Antibody for treating brain tumours and epilepsy correlated thereto

Abstract

The present invention relates to the use of a direct antibody, with a high degree of affinity and specificity, against the extracellular domain of the hemichannels formed by connexin 26 (Cx26, encoded by GJB2), connexin 30 (Cx30, encoded by GJB6) and / or connexin 32 (Cx32, encoded by GJB1), to treat brain tumours and epilepsy correlated thereto or to reduce or inhibit the development thereof.

Description

[0001] ANTIBODY FOR TREATING BRAIN TUMOURS AND EPILEPSY CORRELATED

[0002] THERETO

[0003] The present invention relates to an antibody for treating brain tumours and epilepsy correlated thereto. In particular, the invention relates to the use of a direct antibody, with a high degree of affinity and specificity, against the extracellular domain of the hemichannels formed by connexin 26 (Cx26, encoded by GJB2), connexin 30 (Cx30, encoded by GJB6) and / or connexin 32 (Cx32, encoded by GJB1 ), to treat brain tumours and epilepsy correlated thereto or to reduce or inhibit the development thereof.

[0004] It is well known that brain tumours represent one of the most complex and devastating pathologies, characterised by the uncontrolled growth of anomalous cells within brain tissue. These tumours can be primary, originating directly in the brain, or metastatic, deriving from other parts of the body.

[0005] In this context, it is well known that gliomas with a high degree of malignity represent the main cause of death due to brain tumours both in children and in adults.

[0006] Grade IV gliomas are prevalently glioblastomas (GBMs), highly undifferentiated malignant tumours with a very aggressive progression

[0001] ,

[0007] Glioma stem cells (GSCs) are a subpopulation of cells within gliomas which possess characteristics typical of stem cells, including the ability to self-renew and to differentiate [2, 3], It is believed that these cells play a decisive role in the onset, progression and recurrence of gliomas, including GBMs. In particular, GSCs differentiate into endothelial cells, thus supporting tumour growth through angiogenesis [2], and their resistance to chemotherapy treatment has been demonstrated [3],

[0008] The available data indicate that a vicious cycle is established between glioma cells and adjacent neurons and this promotes an increase in neuronal activity, which in turn favours tumour growth with the consequent destruction of healthy brain tissue [4], The presence of groups of mutually interconnected glioma cells that can migrate together increases the likelihood of recurrence and makes it difficult to remove the tumour mass by surgical resection [5, 6],

[0009] There are known standard therapeutic treatments, which include surgical resection, chemotherapy (mainly with temozolomide) and radiotherapy. However, due to the invasiveness and the poor response to standard treatments, the average survival time of patients with GBM is only 12-16 months, with a 10-year survival rate of 0.17% [7],

[0010] It is likewise known that the interaction between astrocytes and microglia within the tumour mass mediates the transcriptional reprogramming of microglia and myeloid cells towards an anti-inflammatory phenotype and promotes the creation of an overall immunosuppressive microenvironment [8] that may potentially hinder immunotherapy-based therapeutic developments [9],

[0011] The combined treatment with electrical fields (“Tumour Treating Fields” - TTF) and temozolomide, carried out in the context of a phase III clinical trial, represented a breakthrough in the treatment of GBM. However, the median survival time only increased from 16 to 21 months

[0010] ,

[0012] On 19 May 2024, the Clinical Trial Finder of the National Brain Tumor Society (https: / / braintumor.org) reported 449 ongoing trials. However, despite the considerable commitment, the overall survival rate for patients with GBM 5 years after diagnosis is still less than 5%

[0011] ,

[0013] It is also known that GBM can influence normal brain function by invading and damaging normal brain tissue, disrupting normal electrical activity in the brain and potentially leading to epileptic seizures, which are not only a common comorbidity of GBM, but may also be an initial clinical symptom of this cancerous entity.

[0014] In the light of the above, it appears evident that there is a need to provide new therapies for treating brain tumours, in particular gliomas and glioblastomas.

[0015] The solution according to the present invention fits into this context; it aims to provide an antibody for treating brain tumours and the epilepsy correlated thereto.

[0016] According to the present invention, it has now been found that an antibody capable of binding and inhibiting the hemichannels formed by connexins is capable of acting to reduce or inhibit the tumour and the epilepsy correlated thereto upon administration of the antibody itself or of a vector that encodes and expresses that antibody.

[0017] Connexins (Cxs) are a family of 21 transmembrane proteins that represent essential elements in the cell-to-cell communication system, contribute to regulating cell proliferation and survival and have been extensively studied in relation to cancer [12, 13], including malignant gliomas

[0014] , Table 1 shows the connexins of Homo Sapiens'.

[0018] Table 1

[0019] In their typical function, Cxs self-assemble to form hexameric membrane structures, called connexons or hemichannels (HCs), which can in turn form gap junctions (GJs) through the head-to-head binding of two adjacent HCs.

[0020] GJs enable a direct communication between the cytoplasm of adjacent cells, while non-joined HCs mediate the exchange of molecules between the cell cytoplasm and the extracellular environment.

[0021] Since Cxs perform a vast range of functions, which may further vary based on the type of tumour and the experimental conditions, to date the mechanism whereby Cxs suppress or promote neoplastic progression is not exactly known

[0012] , In this regard, the data available in the literature indicate that:

[0022] - Cx26 is a negative prognostic indicator of survival when overregulated in various types of tumours

[0015] , including biopsy samples of human glioma

[0016] ;

[0023] - Cx43 is increased in glioma-associated astrocytes, in particular in the peritumoural region, where it contributes to the dissemination of glioma cells [17, 18];

[0024] - glioma stem cells (GSCs) express larger quantities of Cx46 compared to non-stem tumour cells, which represents a necessary and sufficient condition for maintaining GSCs [19, 20],

[0025] Cx HCs, too, have a role in carcinogenesis and tumour progression, since:

[0026] - Cx HCs mediate the release of signal molecules, such as ATP, which promote cell motility and the establishment of an invasive microenvironment

[0021] ;

[0027] - Cx HCs modulate the interactions between tumour cells and their microenvironment, with effects on the immune response, on the remodelling of the extracellular matrix and on other factors which influence tumour progression [22, 23];

[0028] - the role of Cx HCs in the invasion by the tumour mass has been demonstrated in a model of breast cancer

[0024] ;

[0029] - Cx HCs can promote the formation of new blood vessels both by facilitating the release of pro-angiogenic factors and mediating the Ca2+signalling pathway associated with the migration of endothelial cells [25, 26], whereas the genetic reduction of endothelial connexins compromises angiogenesis

[0027] and inhibits tumour growth

[0028] ;

[0030] - epilepsy often develops in patients with brain tumours, and the two conditions share common pathogenetic mechanisms

[0029] ;

[0031] - Cx HCs play an important role in hyper-excitability and the onset of epilepsy in mouse models of epilepsy / convulsions [30-32] through the deregulation of Ca2+-dependent glutamatergic signalling [29, 32-34],

[0032] According to the present invention it has now surprisingly been found that an antibody capable of inhibiting Cx26, Cx30 and Cx32 HCs is effective in animal models of glioma and in epilepsy associated therewith.

[0033] In particular, according to the present invention the effectiveness of the abEC1.1 antibody, which specifically binds an extracellular epitope conserved in HCs formed by the connexins Cx26, Cx30 and Cx32, has been tested against the brain tumours.

