQGTKKEGCTILFMMLYFFSMASSIWWVILSLTWFLAAGMK WGHEAIEANSQYFHLAAWAVPAIKTITILALGQVDGDVLSGVCFVGLNNVDALRGFVLAPLFV YLFIGTSFLLAGFVSLFRIRTIMKHDGTKTEKLEKLMVRIGVFSVLYTVPATIVIACYFYEQAFRD QWERSWVAQSCKSYAIPCPHLQAGGGAPPHPPMSPDFTVFMIKYLMTLIVGITSGFWIWSG KTLNSWRKFYTRLTNSKQGETTV

[0589] Table 1. CDR1, CDR2 and CDR3 sequences of VHHs targeting LRP6 CDR1 CDR2 CDR3 VHH SIFMINTMA (SEQ ATIRPWSETTYA (SEQ CNAKRPWGTRDEYW (SEQ SEQ ID N°: ID N°: 30) ID N°: 27) ID N°: 24) 18 RIFAIYDIA (SEQ ID AMIRPWTEIDYA (SEQ CNAKRPWGSRDEYW (SEQ SEQ ID N°: N°: 17) ID N°: 16) ID N°: 15) 19 GTFSRYHMG (SEQ SAITWSGGRTYYA CALTWAPTPTN RRSDYAYW SEQ ID N°: ID N°: 31) (SEQ ID N°: 28) (SEQ ID N°: 25) 20 RIFAIYDMA (SEQ ATIRPWSETTYA (SEQ CNAKRPWGTRDEYW (SEQ SEQ ID N°: ID N°: 32)) ID N°: 27) ID N°: 24) 21 RIFAIYDIA (SEQ ID ATIRPWSETTYA (SEQ CNAKRPWGTRDEYW (SEQ SEQ ID N°: N°: 17) ID N°: 27) ID N°: 24) 22 RFSMYDMG (SEQ ASIRWSSGNTWYA CYANIYYTRRAPEEYW (SEQ SEQ ID N°: ID N°: 33) (SEQ ID N°: 29) ID N°: 26) 23

[0590]

[0591] Table 2. CDR1, CDR2 and CDR3 sequences of VHHs targeting LRP5 CDR1 CDR2 CDR3 VHH NINSIETLG (SEQ ANMRGGGYMKYA CYARTQRMGWNSYW (SEQ SEQ ID N°: ID N°: 43) (SEQ ID N°: 42) ID N°: 38) 34

[0592] SEQ ID N°: 43 SEQ ID N°: 42 CNAVTYDGY (SEQ ID N°: SEQ ID N°:

[0593] 39) 35

[0594] SEQ ID N°: 43 SEQ ID N°: 42 CAAQFRN DYG LRYQSTN N YW SEQ ID N°:

[0595] (SEQ ID N°: 40) 36

[0596] SEQ ID N°: 43 SEQ ID N°: 42 CNANYRGNRYW (SEQ ID N°: SEQ ID N°:

[0597] 41) 37

[0598]

[0599] SEQ ID N°:12 (anti-LRP VL) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQKKPGKKPKLLIYAASSLQSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGGGTKVEIK

[0600] SEQ ID N°:13 (anti-LRP VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMHWVRDAPGKGLEWVASISSTSGSKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYSCAKTYYDFWSGYYTFDYWGDGTLVTVS S

[0601] SEQ ID N°:14 (anti-LRP VHH) DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYAD SVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS

[0602] SEQ ID N°:18 (anti-LRP6 VHH) DVQLVESGGGLVQPGGSLRLSCAASGSIFMINTMAWYRQAPGNQRELVATIRPVVSETTYAD AVKGRFTISRDNAKNTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0603] SEQ ID N°:19 (anti-LRP6 VHH) DVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRHPPGNQRELVAMIRPVVTEIDYAD SVKGRFTISRNNAMKTVYLQMNNLKPEDTAVYYCNAKRPWGSRDEYWGQGTQVTVSS

[0604] SEQ ID N°:20 (anti-LRP6 VHH) EVQLVESGGGLVQAGGSLRVSCAASGGTFSRYHMGWFRQAPGKEREFVSAITWSGGRTYY ADFVKGRFTISRDDARNTVYLQMSSLKPEDTAVYYCALTWAPTPTNRRSDYAYWGQGTQVT VSS

