Antibody Delivery

Abstract

The vector comprises a polynucleotide encoding an antibody or antibody fragment for use in a method of treating a disease or disorder of the central nervous system (CNS) in a subject, the vector transduces or transfects cells of the blood-brain barrier (BBB), and the transduced or transfected BBB cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS, preferably into the brain parenchyma. An expression cassette useful in such a vector comprises at least one promoter operably linked from 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, and further comprises an IRES after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment, or a first promoter operably linked to the first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to the second gene encoding the heavy chain of the antibody or antibody fragment. Antibodies and antibody fragments produced in this manner can be of higher quality and exhibit lower levels of aggregation and undesirable immunogenicity.

Description

[Technical field]

[0001] The present invention relates in particular to a means and method for effectively delivering genes encoding antibodies or antibody fragments to cells from the blood-brain barrier (BBB) ​​or the central nervous system (CNS), such as brain endothelial cells, via a vector, such as a viral vector, and producing antibody molecule(s) in the cells, which are released into the CNS, preferably into the brain parenchyma. By delivering antibody genes to the BBB or CNS, particularly to brain endothelial cells, the present invention also relates to a method for increasing antibody concentration in the central nervous system (CNS). Delivery of therapeutic antibodies by this approach is useful for treating various diseases or disorders originating from the CNS, such as neurodegenerative diseases or disorders, movement diseases or disorders, brain-related tumors, psychiatric diseases and CNS neuroinflammation. In one aspect, the present invention derives from the unexpected discovery that transduction of brain endothelial cells in vitro with a vector, such as an adeno-associated virus (AAV) vector, results in the secretion of large amounts of high-quality antibodies into the basolateral space. The present invention also describes surprisingly high antibody expression yields and improved protein quality by alternating chain positions and customizing upstream, mid- and downstream regulatory elements with different secretory peptides. The present invention avoids the difficulties associated with the delivery of therapeutic antibodies to the brain due to the need to cross the blood-brain barrier at sufficient therapeutic doses. The present invention is applicable to any mammalian subject, particularly human subjects, and aims to improve the delivery of therapeutic antibodies to the brain. Thus, the present invention can be used to treat diseases, disorders or conditions in patients suffering from diseases or disorders of the CNS, including, but not limited to, diseases associated with amyloid-beta protein, TDP-43-proteinopathy, alpha-synucleinopathy, tauopathy, trinucleotide repeat disorders including polyglutamine disorders, such as Huntington's disease, brain-related cancers and tumors, epilepsy, psychiatric disorders, neuroinflammatory disorders, neuromuscular disorders, viral-induced encephalitis, and diseases characterized by microglial dysfunction. [Background technology]

[0002] The blood-brain barrier (BBB) ​​is a structural and functional barrier that protects the brain from blood-borne pathogens and toxins but maintains a tightly regulated microenvironment necessary for proper neuronal function to the central nervous system (CNS) [1,2]. The BBB is composed of three cell types: endothelial cells (ECs), which form a physical barrier between the bloodstream and the brain, and pericytes and astrocytes, two mural cells located on the abluminal surface of the EC layer [3]. Although endothelial cells contribute the primary function of the BBB, these three cell types constitute the entire blood-brain barrier in most vertebrates. Their interactions and signaling are critical for maintaining tightly controlled CNS homeostasis.

[0003] Brain endothelial cells have unique properties compared to endothelial cells: they are held together by tight junctions (TJs), a multiprotein junction network that limits the diffusion of compounds through what is considered the paracellular space. While small molecules such as water, oxygen, and small lipid-soluble substances can easily pass from the blood to the brain, tight junctions prevent the diffusion of larger molecules, including antibodies, into the brain [4].

[0004] This tightly controlled brain entry is one of the main challenges in the development of biologics for diseases involving the central nervous system (CNS). Indeed, it has been reported that with peripheral administration, approximately 0.1–0.3% of injected antibodies reach the brain [5][6]. Even when high doses are injected, it is often difficult to obtain sufficient concentrations of antibodies in the brain to elicit a therapeutic response. This drawback has been described as one of the possible causes of failure of passive immunotherapy during clinical trials ([7]).