[0034] The abEC1.1 antibody has already been described in patent applications WO2017128880A1 and US2022251179A1 (WO2020237491A1 ). In particular, WO201 7128880A1 would seem to suggest the use of the antibody to treat deafness, dermatitis or tumours caused by mutations in connexin 26; however, the experimental part of the document focuses on the characterisation of the antibody itself and on the inhibitory effect of the antibody on Cx26 in cochlear tissue. The document US2022251179A1 (WO2020237491 A1 ) focuses instead on the use of the antibody against ectodermic dysplasia.

[0035] Table 2 shows the amino acid sequences of the specific epitope bound by the abEC1 .1 antibody with reference to the human Cx26 protein sequence, but the epitope is the same in Cx30 and Cx32:

[0036] Table 2

[0037] The abovementioned epitope was deduced from in silico simulations based on the crystallographic structures of both the target (PDB ID: 2ZW3) and the antibody (PDB ID: 5WYM) and validated by mutagenesis experiments [35, 36], Furthermore, abEC1.1 :

[0038] - inhibits the Cx26, Cx30 and Cx32 HCs with ICso in the range of nM [35, 37, 38];

[0039] - inhibits the uptake of Ca2+and release of ATP through the Cx HCs [35-41 ] , with implications of vast scope for the modulation of the tumour microenvironment and the containment of tumour growth [42-45];

[0040] - it is effective in vivo, in mouse models of pathologies correlated to Cx HCs [39, 40];

[0041] - inhibits Cx HCs in a solid tumour, as demonstrated by in vivo experiments conducted on a mouse model of melanoma

[0041] ,

[0042] According to the present invention, an anti-proliferative effect in brain tumours using an antibody having hemichannels formed by connexins as its target, in particular connexins Cx26, Cx30 and Cx32, has been demonstrated for the first time.

[0043] In particular, according to the present invention it has been found that the abEC1 .1 antibody is capable of providing, both in vivo and in vitro:

[0044] - an inhibition of the invasiveness of the glioma;

[0045] - a reduction in the expression of GFAP, Ki-67 and phospho-histone H3;

[0046] - an increase in Iba1 ;

[0047] - an attenuation of the epileptiform activity induced by the glioma; and

[0048] - prolonged survival in animal models.

[0049] These results on brain tumours and epilepsy correlated thereto were not predictable on the basis of the already known effects of the antibody and thus represent a surprising result.

[0050] In greater detail, according to the present invention, said antibody was administered in mice via recombinant adeno-associated viral vectors (AAVs) encoding for the antibody itself in an scFv-mFc format comprising a variable singlechain fragment (scFv) linked to a crystallisable fragment (Fc), i.e. a mouse crystallisable fragment (mFc); said vectors were administered by intracerebroventricular (ICV) or retro-orbital injection. The antibody was also administered in mice as such, in an immunoglobulin G1 (lgG1 ) format (in this case as well with mFc), by convection enhanced delivery (CED). CED provides for direct infusion of therapeutic agents into brain tissue under positive pressure, enabling a wide distribution in the area of interest.

[0051] Based on the results obtained according to the present invention, as reported further below, it is possible to observe that ICV administration with a recombinant vector encoding the antibody in mice with glioma surprisingly resulted in:

[0052] - a diffuse expression of the antibody in the brain; - a considerable reduction in the volume of the gliomas compared to untreated mice;

[0053] - a lower tumour invasiveness compared to untreated mice;

[0054] - the absence of necrotic areas;

[0055] - a reduced density of GFAP-positive reactive astrocytes within the tumour mass compared to untreated mice;

[0056] - a reduced proliferation of tumour cells; and

[0057] - an average lengthening of the life of treated mice by 5 days compared to the controls.

[0058] Furthermore, as may be observed from the results reported further below, according to the present invention, intravenous retro-orbital injection of a recombinant viral vector encoding the antibody and capsid capable of passing through the brain-blood barrier (BBB) led to a substantial expression of the antibody itself selectively in the brain astrocytes of mice.

[0059] Based on the results obtained according to the present invention, the administration of the antibody as such by convection enhanced delivery (CED) also led to a noteworthy tumour size reduction and a reduction in tumour invasiveness in treated animals compared to untreated ones.

[0060] Finally, according to the present invention, the effects of the antibody of the invention on epilepsy correlated to the glioma were assessed using a system of 2D primary co-culture of astrocytes and neurons isolated from the hippocampus of mice. In addition to confirming the role of the glioma in generating neuronal rearrangements and aberrant action potential peaks, the experimental results according to the invention show a positive effect of the antibody in normalising both the frequency of miniature postsynaptic excitatory currents and the increase in multiunit activity by acting selectively on the connexins of astrocytes.

[0061] It is therefore a specific object of the present invention a product for use in the treatment of a brain tumour and / or epilepsy associated with a brain tumour, said product being

[0062] - an antibody or an antigen-binding fragment thereof,

[0063] - a nucleotide sequence encoding said antibody or said antigen-binding fragment,

[0064] - an expression vector comprising said nucleotide sequence, or - a pharmaceutical composition comprising said antibody or fragment thereof, said nucleotide sequence or said vector, together with one or more pharmaceutically acceptable excipients and / or adjuvants, wherein said antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said VH comprises a first CDR region VH-CDR1 , comprising or consisting of the sequence GFTFSSYA (SEQ ID NO:2), a second CDR region VH-CDR2 comprising or consisting of the sequence ISHGGSNK (SEQ ID NO:3) and a third CDR region VH-CDR3 comprising or consisting of the sequence ARDFSWRGYYMDV (SEQ ID NO:4); said VL comprises a first CDR region VL-CDR1 , comprising or consisting of the sequence QSISSY (SEQ ID NO:5), a second CDR region VL-CDR2 comprising or consisting of the sequence GAS and a third CDR regionVL-CDR3 comprising or consisting of the sequence QQYGSSPRT (SEQ ID NO:6).

[0065] The antibody or antigen-binding fragment thereof according to the present invention has as its target and binds three connexins: Cx26, Cx30 and Cx32, since these three connexins expose, on the outside of the cell and in the same position, an epitope which strongly interacts with the CDRs of said antibody; said epitope is made up of 5 amino acids (NTQLP, SEQ ID NO:1 ) in the first extracellular loop, and two amino acids (NP) in the second extracellular loop (table 2).

[0066] According to the present invention, said brain tumour is preferably a glioma, such as a grade IV glioma, for example glioblastoma.

[0067] Preferably, the product according to the present invention is for use in the treatment of a glioma and / or glioma-associated epilepsy. For example, according to the present invention, the product can be for use in the treatment of glioblastoma and / or glioblastoma-associated epilepsy.

[0068] According to the present invention, said VH can comprise or consist of the sequence QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVIS HGGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFSWRGY YMDVWGKGTLVTVSS (SEQ ID NO:7), and / or wherein said VL can comprise or consist of the sequence ETTLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYGASTRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR (SEQ ID NO:8).

[0069] According to the present invention, said product can be a whole antibody or an antigen-binding fragment thereof. When the product according to the invention is a whole antibody, this can be, for example, in the form of an IgG.

[0070] According to the present invention, said fragment can be selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.

[0071] In one embodiment of the present invention, said antigen-binding fragment is an scFv and comprises a VH having sequence SEQ ID NO:7 and a VL having sequence SEQ ID NO:8 linked together by a linker, for example a linker having sequence GSGGGGS (SEQ ID NO:9).