[0605] SEQ ID N°:21 (anti-LRP6 VHH) EVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDMAWYRQAPGNQRELVATIRPVVSETTYAD AVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0606] SEQ ID N°:22 (anti-LRP6 VHH) AVQLVESGGGLVQAGGSLRLACAGSGRIFAIYDIAWYRQAPGNQRELVATIRPVVSETTYAD AVKGRFTISRSNAMKTVYLQMNSLKSEDTAIYYCNAKRPWGTRDEYWGQGTLVTVSS

[0607] SEQ ID N°:23 (anti-LRP6 VHH) AVQLVDSGGGLVQAGGSLRLSCAVSGRTFSMYDMGWFRQAPGKEREFVASIRWSSGNTWY ADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYANIYYTRRAPEEYWGQGTLVTVSSS

[0608] SEQ ID N°:34 (anti-LRP5 VHH)EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYYCYARTQRMGVVNSYWGQGTLVTVSS

[0609] SEQ ID N°:35 (anti-LRP5 VHH) QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTLYLQMNSLKPEDTAVYFCNAVTYDGYTIRGQGTLVTVSS

[0610] SEQ ID N°:36 (anti-LRP5 VHH) EVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAD SVQGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAQFRNDYGLRYQSTNNYWGQGTLVTV SS

[0611] SEQ ID N°:37 (anti-LRP5 VHH) QVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTVYLQMNSLKPEDSAVYYCNANYRGNRYWGQGTLVTVSS

[0612] SEQ ID N°: 44 (zGprl24 (25-742)) AGCPELFSSGCSCTEDRSKAHPTPGTRRKVSCGGKELTETPEVSLLPNRTVSLNLSNNRIRML KNGSFAGLSSLEKLDLRNNLISTIMPGAFLGLTALRKLDLSSNRIGCLTPEMFQGLTNLTKLNI SGNIFSSLDPNVFMELHSLKLVNFHSEFLSCDCGLRWVPSFFRSGSARLGDETLCAYPRRLQN KPLRLLRESDLSCEGPLELHTLSLLPSQRQVVFKGDRLPFHCTASLVDKITALHWRQNGQPVT SDPTKGIHLEESVQHDCTFITSELILSNVHVEASGEWECVVSTGRGNTSCSVEIVVLENSASF CPEQKVNNNRGEFRWPRTLAGITSYQHCLQLRYPSLTLGGGVEQKKASRNCDRSGRWEEAD YSQCLYTN DITRI LHTFI LM PVN ASN AVTLAHQVRSYTLEAAG FTDTVDVLYVAQM M H KFM DY VTELRELSEVLVEMGSNLMQVDDQILARAQREERACSSIVYTLETLAWPQLHSHAQDLSRYS RNIVMEAHLIRPAHFTGISCTVYQRREGAAGSQVHDGADLSLEQQLRFRCTTGTHNTSLNAF HLKNAVALATVSLPATLFPPNAPPDCKLQFVAFRNGRFFPFTSNFTGHSDLARRRGISTPVIYA GLDGCSMWNQSDPIIVSLRHTSPGHDPVAAHWNSQALGHHGSWSLDGCQLIHSDVSISTL RCSVLSNYAVLQEIPDFPGSPSIPVEVLHP

[0613] SEQ ID N°: 45 (hADAMTSlO (823-1103)) SLPPYSWHYAPWTKCSAQCAGGSQVQAVECRNQLDSSAVAPHYCSAHSKLPKRQRACNTE PCPPDWVVGNWSLCSRSCDAGVRSRSVVCQRRVSAAEEKALDDSACPQPRPPVLEACHGPT CPPEWAALDWSECTPSCGPGLRHRVVLCKSADHRATLPPAHCSPAAKPPATMRCNLRRCPPA RWVAGEWGECSAQCGVGQRQRSVRCTSHTGQASHECTEALRPPTTQQCEAKCDSPTPGD GPEECKDVNKVAYCPLVLKFQFCSRAYFRQMCCKTCHGH

