ANTIBODY FOR THE TREATMENT OF BRAIN TUMORS AND RELATED EPILEPSY.

Description

ANTIBODY FOR THE TREATMENT OF BRAIN TUMORS AND OF THE EPILEPSY RELATED TO THEM. The present invention relates to an antibody for the treatment of brain tumors and related epilepsy. Specifically, the invention concerns the use of an antibody directed, with a high degree of affinity and specificity, against the extracellular domain of hemichannels formed by connexin 26 (Cx26, encoded by GJB2), connexin 30 (Cx30, encoded by GJB6), and / or connexin 32 (Cx32, encoded by GJB1), for the treatment of brain tumors and related epilepsy or to reduce or inhibit their development. Brain tumors are known to be one of the most complex and devastating oncological diseases, characterized by the uncontrolled growth of abnormal cells within brain tissue. These tumors can be primary, originating directly in the brain, or metastatic, arising from other parts of the body. In this context, it is known that high-grade gliomas are the leading cause of brain tumor death in both children and adults. Grade IV gliomas are predominantly glioblastomas (GBMs), highly undifferentiated malignant tumors with very aggressive progression [1]. Glioma stem cells (GSCs) are a IC / A138842 Barzanò & Zanardo -2 subpopulation of cells within gliomas that possess characteristics of stem cells, including the capacity for self-renewal and differentiation [2, 3]. These cells are thought to play a key role in the initiation, progression and recurrence of gliomas, including GBM. In particular, GSCs differentiate into endothelial cells, thus supporting tumor growth through angiogenesis [2] and their resistance to chemotherapy has been demonstrated [3]. Available data indicate that a vicious circle is established between glioma cells and adjacent neurons that promotes increased neuronal activity, which in turn promotes tumor growth with subsequent destruction of healthy brain tissue [4]. The presence of interconnected clusters of glioma cells that can migrate together increases the likelihood of recurrence and makes removal of the tumor mass by surgical resection difficult [5, 6]. Standard therapeutic treatments are known, which include surgical resection, chemotherapy (mainly with temozolomide), and radiotherapy. However, due to its invasiveness and poor response to standard treatments, the median survival of GBM patients is only 12–16 months, with a 10-year survival rate of 0.17% [7]. It is also known that the interaction between astrocytes and microglia within the tumor mass mediates the IC / A138842 Barzanò & Zanardo ^— transcriptional reprogramming of microglia and myeloid cells towards an anti-inflammatory phenotype and promotes the establishment of an overall immunosuppressive microenvironment [8] which can potentially hinder therapeutic developments based on immunotherapy [9]. Combined treatment with electric fields (Tumor Treating Fields, TTF) and temozolomide, performed in a phase 3 clinical trial, represented a breakthrough in the treatment of GBM. However, the median survival time increased only from 16 to 21 months

[10] . As of May 19, 2024, the National Brain Tumor Society's Clinical Trial Finder (https: / / braintumor.org) reported 449 active trials. However, despite significant efforts, the overall survival of GBM patients 5 years after diagnosis is still less than 5%

[11] . It is also known that GBM can affect 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 can also be an early clinical symptom of this cancerous entity. In light of the above, the need to provide new therapies for the treatment of brain tumors, and in particular gliomas and glioblastomas, appears evident. IC / A138842 Barzanò & Zanardo -4In this context, the solution according to the present invention comes into play, which aims to provide an antibody for the treatment of brain tumors and related epilepsy. According to the present invention, it has now been found that an antibody designed to bind and inhibit hemichannels formed by connexins is designed to act by reducing or inhibiting tumor and related epilepsy through the administration of the antibody itself or of a vector encoding and expressing such antibody. Connexins (Cxs) are a family of 21 transmembrane proteins that are essential elements in the cell-cell communication apparatus, contribute to regulating cell proliferation and survival, and have been extensively studied in cancer [12, 13], including malignant tumors

[14] . Table 1 shows the connections of Homo Sapiens: Table 1 # Gene Nome Proteina OMIM ID NCBI CCDS Database ID Riferimento NCBI Sequence ID 1 GJA1 CX43 121014 CCDS5123.1 NM_000165.5 2 GJA3 CX4 6 121015 CCDS9289.1 NM_021954.4 3 GJA4 CX37 121012 CCDS30669.1 NM_002060.3 4 GJA5 CX4 0 121013 CCDS929.1 NM_181703.4 5 GJA8 CX50 600897 CCDS30834.1 NM 005267.5 IC / A138842 Barzanò & Zanardo 6 GJA9 CX5 9 611923 CCDS432.1 NM_030772.5 7 GJA10 CX62 611924 CCDS5025.1 NM_032602.2 8 GJB1 CX32 304040 CCDS14408.1 NM_001097642.3 9 GJB2 CX2 6 121011 CCDS9290.1 ​​NM_004004.6 10 GJB3 CX31 603324 CCDS384.1 NM_024009.3 11 GJB4 CX30.3 605425 CCDS383.1 NM_153212.3 12 GJB5 CX31.1 604493 CCDS382.1 NM_005268.4 13 GJB6 CX30 604418 CCDS9291.1 NM_006783.5 14 GJB7 CX2 5 611921 CCDS5008.1 NM_198568.3 15 GJC1 CX45 608655 CCDS11487.1 NM_005497.4 16 GJC2 CX47 608803 CCDS1569.1 NM_020435.4 17 GJC3 CX29 / 30. 2 / 31.3 611925 CCDS34697.1 NM_181538.3 18 GJD2 CX3 6 607058 CCDS10040.1 NM_020660.3 19 GJD3 CX31.9 607425 CCDS58547.1 NM_152219.4 20 GJD4 CX40.1 611922 CCDS7191.1 NM_153368.3 21 GJE1 CX2 3 611925 CCDS87449.1 NM_001358410.2 In their canonical function, Cxs 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 HCs from adjacent cells. GJs allow direct communication between the cytoplasms of adjacent cells, while unjoined HCs mediate the exchange of molecules between the cell's cytoplasm and the extracellular environment. Since Cxs exert a broad range of functions, which may further vary depending on tumor type and experimental conditions, IC / A138842 Barzanò & Zanardo ^6 to date the exact mechanism through which Cxs suppress or promote neoplastic progression is not known

[12] . In this regard, the data available in the literature indicate that: - Cx26 is a negative prognostic indicator of survival when upregulated in several tumor types

[15] , including human glioma biopsy specimens

[16] ; - Cx43 is increased in glioma-associated astrocytes, particularly in the peritumoral region, where it contributes to the dissemination of glioma cells [17, 18]; - glioma stem cells (GSCs) express higher amounts of Cx46 than non-stem tumor cells, constituting a necessary and sufficient condition for the maintenance of GSCs [19, 20]. Cxs HCs also play a role in carcinogenesis and tumor progression, as: - Cxs HCs mediate the release of signaling molecules, such as ATP, which promote cell motility and the establishment of a microenvironment invasive