[0072] According to the present invention, the nucleotide sequences encoding said antibody or an antigen-binding fragment thereof can be carried by one or more expression vectors. In particular, according to the present invention, said expression vector can be a viral vector.

[0073] The viral vector according to the present invention can be an adeno- associated virus (AAV), such as, for example, an AAV capsid of the serotype AAV1 , or AAV2, or AAV5, or AAV8, or AAV9, or AAVrh8, or AAVrhW, or AAVHu68, or AAVHSC15, or AAVSNY001 , or AAV.CAP-B10, or AAV.CAP-Mac, or AAV.CAP- Mac9, or BI-hTFR1. The listed vectors have been used in clinical or preclinical studies for their tropism towards the cells of the human central nervous system (SNC) or that of non-human primates, or of other animal models.

[0074] According to the present invention, the nucleotide sequence encoding SEQ ID NO:2 can be GGATTCACCTTCAGTAGCTATGCT (SEQ ID NO: 10), the nucleotide sequence encoding SEQ ID NO:3 can be ATATCACATGGTGGAAGTAATAAA (SEQ ID NO:11 ), the nucleotide sequence encoding SEQ ID NO:4 can be

[0075] GCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTC (SEQ ID NO: 12), the nucleotide sequence encoding SEQ ID NO:5 can be CAGAGTATTAGCAGCTAC (SEQ ID NO: 13), the nucleotide sequence encoding GAS can be GGTGCATCC and the nucleotide sequence encoding SEQ ID NO:6 can be CAGCAGTATGGTAGCTCACCTCGAACT (SEQ ID NO: 14).

[0076] Furthermore, according to the present invention, the nucleotide sequence encoding SEQ ID NO: 7 (VH) can be

[0077] CAGGTACAGCTGCAGCAGTCAGGGGGGGGCGTGGTCCAGCCTGGGAGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGC ACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATAT CACATGGTGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCAC CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA GAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTTTAGTTGGAGAGG GTACTACATGGACGTCTGGGGCAAAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 15) and the nucleotide sequence encoding SEQ ID NO: 8 (VL) can be GAAACGACACTCACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC ACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACA GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTAGCTCACCTCGAACTTTCGGCGGAGGGACCAAGGT GGAAATCAAACGT (SEQ ID NO: 16).

[0078] According to the present invention, the nucleotide sequence encoding said linker having sequence SEQ ID NO:9 can be GGCAGCGGCGGTGGCGGATCC (SEQ ID NO:17).

[0079] According to the present invention, said nucleotide sequence can further comprise a signal sequence for the secretion of the antibody, such as, for example, the human interleukin-2 secretion signal sequence.

[0080] For example, said human interleukin-2 secretion signal sequence (IL2ss) can be ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCAC GAATTCG (SEQ ID NO: 18), coding for MYRMQLLSCIALSLALVTNS (SEQ ID NO:19).

[0081] According to the present invention, said product can be administered by intraparenchymal convection enhanced delivery (CED), when said product consists of said antibody or antigen-binding fragment thereof or said pharmaceutical composition comprising said antibody or fragment thereof.

[0082] As mentioned above, convection enhanced delivery (CED) provides for direct infusion of the product according to the invention into the brain tissue under positive pressure, allowing for a wide distribution thereof in the area of interest.

[0083] Furthermore, according to the present invention, said product can be delivered by intranasal administration, or by intravenous injection, or into cerebrospinal fluid by intracerebroventricular (ICV) or intrathecal or intra-cisterna magna injection, or into the parenchyma by intraparenchymal injection (optionally convection enhanced - CED), when said product consists in said nucleotide sequence, expression vector or pharmaceutical composition comprising said nucleotide sequence or expression vector.

[0084] According to one embodiment of the present invention, said pharmaceutical composition further comprises a drug other than said product, wherein said drug is for treating a brain tumour and / or epilepsy associated with a brain tumour.

[0085] In particular, said drug can be a drug for treating glioma and / or glioma- associated epilepsy. For example, said drug can be a drug for treating glioblastoma and / or epilepsy associated therewith.

[0086] The present invention also concerns a combination of a product with a drug other than said product, for separate or sequential use in the treatment of a brain tumour and / or epilepsy associated with a brain tumour, said drug being for treating a brain tumour and / or epilepsy associated with a brain tumour and said product consisting of

[0087] - an antibody or an antigen-binding fragment thereof,

[0088] - a nucleotide sequence encoding said antibody or said antigen-binding fragment,

[0089] - an expression vector comprising said nucleotide sequence, or

[0090] - a pharmaceutical composition comprising said antibody or fragment thereof, said nucleotide sequence or said vector, together with one or more pharmaceutically acceptable excipients and / or adjuvants, wherein said antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said VH comprises a first CDR region VH-CDR1 , comprising or consisting of the sequence GFTFSSYA (SEQ ID NO:2), a second CDR region VH-CDR2 comprising or consisting of the sequence ISHGGSNK (SEQ ID NO:3) and a third CDR region VH-CDR3 comprising or consisting of the sequence ARDFSWRGYYMDV (SEQ ID NO:4); said VL comprises a first CDR region VL-CDR1 , comprising or consisting of the sequence QSISSY (SEQ ID NO:5), a second CDR region VL-CDR2 comprising or consisting of the sequence GAS and a third CDR region VL-CDR3 comprising or consisting of the sequence QQYGSSPRT (SEQ ID NO:6). Said brain tumour can be a glioma, such as a grade IV glioma, for example glioblastoma.

[0091] Preferably, said drug can be a drug for treating glioma and / or glioma- associated epilepsy. For example, said drug can be a drug for the treatment of glioblastoma and / or epilepsy associated therewith.

[0092] According to the present invention, “separate use” means the administration, at the same time, of the two compounds of the combination according to the invention in distinct pharmaceutical forms, whereas “sequential use” means the successive administration of the two compounds of the combination according to the invention, each in a distinct pharmaceutical form.

[0093] According to the present invention, said product of the combination may be a product as defined in any one of claims 1 -12.

[0094] The present invention will now be described by way of non-limiting illustration according to a preferred embodiment thereof, with particular reference to the examples and the figures in the appended drawings, wherein:

[0095] - Figure 1 shows the tumour growth following intracortical inoculation ofGL261 cells in C57BL / 6J mice. (A) Mouse kept in a stereotaxic apparatus under gas anaesthesia (2% isoflurane). (B) Image taken with a stereomicroscope to document the intracortical inoculation of GL261 cells; a glass microcapillary containing the cells in suspension is inserted through a hole with a diameter of 0.5 mm drilled into the cranial bone (craniotomy) according to predefined stereotaxic coordinates (A.P. -1.00 mm, M.L. ±1.50 mm, D.V. -1.10 mm, relative to Bregma). (C) Confocal immunofluorescence images of a coronal section of the brain obtained from a mouse with a tumour that developed starting from an intracortical inoculation of 4*104GL261 cells; the nuclei were stained with DAPI (4',6-diamidino-2- phenylindole, MolecularProbes D1306) which clearly highlights the tumour area. The section was also stained with F-actin (ActinGreen 488 ReadyProbes, Thermo Fisher Scientific R37110); the arrow indicates a presumed tumour nanotube detected by that staining. Unpublished data from the laboratory of the P.l.