[0614] SEQ ID N°: 46 (hWnt7a linker (171-199)) VEPVRASRNKRPTFLKIKKPLSYRKPMDT

[0615] SEQ ID N°: 47 (hReck CK1-5 (27-340)) GLAPGSAGALCCNHSKDNQMCRDVCEQIFSSKSESRLKHLLQRAPDYCPETMVEIWNCMNS SLPGVFKKSDGWVGLGCCELAIALECRQACKQASSKNDISKVCRKEYENALFSCISRNEMGS VCCSYAGHHTNCREYCQAIFRTDSSPGPSQIKAVENYCASISPQLIHCVNNYTQSYPMRNPTDSLYCCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQCFLESSQSVHP GVTVHPPPSTGLDGAKLHCCSKANTSTCRELCTKLYSMSWGNTQSWQEFDRFCEYNPVEVS MLTCLA

[0616] SEQ ID N°: 48 (Wnt7b linker)

[0617] VEVVRASRLRQPTFLRLKQLRSYQKPMET

[0618] SEQ ID N°: 49 (CK4 motif RECK) CCDRAEDHACQNACKRILMSKKTEMEIVDGLIEGCKTQPLPQDPLWQC

[0619] SEQ ID N°: 50 (CK5 motif RECK)

[0620] CCS KANTSTC RE LCTKLYS M S WG NTQS WQE FD RFCEYN PVE VS M LTC

[0621] SEQ ID N°: 51 (RECK binding domain) HVEPVRASRNKRPTFLKIKKPLSYRKPMDTDLVYIEKSPNYC

[0622] SEQ ID N°: 52 (RECK binding domain) YEWRASRLRQPTFLRIKQLRSYQKPMET

[0623] SEQ ID N°: 53 (ANTI-LRP5 VHH) DVQLVESGGGLVQPGGSLRLSCTSSANINSIETLGWYRQAPGKQRELIANMRGGGYMKYAG SLKGRFTMSTESAKNTMYLQMNSLKPEDTAVYYCYVKLRDDDYVYRGQGTQVTVSS

[0624] SEQ ID N°: 54 (RECK binding domain) XXXVXAXRXXXXXFLXIXXXXXYXKXXXX

[0625] SEQ ID N°: 55 (RECK binding domain)

[0626] VXAXRXXXXXFLXIXXXXXYXK

[0627] SEQ ID N°: 56 (RECK binding domain) XXXVXAXRXXXXXFLXXXXXXXXXKXXXX

[0628] SEQ ID N°: 57 (RECK binding domain)VXAXRXXXXXFLXXXXXXXXXK

[0629] SEQ ID N°: 58 (N-Terminal Domain Gprl24) APGCPLSIRSCKCSGERPKGLSGGVPGPARRRVVCSGGDLPEPPEPGLLPNGTVTLLLSNNKI TGLRNGSFLGLSLLEKLDLRNNIISTVQPGAFLGLGELKRLDLSNNRIGCLTSETFQGLPRLLRL NISGNIFSSLQPGVFDELPALKVVDLGTEFLTCDCHLRWLLPWAQNRSLQLSEHTLCAYPSAL HAQALGSLQEAQLCCEGALELHTHHLIPSLRQVVFQGDRLPFQCSASYLGNDTRIRWYHNRA PVEGDEQAGILLAESLIHDCTFITSELTLSHIGVWASGEWECTVSMAQGNASKKVEIVVLETS ASYCPAERVANNRGDFRWPRTLAGITAYQSCLQYPFTSVPLGGGAPGTRASRRCDRAGRWE PGDYSHCLYTNDITRVLYTFVLMPINASNALTLAHQLRVYTAEAASFSDMMDVVYVAQMIQKF LGYVDQIKELVEVMVDMASNLMLVDEHLLWLAQREDKACSRIVGALERIGGAALSPHAQHIS VNARNVALEAYLIKPHSYVGLTCTAFQRREGGVPGTRPGSPGQNPPPEPEPPADQQLRFRCTT GRPNVSLSSFHIKNSVALASIQLPPSLFSSLPAALAPPVPPDCTLQLLVFRNGRLFHSHSNTSR PG AAG PG KRRG VATPVI FAGTSGCG VG N LTE PVAVSLRH WAEG AE PVAAW WSQEG PG E AG GWTSEGCQLRSSQPNVSALHCQHLGNVAVLMELSAFPREVGGAGAGLHP