[21] ; Cxs HCs modulate interactions between tumor cells and their microenvironment, with effects on the immune response, on extracellular matrix remodeling and other factors influencing tumor progression [22, 23]; - the role of Cxs HCs in invasion by IC / A138842 Barzanò & Zanardo ^7 of the tumor mass has been demonstrated in a breast cancer model

[24] ; - Cxs HCs can promote the formation of new blood vessels both by facilitating the release of pro-angiogenic factors and by mediating the Ca2+ signaling pathway associated with endothelial cell migration [25, 26], while genetic knockdown of endothelial connexins impairs angiogenesis

[27] and inhibits tumor growth

[28] ; - epilepsy often develops in patients with brain tumors and the two conditions share common pathogenic mechanisms

[29] ; - Cxs HCs play an important role in hyper-excitability and epilepsy onset in murine models of epilepsy / seizures [30-32] by deregulating Ca2+-dependent glutamatergic signaling [29, 32-34]. According to the present invention, it has now been surprisingly found that an antibody capable of inhibiting Cx26, Cx30 and Cx32 HCs is effective in animal models of glioma and associated epilepsy. In particular, according to the present invention, the efficacy of the antibody abEC1,1, already described in the patent application US2022251179A1, which specifically binds an extracellular epitope conserved in HCs formed by connexins Cx26, Cx30 and Cx32, was tested. In particular, table 2 shows the amino acid sequences of this epitope, with reference to IC / A138842 Barzanò & Zanardo -8sequence of human Cx26 protein, but the epitope is the same in Cx30 and Cx32: Table 2 SEQ ID NO Amino Acids (single letter) Position of residues in Cx26 SEQ ID NO: 1 NTLQP 54-58 PN 175-176 The above epitope was deduced from in silico simulations based on the crystal structures of both the target (PDB ID: 2ZW3) and the antibody (PDB ID: 5WYM) and validated by mutagenesis experiments [35, 36]. Furthermore, abECl.1: - inhibits Cx26, Cx30 and Cx32 HCs with IC50 in the nM range [35, 37, 38]; - inhibits Ca2+ uptake and ATP release through Cxs HCs [35-41], with far-reaching implications on the modulation of the tumor microenvironment and containment of tumor growth [42-45]; - it is effective in vivo, in murine models of Cxs HCs-related pathologies [39, 40]; - inhibits Cxs HCs in a solid tumor, as demonstrated by in vivo experiments conducted on a murine model of melanoma