[0096] - Figure 2 shows the expression of Cx and GFAP in the peritumoural region in mice with glioma. Images representative of confocal coimmunofluorescence of the expression of some Cx and GFAP in coronal sections of tumours grown in the brains of C57BL / 6J mice and examined two weeks after the intracortical inoculation of 4x104GL261 cells. Each column shows the same section marked with an anti-Cx primary antibody (top row), an anti-GFAP antibody (middle row) and DAP I (bottom row) to better highlight the tumour area. The anti-Cx primary antibodies used in these studies are: Cx26, Thermo Fisher Scientific 13-8100, diluted 1 :100; Cx30, Thermo Fisher Scientific 71 -2200, diluted 1 :50; Cx43, Thermo Fisher Scientific 71 -0700, diluted 1 :50; Cx46, Thermo Fisher Scientific 38-8300, diluted 1 :50. The secondary antibodies are: Donkey anti-Rabbit, Alexa Fluor 555 Thermo Fisher Scientific A-31572, or Donkey anti-Mouse, Alexa Fluor 555, Thermo Fisher Scientific A-31570, both diluted 1 :800). GFAP - glial fibrillar acidic protein - is a marker of reactive astrocytes associated with the tumour [8] (anti-GFAP primary antibody: BD Pharmingen 556327, diluted 1 :50; secondary antibody: Goat antiMouse, Alexa Fluor 488 Thermo Fisher Scientific A11029, diluted 1 :800). Unpublished data from the laboratory of the P.l.

[0097] - Figure 3 shows the expression in healthy mice of the abEC1.1(scFv- mFc) antibody following ICV administration of AAV8-abEC1.1(scFv-mFc) at P0.5. Images representative of confocal immunofluorescence of the expression of the abEC1 .1 (scFv-mFc) antibody in sagittal sections of mouse brains, 33 days after ICV administration of AAV8-abEC1.1 (scFv-mFc) (6x1010GC) at P0.5. Each column shows the same section marked with a secondary antibody, Goat-anti-Mouse conjugated with Alexa Fluor 488 (Thermo Fisher Scientific A11029, diluted 1 :300), which recognises the mFc domain of abEC1.1 (scFv-mFc) (top row) and with DAPI (bottom row) to highlight the presence of cells also in the absence of a signal due to the secondary antibody. Unpublished data from the laboratory of the P.l.

[0098] - Figure 4 shows the effect of the administration ofAAV8-abEC1.1(scFv- mFc) on tumour growth in mice with glioma. (A) Schematic diagram illustrating the chronology of the experimental protocol (P, postnatal day). At P0.5, the C57BL / 6J mice of the treatment group underwent intracerebroventricular (ICV) injection with 6X1 O10GC of AAV8-abEC1.1 (scFv-mFc). Subsequently, the mice of each group, those treated with AAV8-abEC1.1 (scFv-mFc) and the untreated ones, underwent an intracortical inoculation of 4x104GL261 cells at P75; the brain histological analysis was conducted after euthanasia at P90. (B) The DAPI staining of the coronal brain sections highlights tumour development. The images refer to sections taken at intervals of about 250 pm around the site of inoculation of the GL261 cells. (C) On the left: table of the tumour volumes for every type of treatment and corresponding identification code of the animal; on the right: distributions of the tumour volume data which show the mean (wide horizontal bars) ± standard error of the mean (s.e.m.); p-value = 0.0025 (two-sample t-test). The significant differences are indicated by * = p-value < 0.05, ** = p-value < 0.005. Unpublished data from the laboratory of the P.l.

[0099] - Figure 5 shows the effect of the administration ofAAV8-abEC1.1(scFv- mFc) on tumour morphology and infiltration in mice with glioma. The images show the confocal immunofluorescence of tumours originating from GL261 cells in coronal sections of the brain of C57BL / 6J mice treated (bottom row) with 6x1010GC of AAV8-abEC1.1 (scFv-mFc) at P0.5 and corresponding untreated controls (top row). The mice of each group (treated and untreated) underwent intracortical inoculation of 4x104GL261 cells at P75, and the brain analysis was conducted after euthanasia at P90. GFAP - glial fibrillar acidic protein - is a marker of the reactive astrocytes associated with the tumour [8] (anti-GFAP primary antibody: BD Pharmingen 556327, diluted 1 :50; secondary antibody: Goat anti-Mouse: Alexa Fluor 488 Thermo Fisher Scientific A11029, diluted 1 :800). The nuclei were stained with DAP I to better highlight the tumour area. Unpublished data from the laboratory of the P.l.

[0100] - Figure 6 shows the effect of the administration ofAAV8-abEC1.1(scFv- mFc) on the proliferation of tumour cells in mice with glioma. (A) Images representative of the confocal immunofluorescence of tumours originating from GL261 in coronal sections of the brain of mice treated with 6x1010GC of AAV8- abEC1.1 (scFv-mFc) at P0.5 and corresponding untreated controls. The mice of each group (treated and untreated) underwent intracortical inoculation of 4x104GL261 tumour cells at P75, and the histological brain analysis was conducted after euthanasia at P90. The nuclei were stained with DAPI (on the left). Ki-67 (on the right) is a marker of proliferation (anti-Ki-67 primary antibody: Thermo Fisher PAS- 19462, diluted 1 :100; secondary antibody: Donkey anti-Rabbit, Alexa Fluor 488 Thermo Fisher Scientific A-21206, diluted 1 :800). (B) Highly magnified images of the tumour areas delimited by white squares in A. (C) Confocal images representative of coronal brain sections marked only with the secondary antibody in the tumour areas of mice treated with AAV8-abEC1.1 (scFv-mFc) at P0.5 and corresponding untreated controls. (D) Quantification of the Ki-67-positive nuclei (Ki- 67 index, Ref.

[0055] ) from mice treated with AAV8-abEC1.1 (scFv-mFc) at P0.5 (n=5 mice, 3 sections each) and corresponding untreated controls (n=4 mice, 3 sections each) shown as the mean ± standard error of the mean (s.e.m.); p-value = 0.01 (two- sample t-test). The significant differences are indicated by * = p-value < 0.05. Unpublished data from the laboratory of the P.l.

[0101] - Figure 7 shows the effect of the administration ofAAV8-abEC1.1(scFv- mFc) on the survival of mice with glioma. The mice of each group (treated and untreated) underwent intracortical inoculation of 6x104GL261 cells at P75. (A) Average daily trend in weight relative to the day of the inoculation of GL261 cells; comparison between untreated C57BL / 6J mice (n=6, solid line and circle), undergoing ICV injection at P0.5 with 6x1010GC of AAV8-empty, not expressing the antibody (n=14, dotted line and triangle) and undergoing ICV injection at P0.5 with 6X1 O10GC of AAV8-abEC1.1 (scFv-mFc) (n=12, dashed line and square). Below, the p-values calculated point by point for each pair of treatments; the differences are significant below the horizontal dashed line (p=0.05). (B) Kaplan-Mayer curves for each treatment (“Untreated”, solid line; “AAV8-empty”, dotted line; “AAV8- abEC1 .1 ”, dashed line). (C) Distribution of survival time calculated starting from the date of inoculation of GL261 cells for the three groups of treatments (“Untreated”, circle; “Treated with AAV8-empty”, triangle; “Treated with AAV8-abEC1 .1 ”, square); they show the median with the respective interquartile deviation. The significant differences are indicated by * = p-value < 0.05, ** = p-value < 0.005. Unpublished data from the laboratory of the P.l.