[0630] LRP6 Sequences

[0631] ID VHH CDRH1 CDRH2 CDRH3

[0632] 01B01 QVQLQESGGGLVQPGGSLRLS DYDMS TISWNGAGTYYAES YAGNTIATSKSTYDY CAASG FTFD DYD MSWVRQAPG (SEQ ID N°: VKG (SEQ ID N°: (SEQ ID N°: 85) KGLEWLSTISWNGAGTYYAESV 77) 81)

[0633] KGRFTISRDNTKNTLYLQMNSLK PDDTAVYYCAAYAGNTIATSKSTY DYWGQGTQVTVSS (SEQ ID N°:

[0634] 59)

[0635] 01E12 QVQLVESGGGLVQAGGSLSLS DFGMA SINWSGSSTYYED RHSNRVKEYDD (SEQ CVASGRNFDDFGMAWFRQAP (SEQ ID N°: SVKG (SEQ ID N°: ID N°: 86) GKDREFVASINWSGSSTYYEDS 78) 82)

[0636] VKGRFTISRDNAKNTVYLQMNS LKSEDTAVYYCVRRHSNRVKEY DDWGQGTQVTVSS (SEQ ID

[0637] N°: 60)

[0638] 01F11 QVQLVESGGGLVQPGGSLRLS NHAIG (SEQ SIGNSDGSTYYADS QSMMINRNY (SEQ ID CTASGFSLDNHAIGWFRQAPG ID N°: 79) VKG (SEQ ID N°: N°: 87) KEREWVSSIGNSDGSTYYADSV 83)

[0639] KGRFTISRDNAKNALYLQMNSL KPDDTAVYYCNAQSMMINRNY WGQGTQVTVSS (SEQ ID N°:

[0640] 61)

[0641] 02G02 QLQLVESGGGLVQPGGSLRVS GNNMG EITYRGNTNYADSV RSRNLIRNY (SEQ ID CVASGRPFSGNNMGWYRQAP (SEQ ID N°: KG (SEQ ID N°: 84) N°: 88) GKQREWIAEITYRGNTNYADSV 80)

[0642] KGRFIISRDNAKNTVYLQMNSLK PEDTAVYYCNARSRNLIRNYWG

[0643]

[0644] QGTQVTISS (SEQ ID N°: 62)

[0645] GPR124 sequences

[0646] CD CD CD FR FR FR FR CD CD CD FRL FRL FRL FRL ID VH RH RH RH VL

[0647] Hl H2 H3 H4 RL1 RL2 RL3 1 2 3 4 1 2 3

[0648]