[0005] During the past decades, alternative non-invasive approaches such as receptor-mediated transcytosis ([8]), nanoparticle delivery [9]

[10] , intranasal delivery [11,12], or cell-based techniques ([13,14]) have been developed to increase the penetration of macromolecules in the brain. However, limited efficacy has been reported, mainly related to manufacturing challenges

[15] , in vivo target-mediated pharmacokinetics

[16]

[17] , or the suspected antigenicity of modified proteins

[18] .

[0006] In a recent study

[14] , the authors reported a cell-targeted delivery system consisting of ex vivo transfected autologous endothelial progenitor cells (EPCs) capable of homing to the BBB and expressing therapeutic antibodies. However, despite the observed success of ex vivo transfection, limited homing of engineered EPCs after intravenous transplantation is expected

[19] , reducing the therapeutic potential of such an approach. Intra-arterial injection could be a suitable alternative but remains challenging as cell dose, infusion rate, and cell type must be carefully evaluated and optimized

[20] .

[0007] According to gene therapy using viral vectors, genes of interest can be delivered directly into the CNS by using viruses modified to contain the transgene of interest in their genome, based on the introduction of therapeutic genes into specific organs, tissues, or cell types.The viral vectors that have been used in gene delivery therapy are based on, but not limited to, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses.

[0008] Adeno-associated viruses (AAV) provide an efficient and clinically safe platform for gene delivery as evidenced by recent approvals: Luxturna® (voretigene neparvovec-rzyl), the first approved AAV2-based therapy, delivers a functional copy of the RPE65 gene to patients with an inherited retinal disease caused by mutations in both copies of the RPE65 gene, and Zolgensma® (onasemnogene abeparvovec-xioi), an AAV9-vectored therapy, delivers a functional copy of the SMN1 gene to motor neurons in patients with spinal muscular atrophy (SMA).

[0009] AAVs have also been used as vehicles for gene transfer into the nervous system, enabling gene expression, knockdown, and gene editing.

[21] However, most of these applications rely on invasive local injection of AAV vectors (intracerebroventricular, intrathecal, or intracisternal administration) to 1) circumvent the blood-brain barrier and 2) restrict transgene expression temporally and spatially.

[0010] Various wild-type serotypes, including AAV2, transduce the BBB but do not cross it, requiring invasive surgery for delivery to the brain.[22,23] In contrast, intravenously administered AAV9 serotypes overcome the BBB and enter the CNS, resulting in gene transfer to the brain and spinal cord.[24,25]

[0011] Recently reported engineered capsid vectors, such as, but not limited to, AAV-S, AAV-F

[26] or AAV9-based AAV-PHP.eB ([27-29]), provided good brain transduction, with the majority of neurons and astrocytes across many regions of adult mice being transduced using the intravenous route of administration. Similarly, AAV2-based AAV-BR1 (synonymously referred to herein as AAV2-BR1) capsid

[30] , or more recently, AAV9-PHP.V1

[31] , have been reported to selectively transduce brain endothelial cells with long-lasting transgene expression, with the potential to treat neurovascular diseases. Summary of the Invention [Problem to be solved by the invention]

[0012] AAV-mediated expression of whole immunoglobulin (IgG) or antibody fragments lacking the Fc domain has been demonstrated in the CNS for various indications [32-44], but limitations inherent to both formats have also been reported [32,45,46]. Indeed, the packaging size of the AAV expression cassette imposes a design constraint for the entire IgG gene, which requires that all elements required for transcription and translation, including both heavy and light chain genes, be less than 4.7 kb. To date, the majority of constructs consist of a single promoter and therefore require the use of self-processing sequences such as Furin-2A (F2A) between the antibody heavy chain genes (HC) and light chain (LC) [47-49]. Researchers highly prefer this construct due to its high expression titers, small F2A sequence size (only 60-80 base pairs), and equimolar concentrations in antibody chain expression. However, in most cases, the F2A peptide remains bound to either the heavy or light chain, which may cause undesired immunogenicity of the expressed protein or antibody-expressing cells