[0102] - Figure 8 shows the biodistribution of the abEC1.1 antibody following retro-orbital administration of AAV-PHP.eB-abEC1.1(scFv-mFc-Atag) in healthy mice. Selective expression of the antibody in brain astrocytes of 3-month- old mice two weeks after treatment by intravenous retro-orbital injection of AAV- PHP.eB-abEC1 .1 (scFv-mFc-Atag) (3x1011GC) and corresponding untreated control. (A) Sagittal section representative of the distribution of the antibody. (B) Highly magnified images representative of some areas of the brain in which the antibody is particularly expressed. AbEC1.1 marked with the ALFA-tag at the carboxy-terminus was detected using an anti-ALFA-tag primary antibody (NanoTag N1583, diluted 1 :200) and a Donkey anti-Rabbit secondary antibody (Alexa Fluor 488, Thermo Fisher Scientific A-21206, diluted 1 :500). The nuclei were stained with DAPI. Unpublished data from the laboratory of the P.l.

[0103] - Figure 9 shows the colocalisation of abEC1.1 and Cx26 in the peritumoural region following retro-orbital administration of AAV-PHP.eB- abEC1.1(scFv-mFc-Atag) in mice with glioma. (A) Images representative of DAP I staining of tumours originating from intracortical inoculation of 4X104GL261 cells at P75 in coronal brain sections of 3-month-old mice two weeks after treatment by concomitant intravenous retro-orbital injection of AAV-PHP.eB-abEC1.1 (scFv-mFc- Atag) (3*1011GC), and corresponding untreated control. (B) Images representative of immunofluorescence staining of the area delimited by the white square in A (anti- Cx26 primary antibody: Thermo Fisher Scientific 13-8100, diluted 1 :100; Donkey anti-Mouse secondary antibody: Alexa Fluor 555, Thermo Fisher Scientific A-31570, diluted 1 :800. Anti-ALFA-tag primary antibody: NanoTag N1583, diluted 1 :200; Donkey anti-Rabbit secondary antibody: Alexa Fluor 488, Thermo Fisher Scientific A-21206, diluted 1 :500. (C) Highly magnified images of the area delimited by the white square in A; the arrows highlight the colocalisation of Cx26 and abEC1.1 (scFv-mFc-Atag). Unpublished data from the laboratory of the P.l.

[0104] - Figure 10 shows the biodistribution of the antibody after administration via CED of purified abEC1.1-mlGg1-Atag in the striatum in healthy mice. (A) Biodistribution of the abEC1.1 antibody marked with the ALFA-tag at the carboxyterminus (abEC1.1 -mlgG1 -Atag) in coronal brain sections of 3-month-old mice 2 hours after administration via CED of purified abEC1 ,1 -mlgG1 -Atag. (B) Highly magnified images representative of the regions indicated in the white squares show the distribution of the antibody in brain tissue in the injection area and in the contralateral hemisphere. AbEC1.1 -mlgG1 -Atag was detected using an anti-ALFA- tag primary antibody (NanoTag N1583, diluted 1 :200) and a Donkey anti-Rabbit secondary antibody (Alexa Fluor 488, Thermo Fisher Scientific A-21206, diluted 1 :500). The nuclei were stained with DAPI. Unpublished data from the laboratory of the P.l.

[0105] - Figure 11 shows the biodistribution of the purified abEC1.1-mlGg1-Atag antibody after administration via CED in the striatum in mice with glioma. (A) Images representative of the distribution of the abEC1 .1 antibody marked with the ALFA-tag at the carboxy-terminus (abEC1.1 -mlgG1 -Atag) in coronal brain sections of 3-month-old mice inoculated with 6X104GL261 tumour cells in the striatum and treated by repeated intratumoural CED of purified abEC1 ,1 -mlgG1 -Atag (5 mg / ml, 10 pl). The brain sample for the analysis was taken 21 days after the cell implantation, 48 hours after the last treatment. (B) Magnification of the tumour area indicated in the white square in A. (C) Highly magnified image representative of the region indicated in the white square in B, showing the distribution of abEC1.1 - mlgG1 -Atag in the peritumoural area; the antibody was detected using an anti- ALFA-tag primary antibody (NanoTag N1583, diluted 1 :200) and a Donkey antiRabbit secondary antibody (Alexa Fluor 488, Thermo Fisher Scientific A-21206, diluted 1 :500). The nuclei were stained with DAPI. Unpublished data from the laboratory of the P. I.

[0106] - Figure 12 shows the effect of the intratumoural treatments with purified abEC1.1-mlgG1-Atag antibody on the proliferation of tumour cells in mice with glioma. (A) Images representative of confocal immunofluorescence of intrastriatal GL261 tumours (obtained by inoculation of 6x104GL261 cells) in coronal sections of the brain of mice treated at P75 with purified abEC1 ,1 -mlgG1 -Atag (5mg / ml, 10 pl), administered by intratumoural CED repeated every 4 days starting from 7 days after the inoculation of GL261 cells and the corresponding controls injected with saline solution. The nuclei were stained with DAPI (on the left). PH3 (on the right) is a known marker of proliferation (anti-PH3 primary antibody: Thermo Fisher PAS- 17869, diluted 1 :100; secondary antibody: Donkey anti-Rabbit, Alexa Fluor 488 Thermo Fisher Scientific A-21206, diluted 1 :800). (B) Highly magnified images of the tumour areas delimited by the white squares in A. (C) Quantification of the PH3- positive nuclei from mice treated with purified abEC1 .1 -mlgG1 -Atag (n=3 mice, 3 sections each) and corresponding controls injected with saline solution (“Untreated”, n=2 mice, 3 sections each) shown as the mean ± standard error of the mean (s.e.m.). (D) Quantification of the area invaded by the tumour in mice treated with purified abEC1 ,1 -mlgG1 -Atag (n=4 mice, 3 sections each) and corresponding controls injected with saline solution (“Untreated”, n=3 mice, 3 sections each) shown as the mean ± standard error of the mean (s.e.m.). The differences were considered significant if p<0.05, denoted by *; **, p<0.01 ; ***, p<0.001. Unpublished data from the laboratory of the P.I.;

[0107] - Figure 13 shows the quantification of the GFAP- and Iba1 -positive areas in brain sections of mice implanted with GL261 tumours in the striatum and treated with purified abEC1.1 -mlGg1 -Atag via convection enhanced delivery (CED). (A) Expression of GFAP and Iba1 ; scale (bar): 100pm; the images are representative of n=4 mice treated by CED of the purified abEC1 ,1 -mlGg1 -Atag antibody and n=4 untreated controls. (B,C) Boxplots of the quantification of GFAP (B) and Iba1 (C) expression based on data related to (A); the p-values for all the data shown in the figure were derived from the Wilcoxon rank-sum test;

[0108] - Figure 14 shows the analysis of patch-clamp electrophysiology experiments. Quantitative analysis of the frequency (A), amplitude (B) and decay time (C) of the mEPSCs recorded in co-cultures of neurons-astrocytes (CTRL) and neurons-astrocytes-GBM (GBM) treated or not treated with abEC1.1 (scFv-mFc) (1 pM) for 45 minutes. (D) MU frequency analysed in co-cultures of neurons-astrocytes (CTRL) and neurons-astrocytes-GBM (GBM) before and 45 minutes after the application of abEC1.1 (scFv-mFc). Each point represents the mean frequency value of the EPSCs collected from a single neuron; the data are expressed as the mean ± s.e.m. The differences were considered significant if p<0.05, denoted by *; **, p<0.01 ; ***, p<0.001. Unpublished data from the laboratory of the P.l. and collaborators.