[41] . More specifically, according to the present invention, said antibody was administered to mice using recombinant adeno-associated viral (AAV) vectors encoding the antibody itself in scFv-mFc format. IC / A138842 Barzanò & Zanardo -9comprising a single-chain variable fragment (scFv) linked to a murine crystallizable fragment (Fc) (mFc); these vectors were administered by intracerebroventricular (ICV) or retro-orbital injection. The antibody was also administered as is to mice, in immunoglobulin Gl (IgGl, also with mFc), via convection-enhanced delivery (CED). CED involves the direct infusion of therapeutic agents into the brain tissue under positive pressure, allowing for broad distribution in the area of ​​interest. From the results obtained according to the present invention, reported below, it is possible to observe how the ICV administration with recombinant vector encoding the antibody in mice with glioma surprisingly determined: - widespread expression of the antibody in the brain; - a significant reduction in the volume of gliomas compared to untreated mice; - less tumor invasiveness than untreated mice; - the absence of necrotic areas; - a reduced density of GFAP-positive reactive astrocytes within the tumor mass compared to untreated mice; - reduced proliferation of tumor cells; and - an extension of the life of treated mice IC / A138842 Barzanò & Zanardo ^H average of 5 days compared to controls. Furthermore, as can be observed from the results reported below, according to the present invention, the retro-orbital intravenous injection of a recombinant viral vector encoding the antibody and capsid capable of crossing the blood-brain barrier (BBB) ​​led to a consistent expression of the antibody itself selectively in the brain astrocytes of mice. Based on the results obtained according to the present invention, also the administration of the antibody as is via convection-optimized delivery (CED) led to a significant reduction in tumor size and a reduction in tumor invasiveness in treated animals compared to untreated ones. Finally, according to the present invention, the effects of the antibody under investigation on glioma-related epilepsy were evaluated using a 2D primary co-culture system of astrocytes and neurons isolated from the mouse hippocampus. In addition to confirming the role of glioma in generating neuronal rearrangements and aberrant action potential spikes, the experimental results according to the invention show a positive effect of the antibody in normalizing both the frequency of miniature excitatory postsynaptic currents and increasing multiunit activity by selectively acting on astrocytic connexins. It is therefore the specific subject of this IC / A138842 Barzanò & Zanardo - 11 invention a product for use in the treatment of a brain tumor and / or epilepsy associated with a brain tumor, said product being - an antibody or a fragment thereof that binds to the antigen, - 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 a 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). The antigen-binding antibody or fragment thereof according to the present invention targets and binds IC / A138842 Barzanò & Zanardo ^H three connexins: Cx26, Cx30 and Cx32, as these three connexins expose outside the cell and in the same position an epitope that interacts strongly with the CDRs of said antibody; said epitope is composed 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). According to the present invention, said brain tumor is preferably a glioma, such as grade IV glioma, e.g. glioblastoma. Preferably, the product according to the present invention is for use in the treatment of glioma and / or glioma-associated epilepsy. For example, according to the present invention, the product may be for use in the treatment of glioblastoma and / or glioblastoma-associated epilepsy. In accordance with the present invention, said VH may comprise or consist of the sequence QVQLQQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISHGG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFSWRGYYMDVW GKGTLVTVSS (SEQ ID NO:7), and / or wherein said VL may comprise or consist of the sequence ETTLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYGASTRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPRTFGGGTKVEIKR (SEQ ID NO:8). According to the present invention, said product may be a whole antibody or an antigen-binding fragment thereof. When the product according to the invention is a whole antibody, it may be IC / A138842 Barzanò & Zanardo ^13 for example in the form of an IgG. According to the present invention, said fragment can be selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', F(ab')2. In one embodiment of the present invention, said antigen-binding fragment is an scFv and comprises a VH of sequence SEQ ID NO:7 and a VL of sequence SEQ ID NO:8 linked together by a linker, e.g. by a linker of sequence GSGGGGS (SEQ ID NO:9). According to the present invention, the nucleotide sequences encoding said antibody or an antigen-binding fragment thereof can be conveyed by one or more expression vectors. In particular, according to the present invention, said expression vector can be a viral vector. The viral vector according to the present invention may be an adeno-associated virus (AAV), such as an AAV with a capsid of the serotype AAV1, or AAV2, or AAV5, or AAV8, or AAV9, or AAVrh8, or AAVrhlO, 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 cells of the central nervous system (CNS) of humans or non-human primates, or other animal models. According to the present invention, the nucleotide sequence encoding SEQ ID NO:2 may be GGATTCACCTTCAGTAGCTATGCT (SEQ ID NO:10), the nucleotide sequence encoding SEQ ID NO:3 may be IC / A138842 Barzanò & Zanardo 14ATATCACATGGTGGAAGTAATAAA (SEQ ID NO:11), the coding nucleotide sequence SEQ ID NO:4 may be GCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTC (SEQ ID NO:12), the coding nucleotide sequence SEQ ID NO:5 may be CAGAGTATTAGCAGCTAC (SEQ ID NO:13), the coding nucleotide sequence GAS may be GGTGCATCC, and the coding nucleotide sequence SEQ ID NO:6 may be CAGCAGTATGGTAGCTCACCTCGAACT (SEQ ID NO:14). Furthermore, according to the present invention, the coding nucleotide sequence SEQ ID NO: 7 (VH) may be CAGGTACAGCTGCAGCAGTCAGGGGGGGGCGTGGTCCAGCCTGGGAGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGT CCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCACATGGTGGA AGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGC TGTGTATTACTGTGCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTCTGG GGCAAAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 15) and the coding nucleotide sequence SEQ ID NO: 8 (VL) may be GAAACGACACTCACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG CCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCA GCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCC ACTGGCATCCCAGACAGGTTCAGTGGCAGTGGTCTGGGACAGACTTCACTCTCA CCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGG TAGCTCACCTCGAACTTTCGGCGGAGGGACCAAGGTGGAAATCAAACGT ( SEQ ID NO: 16). According to the present invention, the nucleotide sequence encoding for said sequence linker SEQ ID NO:9 may be GGCAGCGGCGGTGGCGGATCC (SEQ ID IC / A138842 Barzanò & Zanardo NO:17). According to the present invention, said nucleotide sequence may further comprise a signal sequence for antibody secretion, such as the human interleukin-2 secretion signal sequence. For example, this human interleukin-2 (IL2ss) secretion signal sequence may be ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGA ATTCG (SEQ ID NO:18), encoding MYRMQLLSCIALSLALVTNS (SEQ ID NO:19). According to the present invention, said product can be administered by convection-optimized intraparenchymal administration (CED), when said product consists of said antibody or antigen-binding fragment thereof or said pharmaceutical composition comprising said antibody or fragment thereof. As mentioned above, convection-optimized delivery (CED) involves the direct infusion of the product according to the invention into the brain tissue under positive pressure, allowing for broad distribution in the area of ​​interest. Furthermore, according to the present invention, said product can be administered by intranasal administration, 