[45] . Internal ribosome entry sites (IRES) for bicistronic antibody expression have been described as an alternative to self-cleaving sequences, but generally result in lower protein expression due to an imbalance in heavy and light chain expression. Although antibody fragments such as scFv or single domain antibodies show some advantages, such as higher protein titers compared to whole IgG molecules, due to monocistronic expression, the above fragments lack Fc effector functions and do not have FcRn binding capacity, which results in a shorter half-life in vivo. In turn, fragments lacking the Fc domain are unable to recruit effector cells to clear pathological complexes in the brain parenchyma, resulting in a reduced efflux of bound antigen from the brain to the blood via reverse transcytosis [32,50-52]. Due to the monovalent binding capacity of such molecules, lower affinities are observed compared to their IgG counterparts. Thus, secreted whole IgG for targeting and clearing extracellular proteinopathy may be the preferred option compared to fragments, but methods need to be developed that circumvent current limitations and allow high quality expression to increase antibody exposure, recruit effector cells, and reduce the risk of undesired immunogenicity of in situ produced IgG. [Means for solving the problem]

[0013] Here, we describe for the first time a delivery method to increase the concentration of (therapeutic) antibodies or antibody fragments in the central nervous system (CNS) by using a vector, such as a viral vector, or another vector, such as a liposome, or a nanoparticle, to deliver a gene encoding an antibody or antibody fragment to cells from the blood-brain barrier (BBB), such as, but not limited to, brain endothelial cells, to produce the therapeutic antibody molecule locally in the CNS, preferably in the brain parenchyma. The antibodies produced by this novel approach can be used to treat CNS-related disorders. In such a scenario, the cells of the BBB (brain endothelial cells and / or pericytes and / or astrocytes) provide long-term expression of high-quality antibodies in the CNS, especially in the brain parenchyma.

[0014] This new strategy circumvents the obstacle of conventional passive immunization strategies that require crossing the blood-brain barrier to reach sufficient therapeutic doses of antibodies within the CNS, and preferably within the brain parenchyma.

[0015] The present invention also relates to improved expression cassettes for producing antibodies and antibody fragments that generate unexpectedly high antibody expression yields and improved protein quality by alternating chain positions with different secretory peptides and by customizing middle and downstream regulatory elements with respect to strategies previously reported in the prior art.

[0016] The present invention is based in part on the discovery that vectors can be used to deliver polynucleotide(s) encoding an antibody or antibody fragment to cells of the blood-brain barrier (BBB). Expression of polynucleotide(s) encoding an antibody or antibody fragment by cells of the BBB results in delivery of the antibody or antibody fragment into the CNS, preferably into the brain parenchyma. This discovery presents a novel strategy that circumvents the limitations of conventional strategies that rely on therapeutic antibodies passing through the BBB to reach sufficient therapeutic doses of the antibody into the CNS, preferably into the brain parenchyma, to treat diseases or disorders of the CNS.

[0017] Thus, in one aspect, the invention provides a vector comprising a polynucleotide(s) encoding an antibody or antibody fragment for use in a method of treating a disease or disorder of the central nervous system (CNS) in a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB) ​​and the transduced or transfected BBB cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

[0018] By targeting the vector containing a polynucleotide encoding the antibody or antibody fragment described herein to the BBB, the amount of antibody or antibody fragment delivered to the CNS, preferably the brain parenchyma, is increased compared to conventional antibody therapeutics that rely on direct delivery of the antibody or antibody fragment to the subject. As described elsewhere, it has been reported that only about 0.1%-0.3% of injected antibodies reach the brain after peripheral administration. In contrast, the inventors have discovered that using an AAV vector to introduce polynucleotide(s) encoding an antibody (MAB1) into a brain endothelial cell line results in non-polarized secretion of MAB1. This means that this technology can be used to express antibodies or antibody fragments in the CNS, preferably in the brain parenchyma.

[0019] The CNS consists of two major components: the brain and the spinal cord. Sensory impulses are transmitted to the CNS and motor impulses travel from the CNS. The CNS also coordinates activity throughout the nervous system. In one embodiment, a vector for use according to the invention transduces or transfects cells of the blood-brain barrier (BBB), and the transduced or transfected BBB cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS. The CNS comprises multiple cell types, and in one embodiment, the antibody or antibody fragment is delivered to at least one (up to all) cell types in the CNS. In one embodiment, the antibody or antibody fragment may be delivered to brain endothelial cells, neurons, pericytes, astrocytes, oligodendrocytes, microglia and ependymal cells. Thus, delivery may be to neurons and non-neuronal (glial) cells of the CNS. In one embodiment, the antibody or antibody fragment is secreted from BBB cells into the CNS. For example, at least 20% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 30% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 40% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 50% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 60% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 70% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 80% of the antibodies or antibody fragments expressed in BBB cells are delivered into the CNS. In one embodiment, at least 90% of the antibodies or antibody fragments expressed in BBB are delivered into the CNS.