[0109] - Figure 15 shows the mEPSC frequency in a co-culture of neurons- astrocytes. (A) Frequency of mEPSCs recorded in co-cultures of astrocytes- neurons exposed to a carrier or abEC1 ,1 (scFv-mFc) (1 pM) for 45 minutes. It should be noted that abEC1.1 selectively influences the mEPSC frequency and not the amplitude (B). Pure hippocampal neurons treated at 14 DIV for 45 minutes with abEC1.1 (scFv-mFc) 1 pM do not show any change in the mEPSC frequency or amplitude (C, D). Each point represents the mean frequency value of the EPSCs recorded for a single neuron; the data are expressed as the mean ± s.e.m. *, p<0.05. Unpublished data from the laboratory of the P.l. and collaborators.

[0110] EXAMPLE 1. Study on the effectiveness of the abEC1. 1 antibody on models of glioma and on epilepsy associated therewith.

[0111] In order to test the effectiveness of abEC1 .1 in the treatment of tumours, the glioma 261 (GL261 ) model was implemented [46-49]; it represents an immunocompetent, orthotopic mouse model of human GBM widely used in preclinical research to study the pathophysiology of GBM, to identify therapeutic interventions, to evaluate new treatment methods (including immunotherapy) and to analyse tumour biology and progression

[0049] ,

[0112] 4*104GL261 cells (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Cell line: GL-261 , DSMZ no. ACC 802) were thus administered by intracortical injection into C57BL / 6J mice reared in the EMMA (European Mouse Mutant Archive) enclosure of the National Research Council (CNR) in Monterotondo, Rome (Fig. 1). Two weeks later, marking with commercial anti-Cx antibodies (which, unlike abEC1.1 , recognise different intracellular epitopes of the respective Cxs) revealed the expression of Cx26 above all in the peritumoural area, whilst Cx30, Cx43 and Cx46 were also expressed within the tumour mass (Fig. 2).

[0113] Once it was confirmed that different Cxs relevant for GBM were expressed in the animal model, the method of administration of abEC1.1 was evaluated. In particular, it was considered that an mAb can be produced directly in an organism following the introduction, into the cells of the organism itself, of a nucleic acid that codes for the mAb, thus enabling a continuous and potentially long-term release of therapeutic antibodies [50, 51 ], The nucleic acid can be introduced using different vectors, including adeno-associated viruses (AAV), which have been extensively studied for gene transfer into various organs, including the central nervous system (CNS), given their safety, tropism towards the CNS and the long-term therapeutic effect

[0052] , The term “AAV-mAb” refers to a specific therapeutic approach which combines AAVs with mAbs for potential applications in the medical realm

[0052] ,

[0114] Experiments were thus carried out using a recombinant AAV8 vector coding for a variant of abEC1.1 which can be secreted; this variant consists of a human domain that binds Cx HCs in the form of a single-chain variable fragment (scFv) linked to a crystallised fragment (Fc) of mice (mFc)

[0040] ,

[0115] The human single-chain variable fragment of abEC1 .1 used as reference for the development of the vector to be administered to the mice includes the nucleotide sequences shown below in Table 3:

[0116] Table 3

[0117] The nucleotide sequences shown in Table 3 code, respectively, for the amino acid sequences shown below in Table 4:

[0118] Table 4

[0119] The nucleotide sequence of the human single-chain variable fragment was optimised for expression in mice under the CAG promoter, while coding in any case for the amino acid sequences shown in Table 4. Furthermore, in order to enable the secretion of the antibody by cells infected by the viral vector, the optimised nucleotide sequence of abEC1 ,1 (scFv-mFc) was fused in phase downstream to the nucleotide sequence of the human interleukin-2 secretion signal (IL2ss), as detailed in table 5:

[0120] Table 5

[0121] The intracerebroventricular (ICV) injection

[0053] of 6x1010genome copies (GC) of AAV8-abEC1.1 (scFv-mFc) into C57BL / 6J baby mice on postnatal day (P) 0.5 (stereotaxic coordinates: A.P. +1.50 mm, M.L. ±0.80 mm, D.V. -3.00 mm, relative to Lambda) subsequently brought about diffuse expression of the antibody produced and secreted in situ, in the brain, by infected cells of the cortex, hippocampus and cerebellum (Fig. 3). The ICV injection consists in the injection of substances into one of the brain ventricles by means of a 100 pl syringe with a 33G needle according to predefined stereotaxic coordinates (A.P. +1 .50 mm, M.L. ±0.80 mm, D.V. -3.00 mm, relative to Lambda).

[0122] Mice of another group subjected to the same treatment, i.e. 6x1010GC of AAV8-abEC1 ,1 (scFv-mFc) on day P0.5, underwent intracortical inoculation of 4x104GL261 cells on day P75, enabling the growth of the tumours up to day P90, when the mice were sacrificed for post-mortem brain analyses (Fig. 4). The results demonstrated not only a considerable reduction in the volume of the gliomas in the mice treated with AAV8-abEC1.1 (scFv-mFc) compared to the untreated controls (Fig. 4), but also a lower tumour invasiveness, as may be deduced from the rounder shape of the profile of the peritumoural area, as well as the absence of necrotic areas and a reduced density of GFAP-positive reactive astrocytes within the tumour mass (Fig. 5).

[0123] Furthermore, the above-described results are correlated with a significant reduction in the immunoreactivity of the KI-67 antigen (Fig. 6), which is a widely used proliferation marker also known as Ki-67 or MKI67 (proliferation marker Ki-67)

[0054] , indicating that the treatment with AAV8-abEC1.1 (scFv-mFc) reduced the proliferation of tumour cells.

[0124] The results shown were confirmed by a study on the survival of C57BL / 6J mice with tumours, divided into 3 groups: untreated animals, animals undergoing ICV injection, at P0.5, of 6x1010GC of AAV8-empty, i.e. the virus with the same serotype ma without the antibody, which serves as a further negative control, and animals undergoing ICV injection, at P0.5, of 6X1 O10GC of AAV8-abEC1.1 (scFv- mFc). After the intracortical inoculation of 6x104GL261 cells, the animals were weighed daily and were sacrificed when their weight had decreased by 20% compared to the weight on the day of the inoculation or when they showed clear signs of suffering. The average weight change trend is shown in Fig. 7 A, where the animals treated with AAV8-abEC1.1 (scFv-mFc) show to have lost weight significantly more slowly compared to the two controls. Furthermore, the median survival measured starting from the day of implantation of the tumour cells showed to be significantly longer for the animals treated with AAV8-abEC1 .1 (scFv-mFc) (31 days, n=12) compared both to the animals injected with AAV8-empty (26.5 days, n=14) and to the untreated animals (25.5 days, n=6); their life was prolonged on average by 5 days (Fig. 7 B, C) compared to the controls.

[0125] The use of an AAV-PHP.eB capsid vector, derived from AAV9, which is capable of passing through the blood-brain barrier (BBB), in C57BL / 6J mice

[0057] was then assessed as a further therapeutic possibility. For these experiments, the sequence coding for the antibody, optimised for expression in mice, was inserted into the plasmid of the AAV-PHP.eB vector, under the short mouse GFAP promoter

[0058] , in the scFv-mFc format with IL2ss and the ALFA-tag marker

[0056] (abbreviated Atag) in the C-terminal portion of the mFc fragment. The amino acid sequences of the scFv portion of the construct are the ones indicated in Table 4. Following an intravenous retro-orbital injection

[0059] of 3X1011GC of AAV-PHP.eB-abEC1.1 (scFv- mFc-Atag) in healthy adult mice, a substantial expression of the antibody was obtained selectively in the brain astrocytes (Fig. 8). By means of immunofluorescence analysis, it was possible to establish that the antibody, produced in situ in the brain by the infected astrocytes and secreted by them, colocalises with Cx26 in the peritumoural region in mice undergoing an intracortical injection of 4X104GL261 cells and concomitant intravenous retro-orbital injection of AAV-PHP.eB-abEC1.1 (scFv-mFc-Atag) (3x1011GC) (Fig. 9).