[0649] A10 GV PD RLT QAVLTQP RFS QV ISV PSASGTP GS QVQLQESG QL DTS GQRVTIS KS PGLVKPSET QE RS CSGSSS QA GT LSLTCTVSG SG QF NIGSNYV VLT SAS GSISGSSYY PG SLK YWYQQL QP WY LAI WSWIRQPP NIY LVK Wl LSS PGTAPKL AA PSA QQ SG GRGLEWIG YS PSE RQ VTA WG LIYRNNQ SG WD SG LP LRS NIYYSGSTY GS LLV TLS pp AD QG RPSGVPD SSS RN DS TP GT ED FG YNPSLKSRL TYY QG LTC GR TAV TM RFSGSKS NIG NQ LS GQ AP EA GG TISVDTSRS GS NP VD TVS GL YY VTV GTSASLAI SN RP GV RVT KLL DY TKL QFSLKLSSV SYY SLK AF GG EWI CA SS SGLRSED YVY S V ISC IY YC TVL TAADTAVYY WS S DI SIS G R (SE EADYYCA (SE (SE (SE (SE (SE (SE (SE CARLLVQG (SE (SE (SE (SE (SE (SE Q AWDDSL Q Q Q Q Q Q Q VDAFDIWG Q Q Q Q Q Q ID SGWFG ID ID ID ID ID ID ID QGTMVTVS ID ID ID ID ID ID N°: GGTKLTV N°: N°: N°: N°: N°: N°: N°: S (SEQ ID N°: N°: N°: N°: N°: N°: 102 L (SEQ ID 103 105 107 110 113 115 117 N°: 63) 89) 91) 93) 95) 97) 99) ) N°: 64) ) ) ) ) ) ) ) C03 GV PD RLT RFS ISV SYVLTQP GS QV DT PSASGTP KS QVQLQESG QL SR GQRVTIS GS PGLVKPSET QE NQ CSGSSS SYV SAS LSLTCTVSG SG FSL NIGSNYV LTQ LAI GSISGSSYY PG KLS YWYQQL PPS WY SG WSWIRQPP NIY LVK Wl SVT PGTAPKL AA AS QQ LQ GKGLEWIG YS PSE RQ AA WG LIYRNNQ SG WD GT LP SE NIYYSGSTY GS LLV TLS PP DT QG RPSGVPD SSS RN DS PG GT DE FG YNPSLKSRL TYY QG LTC GK AVY TM RFSGSKS NIG NQ LN QR AP AD GG TISVDTSRN GS NP VD TVS GL YC VTV GSSASLA SN RP GV VTI KLL YY TKL QFSLKLSSV SYY SLK AF GG EWI AR SS ISGLQSE YVY S V SC IY C TVL TAADTAVYY WS S DI SIS G (SE (SE DEADYYC (SE (SE (SE (SE (SE (SE (SE CARLLVQG (SE (SE (SE (SE (SE Q Q AAWDDS Q Q Q Q Q Q Q VDAFDIWG Q Q Q Q Q ID ID LNGWFG ID ID ID ID ID ID ID QGTMVTVS ID ID ID ID ID N°: N°: GGTKLTV N°: N°: N°: N°: N°: N°: N°: S (SEQ ID N°: N°: N°: N°: N°: 100 102 L (SEQ ID 103 105 108 111 113 116 117 N°: 65) 89) 91) 93) 95) 98) ) ) N°: 66) ) ) ) ) ) ) ) A05 GV RVT PD QV MS SYELTQP RFS QL VD PSASGTP GS QVQLQESG QE TSK GQRVTIS KS PGLVKPSET SG NQ CSGSSS SYE GT LSLSCTVSG PG FSL NIGTNYV LTQ SAS DSISSRSHY LVK NL YWYQQL PPS WY LAI WGWIRQP TIY PSE Wl SSV PGMAPKL AT AS QQ SG PGKGLEWI YS TLS RQ TAA WG LIYSDDQ SG WD GT LP LRS GTIYYSGNT GN LR LS pp DT QG RPSGVPD SSS SD DS PG GM ED FG YYNPSLKSR SR TYY GS CT GK AVY TM RFSGSKS NIG DQ LS QR AP EA GG VTMSVDTS SH NP VD VS GL FC VTV GTSASLAI TNY RP GV VTI KLL DY TKL KNQFSLNL YW SLK AF GD EWI Al SS SGLRSED VY S V SC IY YC TVL SSVTAADTA G S DI SIS G (SE (SE EADYYCA (SE (SE (SE (SE (SE (SE (SE VYFCAILRG (SE (SE (SE (SE (SE Q Q TWDDSL Q Q Q Q Q Q Q SVDAFDIW Q Q Q Q Q ID ID SGWFG ID ID ID ID ID ID ID GQGTMVTV ID ID ID ID ID N°: N°: GGTKLTV N°: N°: N°: N°: N°: N°: N°: SS (SEQ ID N°: N°: N°: N°: N°: 101 102 L (SEQ ID 104 106 109 112 114 115 117

[0650]