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 (possibly optimised for convection, CED), when IC / A138842 Barzanò & Zanardo 16said product consists of said nucleotide sequence, expression vector or pharmaceutical composition comprising said nucleotide sequence or expression vector. According to one embodiment of the present invention, said pharmaceutical composition further comprises a drug other than said product, wherein said drug is for the treatment of a brain tumor and / or epilepsy associated with a brain tumor. Specifically, the drug may be used for the treatment of thyroid cancer and / or thyroid-associated epilepsy. For example, the drug may be used for the treatment of thyroid cancer and / or thyroid-associated epilepsy. The present invention also relates to a combination of a product with a drug other than said product, for separate or sequential use in the treatment of a brain tumor and / or epilepsy associated with a brain tumor, said drug being for the treatment of a brain tumor and / or epilepsy associated with a brain tumor and said product being consisting of - an antibody or an antiqene-binding fragment thereof, - a nucleotide sequence encoding the antibody or the fragment encoding the antibody, - an expression vector comprising said nucleotide sequence, or - a pharmaceutical composition comprising said IC / A138842 Barzanò & Zanardo 17antibody 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). This brain tumor may be a glioma, such as grade IV glioma, for example glioblastoma. Preferably, said drug may be a drug for the treatment of glioma and / or glioma-associated epilepsy. For example, said drug may be a drug for the treatment of glioblastoma and / or associated epilepsy. 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, while sequential use means the administration IC / A138842 Barzanò & Zanardo successive formulations of the two compounds of the combination according to the invention, each in a distinct pharmaceutical form. According to the present invention, said product object of the combination may be a product as defined in any of claims 1-12. The present invention will now be described, for illustrative but not limitative purposes, according to a preferred embodiment thereof, with particular reference to the examples and figures of the attached drawings, in which: - Figure 1 shows tumor growth following intracortical inoculation of GL261 cells in C57BL / 6J mice. (A) Mouse maintained in a stereotaxic apparatus under gas anesthesia (2% isoflurane). (B) Stereomicroscope image to document intracortical inoculation of GL261 cells; a glass microcapillary containing the suspended cells is inserted through a 0.5 mm diameter hole drilled in the cranial bone (craniotomy) at predefined stereotaxic coordinates (AP -1.00 mm, ML ±1.50 mm, DV -1.10 mm, relative to Bregma). (C) Confocal immunofluorescence images of a coronal brain section obtained from a mouse with a tumor developed from intracortical inoculation of 4*104 GL261 cells; The nuclei were stained with DAPI (4',6-diamidino-2-phenylindole, MolecularProbes D1306), which clearly highlights the tumor area. The section was also stained with Factin (ActinGreen 488 ReadyProbes, Termofisher). IC / A138842 Barzanò & Zanardo ^H Scientific R37110); the arrow indicates a putative tumor nanotube revealed by this staining. Unpublished data from the PI's laboratory - Figure 2 shows Cx and GFAP expression in the peritumoral region of glioma mice. Representative confocal co-immunofluorescence images of some Cx and GFAP expression in coronal sections of tumors grown in the brains of C57BL / 6J mice and examined two weeks after intracortical inoculation of 4*104 GL261 cells. Each column shows the same section stained with a primary anti-Cx antibody (top row), with an anti-GFAP antibody (middle row), and with DAPI (bottom row) to better highlight the tumor area. The primary anti-Cx 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.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 fibrillary acidic protein is a marker of tumor-associated reactive astrocytes [8] (primary anti-GFAP antibody: BD Pharmingen 556327, diluted 1:50; secondary antibody Goat anti-Mouse: Alexa Fluor 488 Thermo Fisher Scientific A11029, diluted 1:800). Unpublished data from the laboratory of the PI - Figure 3 shows the expression in healthy mice IC / A138842 Barzanò & Zanardo -20 of abEC1.1(scFv-mFc) antibody following ICV administration of AAV8-abEC1.1(scFv-mFc) at P0.5. Representative confocal immunofluorescence images of abEC1.1(scFvmFc) antibody expression in sagittal sections of mouse brains, 33 days after ICV administration of AAV8-abEC1.1(scFvmFc) (6*1010GC) at P0.5. In each column, the same section is shown stained with a secondary antibody conjugated to Alexa Fluor 488 (ThermoFisher Scientific A11029, diluted 1:300) that recognizes the mFc domain of abEC1.1 (scFv-mFc) (top row) and with DAPI (bottom row) to highlight the presence of cells even in the absence of a signal due to the secondary antibody. Unpublished data from the laboratory of the PI - Figure 4 shows the effect of AAV8-abEC1.1(scFv-mFc) administration on tumor growth in glioma mice. (A) Schematic diagram illustrating the timeline of the experimental protocol (P, postnatal day). At P0.5, C57BL / 6J mice in the treatment group were subjected to intracerebroventricular (ICV) injection with 6*1010GC of AAV8-abEC1.1(scFv-mFc). Subsequently, mice from each group, treated with AAV8-abEC1.1(scFv-mFc) and untreated, were subjected to intracortical inoculation of 4*104 GL261 cells at P75; brain histological analysis was performed after euthanasia at P90. (B) DAPI staining of coronal brain sections highlights tumor development. Images are IC / A138842 Barzanò & Zanardo ^— refer to sections taken at intervals of approximately 250 μm around the site of inoculation of GL261 cells. (C) Left: Table of tumor volumes for each treatment type and corresponding animal identification code; right: Distributions of tumor volume data showing the mean (wide horizontal bars) ± standard error of the mean (sem); p-value = 0.0025 (two-sample t-test). Significant differences are indicated by * = p-value < 0.05, ** = p-value < 0.005. Unpublished data from the PI's laboratory - Figure 5 shows the effect of AAV8-abEC1.1(scFv-mFc) administration on tumor morphology and infiltration in glioma mice. Images show confocal immunofluorescence of tumors originating from GL261 cells in coronal brain sections of C57BL / 6J mice treated (bottom row) with 6*1010GC of AAV8-abEC1.1(scFv-mFc) at P0.5 and their untreated controls (top row). Mice in each group (treated and untreated) were intracortically inoculated with 4*104 GL261 cells at P75, and brain analysis was performed after euthanasia at P90. GFAP, glial fibrillary acidic protein, is a marker of tumor-associated reactive astrocytes [8] (primary anti-Gfap antibody: BD Pharmingen 556327, diluted 1:50; secondary antibody Goat anti-Mouse: Alexa Fluor 488 Thermo Fisher Scientific A11029, diluted 1:800). Nuclei were stained with DAPI to better highlight the tumor area. Unpublished data from IC / A138842 Barzanò & Zanardo ^— PI laboratory - Figure 6 shows the effect of AAV8-abEC1.1(scFv-mFc) administration on tumor cell proliferation in glioma mice. (A) Representative confocal immunofluorescence images of GL261-derived tumors in coronal brain sections of mice treated with 6*1010GC of AAV8-abEC1.1(scFv-mFc) at P0.5 and their untreated controls. Mice in each group (treated and untreated) were intracortically inoculated with 4*104 GL261 tumor cells at P75, and brain histological analysis was performed after euthanasia at P90. Nuclei were stained with DAPI (left). Ki-67 (right) is a proliferation marker (primary antibody antiKi-67: Thermo Fisher PA5-19462, diluted 1:100; secondary antibody: Donkey anti-Rabbit, Alexa Fluor 488 Thermo Fisher Scientific A-21206, diluted 1:800). (B) High-magnification images of the tumor areas delimited by the white squares in A.(C) Representative confocal images of coronal brain sections labeled with secondary antibody only in tumor areas of mice treated with AAV8-abEC1.1(scFvmFc) at P0.5 and their untreated controls. (D) Quantification of Ki-67-positive nuclei (Ki67 index, Ref.