[0126] Furthermore, in order to overcome the obstacle posed by the BBB, experiments of convection enhanced delivery (CED) were conducted using the purified mouse lgG1 variant of the antibody, marked with the ALFA-tag (abEC1.1 - mlgG1 -Atag). In this case, the striatum was selected as the site both for the inoculation of tumour cells and for the subsequent treatments (coordinates from Bregma: AP=+0.86 mm, ML=-1.8 mm, DV=-3.0 mm). As the striatum is located at a greater depth in the brain of the experimental animal, the injection via CED results in a lower likelihood of the injected fluid rising through the injection channel (with consequent dilution in cerebrospinal fluid in the subarachnoid space), which could cause a lower efficiency of the treatment.

[0127] In order to define the timeframes for the repeated treatments, some biodistribution experiments were preliminarily conducted by injecting 10 pl of purified abEC1 ,1 -mlgG1 -Atag into the striatum of healthy mice at a concentration of 5 mg / ml, via CED. The brain of the animals was then removed 2 hours after the CED. The immunofluorescence images obtained from brain sections by means of a confocal microscope show a very intense, concentrated antibody signal in the region of the injection 2 hours after CED (Fig. 10).

[0128] In another group of mice with tumours, 4 treatments with abEC1 ,1-mlgG1 - Atag were then carried out every 4 days starting from the seventh day after the inoculation of 6x104GL261 cells into the striatum. The animals were sacrificed on day 21 after the implantation of the tumour (48 hours after the last treatment) to assess its size and cell proliferation compared to untreated animals (CED of saline solution).

[0129] Based on an analysis of the brain sections of these animals, it was possible to verify the presence of abEC1.1 -mlgG1 -Atag within the tumour microenvironment (Fig. 11). Furthermore, a noteworthy reduction in tumour size was observed in the treated animals; in the central sections, the tumour surface area was reduced by about 3 times compared to the untreated case. These data are also confirmed by the reduction in tumour invasiveness in the treated animals compared to the untreated ones, as quantified by means of the cell proliferation marker phosphohistone-H3 (PH3)

[0060] , which was reduced by about 2.5 times (Fig. 12).

[0130] In addition, in order to evaluate the triggering of an anti-tumour immune response, in another group of mice (GL261 model) the expression levels of GFAP and Iba1 were quantified after the administration in vivo of the abEC1 .1 antibody by convection enhanced delivery (CED) (Fig.13). In particular, quantification of the immunoreactivity to GFAP and Iba1 was achieved using Fiji / lmageJ on confocal images of sections containing brain tumours marked with anti-GFAP or anti-lba1 antibodies (Pharmingen anti-GFAP primary antibody 556327 diluted 1 :50; Wako Chemicals anti-lba1 019-19741 primary antibody diluted 1 :500). The GFAP- and Iba1 -positive pixels were identified by applying a threshold and their number was divided by that of the corresponding tumoural or peritumoural regions. The results obtained showed that, compared to the group of control animals, the treated mice showed an 18% reduction in immunoreactivity to GFAP in the peritumoural area. In contrast, the immunoreactivity to Iba1 increased by 59% in the peritumoural area and by 29% in the tumour mass (Fig. 13), suggesting a potential immuno-mediated antitumour response. These results provide further information about the mechanism underlying the effects of the antibody on GBM in vivo.

[0131] A last but important aspect that was taken into consideration in these studies is the link between GBM and epilepsy

[0061] , and between the latter and Cx HCs [29- 34], since the antitumour therapy can contribute to controlling epileptic manifestations and can thus exert beneficial effects on the treatment both of the glioma / GBM and of epilepsy associated thereto

[0029] ,

[0132] In order to investigate this therapeutic possibility as well, a system of 2D primary co-culture of astrocytes and neurons isolated from the mouse hippocampus was set up. Briefly, cells isolated from litters of mice at an embryonic stage (E) E18 were separately cultured: the hippocampus neurons were plated at a medium density on slides pretreated with poly-L-lysine and cultured in a neuronal culture medium containing B27 supplement and glutamate, whereas the astrocytes were placed in flasks with a culture medium for astrocytes containing 10% foetal bovine serum (FBS). In order to obtain the astrocyte-neuron co-cultures, the astrocytes were detached from the flasks, counted and then placed on the neurons after 7-10 days of in vitro (DIV) culture. The co-culture was maintained for about seven days so as to allow the formation of the leaflet of astrocytes. Finally, we plated GL261 cells onto the co-cultures and made patch-clamp electrophysiological recordings of the cultured neurons within 3 hours after they were placed in culture (Fig. 14).

[0133] These experiments revealed miniature excitatory postsynaptic currents (mEPSCs), which occur at the level of the excitatory synapses between neurons. The term “miniature” refers to the small amplitude of the currents in question, which occur spontaneously, independently of external stimulations. Said currents, recorded in the presence of 1 pM tetrodotoxin (TTX) to prevent the generation of action potentials, are the result of the release of neurotransmitters, such as glutamate, from presynaptic vesicles, also in the absence of action potentials. MEPSCs are considered the basal level of excitatory synaptic activity and are correlated with the random release of neurotransmitters at the level of the individual synapse

[0062] ,

[0134] On the adherent cells we also measured the multi-unit (MU) activity, which represents the ability of each neuron to generate action potentials during a specific time interval. This activity is typically expressed as the number of action potentials per second or in Hertz (Hz)

[0063] ,

[0135] As is shown in Fig. 14, the frequency of mEPSCs (panel A) and MU (panel D) increased significantly when the co-cultures of neurons and astrocytes were incubated for 3 hours with GL261 cells, thus confirming the role of the glioma in generating neuronal rearrangements and aberrant action potential peaks. Electrophysiological recordings in the co-cultures of neurons, astrocytes and GL261 tumour cells after 45 minutes of incubation with abEC1.1 (scFv-mFc) demonstrated a positive effect of the antibody in normalising both the mEPSC frequency and the increase in MU activity. It should be noted that neither the presence of GL261 nor the administration of abEC1.1 (scFv-mFc) in the presence of GL261 caused variations in the amplitude (Fig. 14, B) or in the exponential decay time constant of the mEPSCs (Fig. 14, C).

[0136] As is shown in Fig. 14, the frequency of the mEPSC (panel A) and MU (panel D) increased significantly when the co-cultures of neurons and astrocytes were incubated for 3 hours with GL261 cells, thus confirming the role of the glioma in generating neuronal rearrangements and aberrant action potential peaks. Electrophysiological recordings in the co-cultures of neurons, astrocytes and GL261 tumour cells after 45 minutes of incubation with abEC1 .1 (scFv-mFc) demonstrated a positive effect of the antibody in normalising both the mEPSC frequency and the increase in MU activity. It should be noted that neither the presence of GL261 nor the administration of abEC1.1 (scFv-mFc) in the presence of GL261 caused variations in the amplitude (Fig. 14, B) or in the exponential decay time constant of the mEPSCs (Fig. 14, C).