[0651] N°: 67) 90) 92) 94) 96) 98) ) ) N°: 68) ) ) ) ) ) ) )Reck sequences

[0652] CD CD CD FR FR FR FR CD CD CD FRL FRL FRL FRL ID VH RH RH RH VL

[0653] Hl H2 H3 H4 RL1 RL2 RL3 1 2 3 4 1 2 3

[0654] 01A08 RLT GV QL MS PS QL VD RFS QLQLQESG QE TSK DIVMTQS GS PGRVKPSE SG NQ PSSLSAS GS TLSLTCKVS PG FSL VGDRVTI DIV GT GFSISRGYY RV KM TCRASQS MT DFT WAWVRQA KPS WV RS ISRYLNW QS WY LTI PGKGLEWI NIH AFY ETL RQ VTA YQQKPG PSS QQ SSL GNIHHSGS HS GS SLT AP AD WG KAPKLLM RA LSA KP QP TYYSPSLKS GS GN CK GK TAV QG YAASSLQ SQ QQ SV GK ED FG RLTMSVDT RG TYY YYV VS GL YF TTV SGVPSRF SIS AA SYS GD AP FAT GG SKNQFSLK YY SPS TAL GF EWI CA IVS SGSGSGT RYL SSL TPL RVT KLL YY TKV MRSVTAAD WA LKS DV SIS G R S DFTLTISS N QS T ITC MY C EIK TAVYFCARA (SE (SE (SE (SE (SE (SE (SE LQPEDFA (SE (SE (SE (SE (SE (SE (SE FYGSGNYY Q Q Q Q Q Q Q TYYCQQS Q Q Q Q Q Q Q VTALDVWG ID ID ID ID ID ID ID YSTPLTF ID ID ID ID ID ID ID QGTTVIVSS N°: N°: N°: N°: N°: N°: N°: GGGTKVE N°: N°: N°: N°: N°: N°: N°: (SEQ ID N0: 118 122 126 130 134 138 142 IK (SEQ 146 150 154 158 162 165 168 69) ) ) ) ) ) ) ) ID N°: 70) ) ) ) ) ) ) ) 01F11 RFT GV EV ISR PS QL DN RFS EVQLVESG VES SN DIQLTQS GS GALVQPGG GG NR PSSLSAS GS SLRLSCAAS ALV LYL VGDRVTI DIQ GT GFTVSSNY AEL QP QM TCRASQ LTQ DFT MSWVRQA VIY LPF GG WV NS GISSYLA SPS WY LTI PGKGLEWV SG RV SLR RQ LRA WYQQKP SLS QQ SG SVIYSGGST GS GV LS AP EDT WG GKAPKLLI RA AS KP LQ YYAGSVKG TYY VIS CA GK GV QG YAASTLQ SQ QQ VG GK AE FG RFTISRDNS SN AG KYY AS GL YY TTV SGVPSRF GIS AA SYS DR AP DF QG NNRLYLQM YM SVK GL GFT EW CA TVS SGSGSGT SYL STL SPI VTI KLL ATY TRL NSLRAEDT S G DV VS VS R S DFTLTISG A QS T TC IY YC DIK GVYYCARA (SE (SE (SE (SE (SE (SE (SE LQAEDFA (SE (SE (SE (SE (SE (SE (SE ELLPFRVGV Q Q Q Q Q Q Q TYYCQQS Q Q Q Q Q Q Q VISKYYGLD ID ID ID ID ID ID ID YSSPITFG ID ID ID ID ID ID ID VWGQGTTV N°: N°: N°: N°: N°: N°: N°: QGTRLDI N°: N°: N°: N°: N°: N°: N°: TVSS (SEQ 119 123 127 131 135 139 143 K (SEQ ID 147 151 155 159 163 166 169 ID N°: 71) ) ) ) ) ) ) ) N°: 72) ) ) ) ) ) ) ) 03A04 EVQLVESG EV RFT GV GGLVQPG NS QL ISR DIQMTQS DIQ PS GSLRLACTT GR VES DN PSSLSAS MT RFS SGFTFSNY SIIA TIF GG WV SN AGDRVTI QS WY GS AMSWVRQ SD GV GL RQ DTL TCRASQS PSS QQ GS TPGGRLEW VST VMI VQ TP YL WG INTYLNW RA LSA KP GT VSSIIASDV YYA GE PG GG QM QG YQQKPG SQ AA SA GK DFT FG STYYADSVR NY DS YG GS RLE HS TLV KAPKLLIY SIN SN HQ GD AP LTI QG GRFTISRDN AM VR FD LRL WV LR TVS AASNLQS TYL LQ TYT RVT KLL SSL TKL SNDTLYLQ S G S AC S DE S GVPSRFS N S THT ITC IY QP EIK MHSLRDED (SE (SE (SE TTS (SE DT (SE GSGSGT (SE (SE (SE (SE (SE ED (SE TAVYFCAK Q Q Q GFT Q AVY Q DFTLTISS Q Q Q Q Q FAT Q NSGRTIFGV ID ID ID FS ID FC ID LQPEDFA ID ID ID ID ID YY ID VMIGEYGF N°: N°: N°: (SE N°: AK N°: TYYCHQT N°: N°: N°: N°: N°: C N°: DSWGQGT 120 124 128 Q 136 (SE 144 YTTHTFG 148 152 156 160 163 (SE 170