[55] ) from mice treated with AAV8-abEC1.1(scFvmFc) at P0.5 (n=5 mice, 3 sections each) and their untreated controls (n=4 mice, 3 sections each) shown as mean ± standard error of the mean (s.e.m.); p-value = 0.01 (two-sample t-test). IC / A138842 Barzanò & Zanardo ^— Significant differences are indicated by * = p-value < 0.05. Unpublished data from the PI laboratory. - Figure 7 shows the effect of AAV8-abEC1.1(scFv-mFc) administration on the survival of glioma mice. Mice in each group (treated and untreated) were intracortically inoculated with 6*104 GL261 cells at P75. (A) Mean daily weight change on the day of GL261 cell inoculation; comparison between untreated C57BL / 6J mice (n=6, solid line and circle), injected ICV at P0.5 with 6^1010GC of AAV8empty, not expressing the antibody (n=14, dotted line and triangle), and injected ICV at P0.5 with 6χ1010GC of AAV8-abEC1.1(scFv-mFc) (n=12, dashed line and square). Below are p-values ​​calculated point by point for each treatment pair; differences are significant below the horizontal dashed line (p=0.05). (B) Kaplan-Mayer plots for each treatment (Untreated, solid line; AAV8-empty, dotted line; AAV8abECl.l, dashed line).(C) Distribution of survival time calculated from the date of GL261 cell inoculation for the three treatment groups (Untreated, circle; Treated with AAV8-empty, triangle; Treated with AAV8-abEC1.1, square) showing the median with its interquartile range. Significant differences are indicated by * = p-value < 0.05, ** = p-value < 0.005. Unpublished data from the PI's laboratory. - Figure 8 shows the biodistribution IC / A138842 Barzanò & Zanardo -24of the abEC1.l 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) (3^1011GC) and the untreated control. (A) Representative sagittal section of the antibody distribution. (B) Representative high-magnification images of some brain areas in which the antibody is particularly expressed. Carboxy-terminally alpha-tagged abEC1.1 was detected using an anti-alphatag primary antibody (NanoTag N1583, diluted 1:200) and a donkey anti-rabbit secondary antibody (Alexa Fluor 488, ThermoFisher Scientific A-21206, diluted 1:500). Nuclei were stained with DAPI. Unpublished data from the PI's lab - Figure 9 shows colocalization of abEC1.Cx26 in the peritumoral region following retro-orbital administration of AAV-PHP.eBabEC1.1(scFv-mFc-Atag) in glioma mice. (A) Representative DAPI staining images of tumors originating from intracortical inoculation of 4*104 GL261 cells at P75 in coronal brain sections of 3-month-old mice, two weeks after treatment by concomitant retro-orbital intravenous injection of AAV-PHP.eB-abEC1.1(scFv-mFc-Atag) (3^1011GC), and their untreated control. (B) Representative staining images by. IC / A138842 Barzanò & Zanardo ^— immunofluorescence of the area delimited by the white square in A (primary antibody anti-Cx26: Thermo Fisher Scientific 13-8100, diluted 1:100; secondary antibody Donkey anti-Mouse: Alexa Fluor 555, Thermo Fisher Scientific A-31570, diluted 1:800. Primary antibody anti-ALFA-taq: NanoTaq N1583, diluted 1:200; secondary antibody Donkey anti-Rabbit: Alexa Fluor 488, ThermoFisher Scientific A-21206, diluted 1:500. (C) High-resolution images of the area delimited by the white square in A; arrows highlight the colocalization of Cx26 and abEC1.1(scFv-mFc-Ataq). Unpublished data from the laboratory of the PI - Figure 10 shows the biodistribution of the antibody after CED administration of purified abEC1.1-mIGg1-Atag in the striatum of healthy mice. (A) Biodistribution of the carboxy-terminal alpha-taq-labeled abEC1.1 antibody (abEC1.1mIqG1-Ataq) in coronal brain sections of 3-month-old mice 2 hours after CED administration of purified abEC1.1-mIqG1-Ataq. (B) Representative high-resolution images of the regions indicated in the white boxes show the distribution of the antibody in brain tissue at the injection site and in the contralateral hemisphere. abEC1.1mIqG1-Ataq was detected using a primary antibody against ALFA-taq (NanoTaq N1583, diluted 1:200) and a secondary antibody against Donkey Rabbit (Alexa Fluor 488, ThermoFisher Scientific A-21206, diluted 1:500). Nuclei were stained with DAPI. Unpublished data from the laboratory of the PI IC / A138842 Barzanò & Zanardo ^— - Figure 11 shows the biodistribution of purified abEC1.1-mIGg1-Atag antibody after CED administration in the striatum of glioma mice. (A) Representative images of distribution of carboxy-terminal alpha-tag-labeled abEC1.1 antibody (abEC1.1-mIgG1-Atag) in coronal brain sections of 3-month-old mice inoculated with 6*104GL261 tumor cells in the striatum and treated with repeated intratumoral CEDs of purified abEC1.1-mIgG1-Atag (5 mg / ml, 10 μl). Brain sampling for analysis was performed 21 days after cell implantation, 48 hours after the last treatment. (B) Magnification of the tumor area indicated in the white box in A. (C) High-magnification representative image of the region indicated in the white box in B, showing the distribution in the peritumoral zone of abEC1.1-mIgG1Atag; the antibody was detected using an anti-ALFA-tag primary antibody (NanoTag N1583, diluted 1:200) and a Donkey anti-Rabbit secondary antibody (Alexa Fluor 488, ThermoFisher Scientific A21206, diluted 1:500). Nuclei were stained with DADI. Unpublished data from the laboratory of the PI - Figure 12 shows the effect of intratumoral treatments with purified abEC1.1-mIgG1-Atag antibody on tumor cell proliferation in glioma mice. (A) Representative confocal immunofluorescence images of intrastriatal GL261 tumors (obtained by inoculation of 6*104 GL261 cells) in coronal brain sections of mice. IC / A138842 Barzanò & Zanardo ^— a P75 treated with purified abEC1.1-mIgG1-Atag (5 mg / ml, 10 μA), administered via intratumoral CED repeated every 4 days starting 7 days after inoculation of GL261 cells and relative controls injected with saline solution. Nuclei were stained with DAPI (left). PH3 (right) is a known proliferation marker (primary antibody anti-PH3: Thermo Fisher PA5-17869, diluted 1:100; secondary antibody: Donkey antiRabbit, Alexa Fluor 488 Thermo Fisher Scientific A21206, diluted 1:800). (B) High-magnification images of the tumor areas delimited by the white squares in A. (C) Quantification of PH3-positive nuclei from mice treated with purified abEC1.1-mIgG1-Atag (n=3 mice, 3 sections each) and their saline-injected controls (Untreated, n=2 mice, 3 sections each) shown as mean ± standard error of the mean (s.e.m.). (D) Quantification of tumor invasion area in mice treated with purified abEC1.1-mIgG1-Atag (n=4 mice, 3 sections each) and saline-injected controls (Untreated, n=3 mice, 3 sections each) shown as mean ± standard error of the mean (sem). Differences were considered significant if p<0.05, denoted by *; **, p<0.01; ***, p<0.001. Unpublished data from the PI's laboratory. - Figure 13 shows the analysis of patch-clamp electrophysiology experiments. Quantitative analysis of the frequency (A), amplitude (B), and decay time (C) of mEPSCs recorded in IC / A138842 Barzanò & Zanardo ^— co-cultures of neurons-astrocytes (CTRL) and neuroniastrocytes-GBM (GBM) treated or not with abEC1.1(scFv-mFc) (1 μM) for 45 min. (D) MU frequency analyzed in co-cultures of neurons-astrocytes (CTRL) and neurons-astrocytes-GBM (GBM) before and 45 min after abEC1.1(scFv-mFc) application. Each point represents the mean value of the EPSC frequency collected from a single neuron; data are expressed as mean ± s.e.m. Differences were considered significant if p<0.05, denoted by *; **, p<0.01; ***, p<0.001. Unpublished data from the laboratory of the PI and collaborators. - Figure 14 shows the frequency of mEPSCs in neuron-astrocyte co-cultures. (A) Frequency of mEPSCs recorded from astrocyte-neuron co-cultures exposed to vehicle or abEC1.1(scFv-mFc) (1 μM) for 45 min. Note that abEC1.1 selectively affects mEPSC frequency and not amplitude (B). Pure hippocampal neurons at DIV 14 treated for 45 min with abEC1.1(scFv-mFc) 1 μM show no change in mEPSC frequency and amplitude (C, D). Each point represents the mean value of EPSC frequency recorded from a single neuron; data are expressed as mean ± s.e.m. *, p<0.05. Unpublished data from the laboratory of the PI and collaborators. EXAMPLE 1. Study on the efficacy of the anti-tumor drug abEC1.l on glioma models and associated epilepsy. With reference to the obligations set forth in Article 170-bis of the Property Code IC / A138842 Barzanò & Zanardo ^— Industrial, with regard to paragraph 2, the source of the biological material of animal origin and the cell lines is indicated further down in the experimental section. With regard to paragraph 4, it is declared that the use of genetically modified microorganisms occurred in compliance with the obligations regarding such modifications, deriving from national or EU regulations. To test the efficacy of abEC1.1 in the treatment of tumors, the glioma 261 (GL261) model was implemented [46-49] which is an orthotopic and immunocompetent mouse model of human GBM widely used in preclinical research to study the pathophysiology of GBM, to identify therapeutic interventions, to evaluate new treatment modalities (including immunotherapy) and to analyze tumor biology and progression