[0137] In order to demonstrate that the effect of the mAb is mediated by astrocytes, the changes induced by abEC1.1 (scFv-mFc) on the transmission of excitation in ( / ) non-stimulated co-cultures of neurons and astrocytes and ( / / ) non-stimulated pure neuron cultures were compared. Whereas the co-cultures exposed to abEC1.1 (scFv-mFc) demonstrate a considerable reduction in the mEPSC frequency (Fig. 15, A-B), the mEPSC frequency as measured in hippocampus neurons at 14 DIV in the presence of abEC1.1 (scFv-mFc) shows no changes (Fig. 15, C-D). These results indicate that the effects induced by abEC1.1 (scFv-mFc) on glutamatergic synaptic activity depend on its selective action on the Cxs of the astrocytes, whereas abEC1.1 (scFv-mFc) does not in itself modify neuronal activity.

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[0201] The present invention has been described by way of non-limiting illustration according to the preferred embodiments thereof, but it is understood that variations and / or modifications may be introduced by the person skilled in the art without going outside the relevant scope of protection, as defined by the appended claims.

Claims

CLAIMS1 ) Product for use in the treatment of a brain tumour and / or epilepsy associated with a brain tumour, said product being- an antibody or an antigen-binding fragment thereof,- a nucleotide sequence encoding said antibody or said antigen-binding fragment,- an expression vector comprising said nucleotide sequence, or- a pharmaceutical composition comprising said antibody or fragment thereof, said nucleotide sequence or said vector, together with one or more excipients and / or adjuvants, wherein said antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said VH comprises a first CDR region VH-CDR1 , comprising or consisting of the sequence GFTFSSYA (SEQ ID NO:2), a second CDR region VH-CDR2 comprising or consisting of the sequence ISHGGSNK (SEQ ID NO:3) and a third CDR region VH-CDR3 comprising or consisting of the sequence ARDFSWRGYYMDV (SEQ ID NO:4); said VL comprises a first CDR region VL-CDR1 , comprising or consisting of the sequence QSISSY (SEQ ID NO:5), a second CDR region VL-CDR2 comprising or consisting of the sequence GAS and a third CDR region VL-CDR3 comprising or consisting of the sequence QQYGSSPRT (SEQ ID NO:6).2) Product according to claim 1 for use according to claim 1 , wherein said brain tumour is a glioma, such as a grade IV glioma, for example glioblastoma.3) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein said VH comprises or consists of the sequence QVQLQQSGGGWQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVIS HGGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFSWRGY YMDVWGKGTLVTVSS (SEQ ID NO:7), and / or wherein said VL comprises or consists of the sequence ETTLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYGASTRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR (SEQ ID NO:8).4) Product according to any one of the preceding claims for use according toany one of the preceding claims, wherein said antigen-binding fragment is selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2.5) Product according to claim 4 for use according to claim 4, wherein said fragment is an scFv and comprises a VH having sequence SEQ ID NO:7 and a VL having sequence SEQ ID NO:8 linked together by a linker, for example a linker having sequence GSGGGGS (SEQ ID NO:9).6) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein said expression vector is a viral vector.7) Product according to claim 6, for use according to claim 6, wherein said viral vector is an adeno-associated virus (AAV), such as, for example, an AAV capsid of the serotype AAV1 , or AAV2, or AAV5, or AAV8, or AAV9, or AAVrh8, or AAVrhIO, or AAVHu68, or AAVHSC15, or AAVSNY001 , or AAV.CAP-B10, or AAV.CAP-Mac, or AAV.CAP-Mac9, or BI-hTFR1.8) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein the nucleotide sequence encoding SEQ ID NO:2 is GGATTCACCTTCAGTAGCTATGCT (SEQ ID NO: 10), the nucleotide sequence encoding SEQ ID NO:3 is ATATCACATGGTGGAAGTAATAAA (SEQ ID NO: 11 ), the nucleotide sequence encoding SEQ ID NO:4 is GCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTC (SEQ ID NO: 12), the nucleotide sequence encoding SEQ ID NO:5 is CAGAGTATTAGCAGCTAC (SEQ ID NO: 13), the nucleotide sequence encoding GAS is GGTGCATCC and the nucleotide sequence encoding SEQ ID NO:6 is CAGCAGTATGGTAGCTCACCTCGAACT (SEQ ID NO: 14).9) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein the nucleotide sequence encoding SEQ ID NO: 7 (VH) isCAGGTACAGCTGCAGCAGTCAGGGGGGGGCGTGGTCCAGCCTGGGAGGTC CCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGC ACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATAT CACATGGTGGAAGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCAC CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA GAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATTTTAGTTGGAGAGG GTACTACATGGACGTCTGGGGCAAAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO:15) and the nucleotide sequence encoding SEQ ID NO: 8 (VL) isGAAACGACACTCACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAA GAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTG GTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCC ACCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACA GACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTA CTGTCAGCAGTATGGTAGCTCACCTCGAACTTTCGGCGGAGGGACCAAGGT GGAAATCAAACGT (SEQ ID NO: 16).10) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein said nucleotide sequence further comprises a signal sequence for the secretion of the antibody, such as, for example, the sequence of the human interleukin-2 secretion signal.11 ) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein said product is administered by intraparenchymal convection optimised delivery (CED), when said product consists of said antibody or fragment thereof or said pharmaceutical composition comprising said antibody or fragment thereof.12) Product according to any one of claims 1 -10 for use according to any one of claims 1 -10, wherein said product is administered intranasally, or by intravenous injection, or into the cerebrospinal fluid by intracerebroventricular (ICV) or intrathecal or intra-cisterna magna injection, or into the parenchyma by intraparenchymal injection, when said product consists of said nucleotide sequence, expression vector or pharmaceutical composition comprising said nucleotide sequence or expression vector.13) Product according to any one of the preceding claims for use according to any one of the preceding claims, wherein said pharmaceutical composition further comprises a drug other than said product, wherein said drug is for treating a brain tumour and / or epilepsy associated with a brain tumour.14) Combination of a product with a drug other than said product, for separate or sequential use in the treatment of a brain tumour and / or epilepsy associated with a brain tumour, said drug being for treating a brain tumour and / or epilepsy associated with a brain tumour and said product being- an antibody or an antigen-binding fragment thereof,- a nucleotide sequence encoding said antibody or said antigen-binding fragment,- an expression vector comprising said nucleotide sequence, or- a pharmaceutical composition comprising said antibody or fragment thereof, said nucleotide sequence or said vector, together with one or more excipients and / or adjuvants, wherein said antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), and wherein said VH comprises a first CDR region VH-CDR1 , comprising or consisting of the sequence GFTFSSYA (SEQ ID NO:2), a second CDR region VH-CDR2 comprising or consisting of the sequence ISHGGSNK (SEQ ID NO:3) and a third CDR region VH-CDR3 comprising or consisting of the sequence ARDFSWRGYYMDV (SEQ ID NO:4); said VL comprises a first CDR region VL-CDR1 , comprising or consisting of the sequence QSISSY (SEQ ID NO:5), a second CDR region VL-CDR2 comprising or consisting of the sequence GAS and a third CDR region VL-CDR3 comprising or consisting of the sequence QQYGSSPRT (SEQ ID NO:6).15) Combination according to claim 14, for use according to claim 14, wherein said brain tumour is a glioma, such as a grade IV glioma, for example glioblastoma.

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