[0655]

[0656] LVTVSS ) ) ) ID ) Q ) QGTKLEI ) ) ) ) ) Q )(SEQ ID N0: N°: ID K (SEQ ID ID 73) 132 N°: N°: 74) N°:

[0657] ) 140 165 ) ) 07B05 RVT GIP QV ISV QSVLTQP DR QVQLVESG QL DM PSVSAAP FTG PGLVKPSQ VES SK GQHVTIS QS SKS TLSLTCTVS GP NQ CSGSSS VLT GT GGSISSGD GL FSL NVGKNF QP SAT YYWSWIRQ YIY VKP Wl KLS VSWYQQ PSV WY LGI HPGKGLE YS SQ RQ SVT LPGTVPK SG SA QQ TGL WIGYIYYSG GS ERL TLS HP AA WG LLIYDNN SSS GT AP LP QT STYHNPSL SG TYH KG LTC GK DT QG RRPSGIP NV DN WD GQ GT GD FG KSRVTISVD DY NP LD TVS GL AVY TM DRFTGSK GK NR TSL HV VPK EA GG MSKNQFSL YW SLK AF GG EWI YC VTV SGTSATL NF RP SA TIS LLI DY TRL KLSSVTAAD S S DI SIS G AR SS GITGLQT VS S W C Y YC TVL TAVYYCARE (SE (SE (SE (SE (SE (SE (SE GDEADYY (SE (SE (SE (SE (SE (SE (SE RLKGLDAF Q Q Q Q Q Q Q CGTWDT Q Q Q Q Q Q Q DIWGQGT ID ID ID ID ID ID ID SLSAWF ID ID ID ID ID ID ID MVTVSS N°: N°: N°: N°: N°: N°: N°: GGGTRLT N°: N°: N°: N°: N°: N°: N°: (SEQ ID N0: 121 125 129 133 137 141 145 VL (SEQ 149 153 157 161 164 167 171

[0658]

[0659] 75) ) ) ) ) ) ) ) ID N°: 76) ) ) ) ) ) ) )

[0660] SEQ ID N°: 172 (5-mer linker)

[0661] GSGGS

Claims

noCLAIMS1. A pharmaceutical composition comprising a therapeutically active amount of a soluble antibody or antibody fragment, or a nucleic acid encoding for said soluble antibody or antibody fragment, for use in the treatment and / or prevention of epilepsy or seizure in a patient, wherein said antibody or antibody fragment is a modulator of the Wnt7 signaling pathway, preferably an agonist of the Wnt7 signaling pathway, and wherein said antibody or antibody fragment comprises one or more Lipoprotein Receptor- related Protein (LRP) binding domains, and further one or more Gprl24 and / or Reck binding domains.

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

3. The pharmaceutical composition for use according to claim 1 or 2, wherein said antibody or antibody fragment is tetravalent, said antibody or antibody fragment preferably comprises:i) two regions that each specifically bind to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes; and ii) two regions that each specifically bind to a set of one or more Reck or Gprl24 epitopes.

4. The pharmaceutical composition for use according to claim 1 or 2, wherein said antibody or antibody fragment is bivalent, said antibody or antibody fragment preferably comprises:i) one region that specifically binds to a set of one or more LRP epitopes, preferably a set of one or more LRP epitopes such as LRP5 and / or LRP6 epitopes; andii) one region that specifically binds to a set of one or more Reck or Gprl24 epitopes.