[49] . 4^104 GL261 cells (Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Cell line: GL-261, DSMZ no. ACC 802) were then injected intracortically into C57BL / 6J mice bred in the EMMA (European Mouse Mutant Archive) facility of the National Research Council (CNR), Monterotondo, Rome (Fig. 1). Two weeks later, labelling with commercial anti-Cx antibodies (which, unlike abEC1.1, recognise different intracellular epitopes of the respective Cxs) revealed the expression of Cx26 mainly at the level of the peri-tumoral area, while IC / A138842 Barzanò & Zanardo ^— Cx30, Cx43 and Cx46 were also expressed within the tumor mass (Fig. 2). Once it was confirmed that different GBM-relevant Cxs were expressed in the animal model, the delivery route of abEC1.l was evaluated. In particular, it was reasoned that a mAb can be produced directly in an organism following the introduction of a nucleic acid encoding the mAb into the organism's cells, thus allowing a continuous and potentially long-term delivery 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, CNS tropism and long-term therapeutic effect

[52] . The term AAV-mAb refers to a specific therapeutic approach combining AAVs with mAbs for potential medical applications

[52] . Experiments were then conducted using a recombinant AAV8 vector encoding a secreted variant of abEC1.1, consisting of a human HCs-binding domain of Cxs in the form of a single-chain variable fragment (scFv) linked to a murine crystallizable fragment (Fc) (mFc)

[40] . The human single-chain variable fragment of abEC1.1 used as a reference for the development of the vector to be administered to mice includes the IC / A138842 Barzanò & Zanardo following nucleotide sequences reported in Table 3: Table 3 SEQ ID NO:15 VH CAGGTACAGCTGCAGCAGTCAGGGGGGGGCGT GGTCCAGCCTGGGAGGTCCCTGAGACTCTCCT GTGCAGCCTCTGGATTCACCTTCAGTAGCTAT GCTATGCACTGGGTCCGCCAGGCTCCAGGCAA GGGGCTGGAGTGGGTGGCAGTTATATCACATG GTGGAAGTAATAAATACTACGCAGACTCCGTG AAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCC TGAGAGCTGAGGACACGGCTGTGTATTACTGT GCGAGAGATTTTAGTTGGAGAGGGTACTACAT GGACGTCTGGGGCAAAGGCACCCTGGTCACCG TCTCCTCA SEQ ID NO:17 Linker GGCAGCGGCGGTGGCGGATCC SEQ ID NO:16 VL GAAAC GACACTCACGCAGTCTCCAGCCACCCT GTCTTTGTCTCCAGGGGAAAGAGCCACCCTCT CCTGCAGGGCCAGTCAGAGTATTAGCAGCTAC TTAGCCTGGTACCAGCAGAAACCTGGCCAGGC TCCCAGGCTCCTCATCTATGGTGCATCCACCA GGGCCACTGGCATCCCAGACAGGTTCAGTTGGC AGTGGGTCTGGGACAGACTTCACTCTCACCAT CAGCAGACTGGAGCTGAAGAT TT TGCAGTGT ATTACTGTCAGCAGTATGGTAGCTCACCTCGA ACTTTCGGCGGAGGGACCAAGGTGGAAATCAA ACGT The nucleotide sequences reported in Table 3 code respectively for the following amino acid sequences reported in Table 4: Table 4 IC / A138842 Barzanò & Zanardo SEQ ID NO:7 VH QVQLQQSGGGVVQPGRSLRLSCAASGFTFSSY AMHWVRQAPGKGLEWVAVISHGGSNKYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ARDFSWRGYYMDVWGKGTLVTVS S SEQ ID NO:9 Linker GSGGGGS SEQ ID NO:8 VL ETTLTQSPATLSLSPGERATLSCRASQSISSY LAWYQQKPGQAPRLLIYGASTRATGIPDRFSG SGSGTDFTLTISRLEPEDFAVYYCQQYGSSPR TFGGGTKVEIKR The nucleotide sequence of the human single-chain variable fragment was optimized for expression in mice under the GAG ​​promoter, encoding 5 amino acid sequences reported in Table 4. Furthermore, to allow antibody secretion by cells infected by the viral vector, the optimized nucleotide sequence of abEC1.1(scFv-mFc) was fused downstream to the 10 nucleotide sequence of the human interleukin-2 (IL2ss) secretion signal, as detailed in Table 5: Table 5 SEQ ID NO:18 IL2ss (DNA nucleotide sequence) ATGTACAGGATGCAACTCCTG TCTTGCATTGCACTAAGTCTT GCACTTGTCACGAATTCG SEQ ID NO:19 IL2ss (amino acid sequence) MYRMQLLSCIALSLALVTNS Intracerebroventricular injection (ICV)

[53] of IC / A138842 Barzanò & Zanardo ^H 6x1010genomic copies (GC) of AAV8-abEC1.1(scFv-mFc) in C57BL / 6J mice at day 0.5 postnatal (P) (stereotaxic coordinates: AP +1.50 mm, ML ±0.80 mm, DV -3.00 mm, relative to Lambda), subsequently determined a widespread expression of the antibody produced and secreted in situ, in the brain, by infected cells of the cortex, hippocampus and cerebellum (Fig. 3). ICV injection consists in the injection of substances into one of the cerebral ventricles using a 100 gl syringe with a 33G needle at predefined stereotaxic coordinates (AP +1.50 mm, ML ±0.80 mm, DV -3.00 mm, relative to Lambda). Mice from another group subjected to the same treatment, i.e. 6x1010GC of AAV8-abEC1.1(scFv-mFc) at day P0.5, were intracortically inoculated with 4x104 GL261 cells at day P75, allowing tumor growth until day P90, when mice were sacrificed for postmortem brain analyses (Fig. 4). The results demonstrated not only a significant reduction in glioma volume in mice treated with AAV8-abEC1.1(scFv-mFc) compared to untreated controls (Fig. 4), but also a lower tumor invasiveness, as deduced from the more rounded shape of the peritumoral profile, as well as from the absence of necrotic areas and a reduced density of GFAP-positive reactive astrocytes within the tumor mass (Fig. 5). Furthermore, the results described above correlate with a significant reduction in immunoreactivity IC / A138842 Barzanò & Zanardo ^— of the KI-67 antigen (Fig. 6), a widely used proliferation marker also known as Ki-67 or MKI67 (Ki-67 Proliferation Marker)