[0020] In a preferred embodiment, the antibody or antibody fragment is delivered into the brain parenchyma. As used herein, "brain parenchyma" refers to the functional tissue of the brain composed of neurons and glial cells. The vector for use according to the present invention transduces or transfects cells of the blood-brain barrier (BBB), and the transduced or transfected BBB cells express the antibody or antibody fragment, resulting in the delivery of the antibody or antibody fragment into the brain parenchyma. In one embodiment, the antibody or antibody fragment is secreted from the BBB cells into the brain parenchyma. For example, at least 20% of the antibody or antibody fragment expressed in the BBB cells is delivered into the brain parenchyma. In one embodiment, at least 30% of the antibody or antibody fragment expressed in the BBB cells is delivered into the brain parenchyma. In one embodiment, at least 40% of the antibody or antibody fragment expressed in the BBB cells is delivered into the brain parenchyma. In one embodiment, at least 50% of the antibody or antibody fragment expressed in the BBB cells is delivered into the brain parenchyma. In one embodiment, at least 60% of the antibody or antibody fragment expressed in BBB cells is delivered into the brain parenchyma. In one embodiment, at least 70% of the antibody or antibody fragment expressed in BBB cells is delivered into the brain parenchyma. In one embodiment, at least 80% of the antibody or antibody fragment expressed in BBB cells is delivered into the brain parenchyma. In one embodiment, at least 90% of the antibody or antibody fragment expressed in BBB cells is delivered into the brain parenchyma.

[0021] As already described elsewhere, the BBB is a structural and functional barrier that protects the brain from blood-borne pathogens and toxins. The BBB is composed of three types of cells: endothelial cells, pericytes and astrocytes. Although our findings are primarily related to the endothelial cells of the BBB, the observations are equally applicable to other cells of the BBB. Thus, in one embodiment, the vector transduces or transfects a polynucleotide(s) encoding an antibody or an antibody fragment into a cell type selected from endothelial cells, pericytes and astrocytes. The vector can be transduced or transfected into one specific cell type of the BBB. For example, the vector can be transduced or transfected into pericytes of the BBB. In a further example, the vector can be transduced or transfected into astrocytes of the BBB. As described above, after transduction or transfection, the antibody or antibody fragment is expressed.

[0022] In a preferred embodiment, the vector transfects or transduces endothelial cells of the BBB. Transfection or transduction of BBB endothelial cells with the polynucleotides described herein is preferred because BBB endothelial cells have a slow turnover rate and may prove optimal for long-term expression of antibodies or antibody fragments in vivo in the CNS, preferably in the brain parenchyma.

[0023] Alternatively, the vector can be transduced or transfected into multiple cell types of the BBB. For example, the vector can be transduced or transfected into endothelial cells and astrocytes of the BBB. Alternatively, the vector can be transduced or transfected into endothelial cells and pericytes of the BBB. As a further option, the vector can be transduced or transfected into astrocytes and pericytes of the BBB. In another option, the vector can be transduced or transfected into endothelial cells, astrocytes and pericytes of the BBB. As described above, after transduction or transfection, the antibody or antibody fragment is expressed.

[0024] In some embodiments, a vector comprising a polynucleotide(s) encoding an antibody or antibody fragment may selectively target cells of the BBB. Targeting to specific cells of the BBB can be achieved, for example, by using a neurotropic vector. A neurotropic vector may be a viral vector, which term includes engineered versions. Viral vectors are typically replication-incompetent. The polynucleotide(s) may be integrated into the genome of cells of the BBB. One example of a neurotropic vector is herpes simplex virus (HSV). As used herein, "neurotropic vector" refers to a vector that preferentially transduces or transfects BBB cells and / or CNS cells, respectively, compared to non-BBB cells and / or non-CNS cells. Thus, in one embodiment, the vector comprises a neurotropic vector. In another embodiment, the vector comprises a modified HSV.

[0025] An alternative route to selectively target cells of the BBB is to utilize vectors that express or contain (particularly on their surface, such as in a viral capsid) a peptide, a small molecule (SME), an antibody or antibody fragment thereof, a protein, a nanoparticle, a lipid, an oligonucleotide, an aptamer or a cationic molecule that targets the vector to cells of the BBB.