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

6. The pharmaceutical composition for use according to any one of the previous claims, wherein the one or more LRP binding domains are LRP5 and / or LRP6 binding domains, and wherein said LRP6 binding domains comprise:i) a CDR3 region as depicted in SEQ ID N°: 85, 86, 87, or 88 or consisting of an amino acid sequence with maximally two amino acids different from SEQ ID N°: 85, 86, 87, or 88,Illii) a CDR.2 region as depicted in SEQ ID N°: 81, 82, 83, or 84, and / or iii) a CDR1 region as depicted in SEQ ID N°: 77, 78, 79, or 80; preferably wherein said antibody or antibody fragment comprises an anti-LRP VHH comprising a sequence according to SEQ ID N° 59, 60, 61, or 62.

7. The pharmaceutical composition for use according to any one of the previous claims, wherein said antibody or antibody fragment comprises an anti-Gprl24 IgG or scFv comprising a VH sequence according to SEQ ID N° 63, 65, or 67, and a VL sequence according to SEQ ID N° 64, 66, or 68, optionally separated by a linker sequence; preferably a VH sequence according to SEQ ID N° 63 and a VL sequence according to SEQ ID N° 64, VH sequence according to SEQ ID N° 65 and a VL sequence according to SEQ ID N° 66, or a VH sequence according to SEQ ID N° 67 and a VL sequence according to SEQ ID N° 68.

8. The pharmaceutical composition for use according to any one of the previous claims, wherein said antibody or antibody fragment comprises anti-Reck IgG or scFv comprising a VH sequence according to SEQ ID N° 69, 71, 73, or 75, and a VL sequence according to SEQ ID N° 70, 72, 74, or 76, optionally separated by a linker sequence; preferably comprising a VH sequence according to SEQ ID N° 69 and a VL sequence according to SEQ ID N° 70, a VH sequence according to SEQ ID N° 71 and a VL sequence according to SEQ ID 72, a VH sequence according to SEQ ID N° 73 and a VL sequence according to SEQ ID N° 74, or a VH sequence according to SEQ ID N° 75 and a VL sequence according to SEQ ID N° 76.

9. The pharmaceutical composition for use according to any one of the previous claims, wherein said antibody or antibody fragment does not comprise a Fzd binding domain, and / or is not able to find to Fzd.

10. Pharmaceutical composition for use according to any one of the previous claims, wherein said epilepsy is generalized epilepsy.

11. Pharmaceutical composition for use according to any one of the previous claims 1-9, wherein said epilepsy is focal epilepsy, preferably focal epilepsy chosen from temporal lobe epilepsy (TLE) and non-TLE focal epilepsy.

12. Pharmaceutical composition for use according to any one of the previous claims 1-9, wherein said patient exhibits one or more symptoms of epilepsy, including epileptic seizure; preferably wherein said epilepsy is generalized epilepsy and wherein said patient exhibits one or more symptoms of epilepsy including generalized epileptic seizure, or wherein said epilepsy is focal epilepsy and wherein said patient exhibits one or more symptoms of epilepsy including focal epileptic seizure.

13. Pharmaceutical composition for use according to claim 12, wherein said epileptic seizure is recurrent epileptic seizure that is refractory to treatment.

14. Pharmaceutical composition for use according to any one of the previous claims, wherein said pharmaceutical composition is administered to said patient, wherein said administration comprises one or more doses of between 1 and 10 mg of antibody or antibody fragment in said pharmaceutical composition / kg body weight of said patient.

15. Pharmaceutical composition for use according to any one of the previous claims, wherein said pharmaceutical composition is administered to said patient, and wherein administration of the pharmaceutical composition reduces seizure severity, as measured by reduction in Racine stage, preferably by at least one stage.

16. Pharmaceutical composition for use according to any one of the previous claims 1-9, wherein said epilepsy is focal epilepsy, preferably temporal lobe epilepsy, preferably induced using the kainic acid model, and wherein a dose of said pharmaceutical composition is administered to a patient, wherein said dose comprises between 4 and 6 mg of antibody or antibody fragment / kg body weight of the patient, wherein said administration reduces seizure severity, as measured by reduction in Racine score (stage), preferably by at least one stage.

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