[54] , indicating that treatment with AAV8abEC1.1(scFv-mFc) reduced tumor cell proliferation. The results shown were confirmed by studying the survival of tumor-bearing C57BL / 6J mice, dividing them into 3 groups: untreated animals, animals subjected to ICV injection at P0.5 with 6*1010GC of AAV8empty, i.e., the virus with the same serotype but without the antibody, which serves as an additional negative control, and animals subjected to ICV injection at P0.5 with 6*1010GC of AAV8-abEC1.1(scFv-mFc). After intracortical inoculation of 6^104 GL261 cells, the animals were weighed daily and sacrificed when their weight had decreased by 20% compared to the weight on the day of inoculation or when they showed evident signs of distress. The mean trend of weight change is reported in Fig. 7 A, in which animals treated with AAV8-abEC1.1(scFv-mFc) appear to lose weight significantly more slowly than the two controls.Furthermore, median survival measured from the day of tumor cell implantation was significantly longer for animals treated with AAV8-abEC1.1(scFv-mFc) (31 days, n=12) than for both animals injected with AAV8-empty (26.5 days, n=14) and untreated animals (25.5 days, n=6), prolonging life by an average of 5 days (Fig. 7. IC / A138842 Barzanò & Zanardo ^— B, C) compared to controls. As a further therapeutic possibility, the use of a vector with AAVPHP.eB capsid, derived from AAV9, which is able to cross the blood-brain barrier (BBB) ​​in C57BL / 6J mice

[57] was then evaluated. For these experiments, the coding sequence for the antibody optimized for expression in mice was inserted into the plasmid of the AAV-PHP.eB vector, under the short murine GFAP promoter

[58] , in scFv-mFc format with IL2ss and ALFA-tag

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

[59] of 3^1011GC of AAV-PHP.eBabEC1.1(scFv-mFc-Atag) in healthy adult mice, consistent expression of the antibody selectively in brain astrocytes was obtained (Fig. 8).By immunofluorescence analysis, it was possible to establish that the antibody, produced in situ in the brain by infected astrocytes and secreted by them, colocalizes with Cx26 in the peritumoral region in mice subjected to intracortical injection of 4*104 GL261 cells and concomitant intravenous retro-orbital injection of AAV-PHP.eB-abEC1.1(scFv-mFc-Atag) (3^1011GC) (Fig. 9). Furthermore, to overcome the obstacle posed by the BBB, convection-optimized delivery (CED) experiments were conducted using the purified murine IgG1 variant of the antibody, labelled IC / A138842 Barzanò & Zanardo ^— with ALFA-tag (abEC1.1-mIgG1-Atag). In this case, the striatum was chosen as the site for both tumor cell inoculation and subsequent treatments (coordinates from Bregma: AP=+0.86 mm, ML=-1.8 mm, DV=-3.0 mm). Since the striatum is located deeper in the brain of the experimental animal, injection via the CED presents a lower probability of the injected fluid rising through the injection channel (with consequent dilution in the cerebrospinal fluid in the subarachnoid space), which could cause a lower treatment efficiency. To define the timing of repeated treatments, biodistribution experiments were preliminarily conducted by injecting 10 gl of purified abEC1.1-mIgG1-Atag at a concentration of 5 mg / ml into the striatum of healthy mice via CED. The animals' brains were then harvested 2 hours after CED. Immunofluorescence images obtained by confocal microscopy of brain sections show a very intense and concentrated antibody signal in the injection region 2 hours after CED (Fig. 10). In another group of tumor-bearing mice, 4 treatments every 4 days with abEC1.1mIgG1-Atag were then performed starting from the seventh day after the inoculation of 6*104 GL261 cells into the striatum. The animals were sacrificed on day 21 after tumor implantation (48 hours after the last treatment) to evaluate their size and IC / A138842 Barzanò & Zanardo ^— cell proliferation compared to untreated animals (CED of saline solution). Analysis of brain sections of these animals revealed the presence of abEC1.1mlgGl-Atag within the tumor microenvironment (Fig. 11). Furthermore, a significant reduction in tumor size was observed in the treated animals, with the surface area in the central sections being approximately 3 times smaller than in the untreated case. This finding was also confirmed by the reduction in tumor invasiveness in the treated animals compared to the untreated ones, quantified by the cell proliferation marker phosphohistone-H3 (PH3)

[60] , which was approximately 2.5 times smaller (Fig. 12). A last but important aspect that has been taken into consideration in these studies is the link between GBM and epilepsy

[61] , and between the latter and Cxs HCs

[2934] , since antitumor therapy may contribute to the control of epileptic manifestations and may therefore exert beneficial effects on the treatment of both glioma / GBM and associated epilepsy

[29] . To explore this therapeutic possibility, a 2D primary co-culture system of astrocytes and neurons isolated from the mouse hippocampus was established. Briefly, cells isolated from litters of E18 embryonic stage (E) mice were cultured separately: hippocampal neurons were plated at medium density on slides pretreated with IC / A138842 Barzanò & Zanardo ^— poly-L-lysine and cultured in neuronal culture medium containing B27 supplement and glutamate, while astrocytes were plated in flasks with astrocyte culture medium containing 10% fetal bovine serum (FBS). To obtain astrocyte-neuron cocultures, astrocytes were detached from the flasks, counted, and then placed on neurons at 710 days of in vitro culture (DIV). The coculture was maintained for approximately seven days to allow astrocyte sheet formation. Finally, we plated GL261 cells on the cocultures and performed patch-clamp electrophysiological recordings on the cultured neurons within 3 hours of culture (Fig. 13). These experiments have detected miniature excitatory postsynaptic currents (mEPSCs) occurring at excitatory synapses between neurons. The term miniature refers to the small amplitude of these currents, which occur spontaneously, independently of external stimulation. These currents, recorded in the presence of 1 μM tetrodotoxin (TTX) to prevent the generation of action potentials, result from the release of neurotransmitters, such as glutamate, from presynaptic vesicles, even in the absence of action potentials. mEPSCs are considered the basal level of excitatory synaptic activity and correlate with the random release of neurotransmitters at the level of a single synapse

[62] . We also measured on adhesion cells IC / A138842 Barzanò & Zanardo ^— 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 Hertz (Hz)