[0026] In one embodiment, the vector expresses a peptide on the vector surface that targets the vector to cells of the BBB. In other words, the peptide, small molecule, antibody or antibody fragment thereof, protein, nanoparticle, lipid, oligonucleotide, aptamer, or cationic molecule expressed or configured on the vector surface confers targeting specificity to cells of the BBB. In another embodiment, the peptide or other enumerated molecule expressed or included on the vector surface targets the vector to a specific cell type(s) of the BBB. Examples of suitable peptides, as well as methods for generating and testing such peptides, including phage display methods, are known in the art. For example, the peptide may include a ligand or receptor targeting peptide involved in cell transcytosis. Such peptides allow the uptake of the vector into cells of the BBB using receptor-mediated transcytosis. In one embodiment, the peptide targets a receptor selected from the transferrin receptor, insulin receptor, and low density lipoprotein receptor. In a further embodiment, the peptide includes a transferrin peptide. An exemplary BBB targeting peptide is the NRGTEWD ​​(SEQ ID NO: 15) peptide contained in the AAV2 strain, and AAV-BR1 peptides such as these may be incorporated into non-viral vectors in some embodiments.

[0027] Thus, the vector can carry a mutation, such as an insertion, deletion or substitution in a vector surface protein, such as a viral capsid protein, that results in targeting of the vector to cells of the BBB. In certain embodiments, the vector can contain a mutation that targets the vector to cells of the BBB.

[0028] Alternatively or additionally, a neurotropic viral vector containing a polynucleotide(s) encoding an antibody or antibody fragment can be used to selectively target cells of the BBB.

[0029] The term "vector" is well known in the art and is appropriately used in the context of the present invention to transport (by transduction or transfection) a polynucleotide(s) into a host cell. This definition includes both non-viral and viral vectors. Viral or non-viral vectors can target cells derived from the BBB or CNS, such as, but not limited to, brain endothelial cells, to produce therapeutic antibody molecules locally within the CNS, preferably within the brain parenchyma. Antibodies, particularly therapeutic antibodies, produced by this novel approach can be used to treat CNS-related diseases. In such a scenario, brain endothelial cells and / or pericytes and / or astrocytes act as reservoirs to provide high quality and long-term expression of antibodies into the CNS, preferably within the brain parenchyma.

[0030] Non-viral vectors include, but are not limited to, organic nanomaterials, such as liposomes, exosomes, dendrimers, and micelles, or inorganic nanomaterials, such as gold nanoparticles, silica nanoparticles, and carbon nanotubes. In one embodiment, the non-viral vector expresses on the vector surface a peptide, a small molecule (SME), an antibody or antibody fragment thereof, a protein, a nanoparticle, a lipid, an oligonucleotide, an aptamer, or a cationic molecule that targets the vector to cells of the BBB.

[0031] Viral vectors include, but are not limited to, wild-type viruses and engineered (e.g., modified) viruses. Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, retrovirus, rhinovirus, lentivirus, hepatitis, HSV and any virus-like particle. As known in the art, virus-like particle (VLP) is a multiprotein structure that mimics the composition and conformation of authentic natural virus, but lacks viral genome. The present invention originates from the unexpected discovery that transduction of brain endothelial cells with AAV vectors results in high-quality and high-volume antibody secretion by brain endothelial cells. Thus, in one embodiment, the viral vector is AAV. The AAV may be of any suitable serotype, including, but not limited to, AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12), or any other wild-type serotype or engineered AAV. More specifically, the AAV may be selected from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9) and AAV serotype 10 (AAV10). In another embodiment, the viral vector is selected from AAV2, AAV8, AAV9 and AAVrh.10 (AAV rhesus isolate 10).

[0032] In a further embodiment, the viral vector is an engineered AAV. The engineered AAV can be an engineered AAV2, an engineered AAV9, an engineered AAV1 or an engineered AAV10. In a preferred embodiment, the engineered AAV2 is AAV-BR1. In another preferred embodiment, the engineered AAV9 is AAV-S, AAV-F, AAV-PHP.eB or AAV9-PHP-V. In a further preferred embodiment, the engineered AAV1 is AAV1RX, AAV1R6 or AAV1R7. Further details of engineered AAV1 are provided in Albright BH et al. [53,54], which are incorporated herein by reference. In a most preferred embodiment, the vector is AAV-BR1 or AAV9-PHP-V1, which are specific for BBB endothelial cells.

[0033] In further embodiments, the vector comprises viral and non-viral elements. Virosomes are an example of a vector that comprises both viral and non-viral elements. A further example is a viral vector mixed with a cationic lipid.

[0034] All vectors described herein include a polynucleotide(s) encoding an antibody or antibody fragment. The polynucleotide may include DNA or RNA. The polynucleotide may include additional components to aid in the expression (e.g., translation) of the sequence encoding the antibody or antibody fragment in, for example, cells of the BBB. For example, the polynucleotide encoding the antibody or antibody fragment is included in an expression cassette. In one embodiment, the expression cassette includes, consists essentially of, or consists of a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0035] In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 80% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 85% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 90% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 91% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 92% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 93% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 94% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 95% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 96% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 97% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 98% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In a further embodiment, the expression cassette comprises a nucleotide sequence having at least 99% identity to a nucleotide sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0036] As used herein, "% identity" is used to describe the sequence similarity between two sequences, such as nucleotide sequences and amino acid sequences. This can be determined by comparing two sequences aligned in an optimal manner, and the nucleotide sequence being compared can include additions or deletions to the reference sequence to achieve optimal alignment between the two reference sequences. The percentage of identity is calculated by determining the number of identical positions where the residues are identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window to obtain the percentage of identity between these two sequences, and multiplying the result by 100. For example, the BLAST program "BLAST 2 sequences" available at the site https: / / blast.ncbi.nlm.nih.gov / Blast.cgi (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) can be used, the parameters used are those given by default (specifically, the parameters "open gap penalty": 5, and "extended gap penalty": 2, the matrix selected is, for example, the matrix "BLOSUM 62" proposed by the program), and the percentage of identity between the two sequences to be compared is calculated directly by the program.

[0037] The expression cassette may comprise a sequence that provides or codes for one or more, preferably all, of the promoter, ribosome binding site, start codon, stop codon, and transcription termination sequence that are operably linked to the polynucleotide that codes for antibody or antibody fragment.Preferably, the expression cassette may further comprise a nucleic acid that codes for a post-transcriptional regulatory element.Suitably, the expression cassette may further comprise a nucleic acid that codes for a polyA (polyadenylation) element.

[0038] As used herein, the phrase "promoter" generally refers to a region of DNA located upstream of a polynucleotide sequence to be transcribed (e.g., a polynucleotide sequence encoding an antibody or antibody fragment) that is necessary for transcription to occur, e.g., initiates transcription. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter, EF1A (human eukaryotic translation elongation factor 1 alpha 1), CAG (CMV early enhancer fused to a modified chicken β-actin promoter), CBh (CMV early enhancer fused to a modified chicken β-actin promoter), SV40 (simian virus 40 enhancer / early promoter), GFAP (human glial fibrillary acidic protein promoter), ATP1A2_1 (Na,K ATPase alpha 2), CLDN_5 (claudin 5), ADRB2_1 (adrenergic receptor beta 2), TNFRSR6B_1 (TNF receptor superfamily member 6b), PDYN_1 (prodynorphin), GH1_1 (human growth hormone), OPALIN_1 (opaline), SYN1_1 (synapsin 1), CAMK2A_1 (calcium / calmodulin-dependent protein kinase II alpha), NEFH_1 (neurofilament heavy chain polypeptide), NEUROD6_1 (neuronal differentiation factor 6) or OLIG2_1 (oligodendrocyte transcription factor 2). In a preferred embodiment, the promoter is a cytomegalovirus (CMV) promoter, a CMV early enhancer fused to either CBh, GFAP, ATP1A2_1, CLDN_5, ADRB2_1, TNFRSF6B_1, PDYN_1, GH1_1, OPALIN_1, SYN1_1, CAMK2A_1, NEFH_1, NEUROD6_1 or OLIG2_1 promoter. In a more preferred embodiment, the promoter is CBh, CMV, or a CMV early enhancer fused to either GFAP or OLIG2. In an even more preferred embodiment, the promoter is a CMV promoter or a CBh promoter.