[63] . As highlighted in Fig. 13, the frequency of mEPSCs (panel A) and MUs (panel D) was significantly increased when co-cultures of neurons and astrocytes were incubated for 3 hours with GL261 cells, confirming the role of glioma in generating neuronal rearrangements and aberrant action potential spikes. Electrophysiological recordings in co-cultures of neurons, astrocytes, and GL261 tumor cells after 45 minutes of incubation with abEC1.1(scFv-mFc) demonstrated a positive effect of the antibody in normalizing both the frequency of mEPSCs and the increase in MU activity. Note that neither the presence of GL261 nor the administration of abEC1.1(scFv-mFc) in the presence of GL261 caused changes in the amplitude (Fig. 13, B) or in the exponential decay time constant of mEPSCs (Fig. 13, C). As highlighted in Fig. 13, the frequency of mEPSCs (panel A) and MUs (panel D) was significantly increased when neuron-astrocyte cocultures were incubated for 3 hours with GL261 cells, confirming the role of glioma in generating neuronal rearrangements and aberrant action potential spikes. Electrophysiological recordings in neuron cocultures, IC / A138842 Barzanò & Zanardo ^— Astrocytes and GL261 tumor cells after 45 minutes of incubation with abEC1.1(scFv-mFc) demonstrated a positive effect of the antibody in normalizing both the frequency of mEPSCs and the increase in MU activity. Note that neither the presence of GL261 nor the administration of abEC1.1(scFv-mFc) in the presence of GL261 determined changes in the amplitude (Fig. 13, B) or in the exponential decay time constant of mEPSCs (Fig. 13, C). In order to demonstrate that the effect of the mAb is mediated by astrocytes, we compared the changes induced by abEC1.1(scFv-mFc) on excitatory transmission in (i) unstimulated neuron-astrocyte co-cultures and (ii) unstimulated pure neuron cultures. While co-cultures exposed to abEC1.1(scFv-mFc) demonstrated a consistent reduction in mEPSC frequency (Fig. 14, AB), the mEPSC frequency measured in hippocampal neurons at DIV 14 in the presence of abEC1.1(scFv-mFc) did not undergo changes (Fig. 14, CD). These results indicate that the effects induced by abEC1.1(scFv-mFc) on glutamatergic synaptic activity depend on its selective action on astrocytic Cxs, while abEC1.1(scFv-mFc) does not modify neuronal activity per se. Bibliography .Louis, DN, et al., The 2021 WHO Classification of Tumors of the Central Nervous System: a summary. Neuro-Oncology, 2021. 23(8): p. 1231-1251. 2. 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The present invention has been described for illustrative but not limitative purposes, according to its preferred embodiments, but it is to be understood that variations and / or modifications may be made by those skilled in the art without thereby departing from the relevant scope of protection, as defined by the attached claims.

Claims

CLAIMS 1) Product for use in the treatment of a brain tumor and / or epilepsy associated with a brain tumor, 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 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 IC / A138842 Barzanò & Zanardo ^— according to claim 1, wherein said brain tumor is a glioma, such as grade IV glioma, e.g. glioblastoma. 3) A product according to any preceding claim for use according to any preceding claim, wherein said VH comprises or consists of the sequence QVQLQQSGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISHGG SNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDFSWRGYYMDVW GKGTLVTVSS (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 preceding claim for use according to any preceding claim, wherein said antigen-binding fragment is selected from scFv, (scFv)2, scFv-Fc, Fab, Fab', 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 of sequence SEQ ID NO:7 and a VL of sequence SEQ ID NO:8 linked together by a linker, for example by a linker of sequence GSGGGGS (SEQ ID NO:9). 6) Product according to any preceding claim for use according to any preceding claim, wherein said expression vector is a viral vector. IC / A138842 Barzanò & Zanardo ^— 7) Product according to claim 6, for use according to claim 6, wherein said viral vector is an adeno-associated virus (AAV), such as a capsid-enveloped AAV of serotype AAV1, or AAV2, or AAV5, or AAV8, or AAV9, or AAVrh8, or AAVrhlO, or AAVHu68, or AAVHSC15, or AAVSNY001, or AAV.CAP-B10, or AAV.CAP-Mac, or AAV.CAP-Mac9, or BI-hTFR1. 8) A product according to any preceding claim for use according to any preceding claim, wherein the nucleotide sequence coding for SEQ ID NO:2 is GGATTCACCTTCAGTAGCTATGCT (SEQ ID NO:10), the nucleotide sequence coding for SEQ ID NO:3 is ATATCACATGGTGGAAGTAATAAA (SEQ ID NO:11), the nucleotide sequence coding for SEQ ID NO:4 is GCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTC (SEQ ID NO:12), the nucleotide sequence coding for SEQ ID NO:5 is CAGAGTATTAGCAGCTAC (SEQ ID NO:13), the nucleotide sequence coding for GAS is GGTGCATCC, and the nucleotide sequence coding for SEQ ID NO:6 is CAGCAGTATGAGCTCACCTCGAACT (SEQ ID NO:14). 9) Product according to any of the preceding claims for use according to any of the preceding claims,where the coding nucleotide sequence SEQ ID NO: 7 (VH) is CAGGTACAGCTGCAGCAGTCAGGGGGGGGCGTGGTCCAGCCTGGGAGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGT CCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCACATGGTGGA AGTAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGC IC / A138842 Barzanò & Zanardo ^— TGTGTATTACTGTGCGAGAGATTTTAGTTGGAGAGGGTACTACATGGACGTCTGG GGCAAAGGCACCCTGGTCACCGTCTCCTCA (SEQ ID NO: 15) and the coding nucleotide sequence SEQ ID NO: 8 (VL) is GAAACGACACTCACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAG CCACCCTCTCCTGCAGGGCCAGTCAGAGTATTAGCAGCTACTTAGCCTGGTACCA GCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCACCAGGGCC ACTGGCATCCCAGACAGGTTCAGTGGCAGTGGTCTGGGACAGACTTCACTCTCA CCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGG TAGCTCACCTCGAACTTTCGGCGGAGGGACCAAGGTGGAAATCAAACGT ( SEQ ID NO: 16)., 10) A product according to any preceding claim for use according to any preceding claim, wherein said nucleotide sequence further comprises a signal sequence for secretion of the antibody, such as the human interleukin-2 secretion signal sequence. 11) Product according to any preceding claim for use according to any preceding claim, wherein said product is administered by convection-optimized intraparenchymal administration (CED), wherein said product consists of said antibody or fragment thereof or said pharmaceutical composition comprising said antibody or fragment thereof. 12) Product according to any of claims 1-10 for use according to any 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 intracisterna 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) A product according to any preceding claim for use according to any preceding claim, wherein said pharmaceutical composition further comprises a drug other than said product, wherein said drug is for the treatment of a brain tumor and / or epilepsy associated with a brain tumor. 14) Combination of a product with a drug other than said product, for separate or sequential use in the treatment of a brain tumor and / or epilepsy associated with a brain tumor, said drug being for the treatment of a brain tumor and / or epilepsy associated with a brain tumor, 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, IC / A138842 Barzanò & Zanardo ^— 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) The combination of claim 14, for use according to claim 14, wherein said brain tumor is a glioma, such as grade IV glioma, e.g. glioblastoma.