[0039] The term "promoter" includes synthetic promoters. The term "synthetic promoter" as used herein relates to a promoter that does not occur in nature. For example, functional variants of naturally occurring promoters can be used according to the present invention. A "functional variant" of a promoter in the context of the present invention is a variant of a reference promoter that retains the ability to function in the same manner as the reference promoter. In a further embodiment, a truncated form of a naturally occurring promoter is used. In a preferred embodiment, the promoter is operably linked to an enhancer, such as the CMV early enhancer. A truncated or modified naturally occurring promoter can be used to facilitate the insertion of a relatively large antibody coding sequence into a vector, particularly a viral vector.

[0040] As described in detail elsewhere, vectors can specifically target cells of BBB (and / or CNS).However, in other examples, vectors do not specifically target BBB (and / or CNS).For example, many wild-type viral vectors target any tissue or cell type.In such cases, BBB-specific or CNS-specific promoters can be used to drive the expression of polynucleotides encoding antibodies or antibody fragments in cells of BBB or CNS in a preferential or dominant manner compared to other tissues.

[0041] In one embodiment, the polynucleotide comprises a GFAP (human glial fibrillary acidic protein) promoter operably linked to a polynucleotide encoding an antibody or antibody fragment. In a further embodiment, the GFAP promoter is operably linked to a CMV early enhancer. In this case, there is preferential or predominant expression of the antibody or antibody fragment in astrocytes.

[0042] In one embodiment, the polynucleotide comprises a promoter selected from ATP1A2_1 (Na,K ATPase alpha 2), CLDN_5 (claudin 5), ADRB2_1 (adrenergic receptor beta 2) and TNFRSF6B_1 (TNF receptor superfamily member 6b) operably linked to a polynucleotide encoding an antibody or antibody fragment. In a further embodiment, the promoter is operably linked to a CMV early enhancer. In this case, there is preferential or predominant expression of the antibody or antibody fragment in endothelial cells of the BBB.

[0043] In one embodiment, the polynucleotide operably linked to the polynucleotide encoding the antibody or antibody fragment comprises a promoter selected from PDYN_1 (prodynorphin), GH1_1 (human growth hormone) and OPALIN_1 (opalin). In a further embodiment, the promoter is operably linked to a CMV early enhancer. In this case, there is preferential or predominant expression of the antibody or antibody fragment in brain cells.

[0044] In one embodiment, the polynucleotide comprises a promoter selected from SYN1_1 (synapsin 1), CAMK2A_1 (calcium / calmodulin-dependent protein kinase II alpha), NEFH_1 (neurofilament heavy chain polypeptide), NEUROD6_1 (neuronal differentiation factor 6) operably linked to a polynucleotide encoding an antibody or antibody fragment. In a further embodiment, the promoter is operably linked to a CMV early enhancer. In this case, there is preferential or predominant expression of the antibody or antibody fragment in neurons.

[0045] In one embodiment, the polynucleotide comprises an OLIG2_1 (oligodendrocyte transcription factor 2) promoter operably linked to a polynucleotide encoding an antibody or antibody fragment. In a further embodiment, the OLIG2_1 promoter is operably linked to a CMV early enhancer. In this case, there is preferential or predominant expression of the antibody or antibody fragment in oligodendrocytes.

[0046] The term "operably linked" as used herein refers to the configuration of various polynucleotide elements relative to each other such that the elements are functionally connected and can interact with each other in the intended manner. Such elements may include, but are not limited to, promoters, enhancers and / or regulatory elements, polyadenylation sequences, one or more introns and / or exons, and the coding sequence of the gene of interest to be expressed. When the polynucleotide elements are appropriately oriented or operably linked, they can act together to modulate each other's activity, ultimately affecting the expression level of the product (e.g., an antibody or antibody fragment). "Modulate" means to increase, decrease, or maintain the activity level of a particular element. As will be understood by those skilled in the art, operably linked includes functional activity and does not necessarily relate to natural positional linkage.

[0047] As mentioned above, the expression cassette may include a sequence that provides or codes for a ribosome binding site (RBS). In a preferred embodiment, the RBS is an internal ribosome entry site (IRES). In one embodiment, the IRES is derived from an encephalomyocarditis virus. In a further embodiment, the IRES comprises SEQ ID NO: 1 or SEQ ID NO: 8. IRES are particularly advantageous in expression cassettes that include more than one gene encoding an antibody or antibody fragment. For example, the expression cassette includes a gene encoding a light chain of an antibody or antibody fragment and a gene encoding a heavy chain of an antibody or antibody fragment.

[0048] According to all aspects of the present invention, the expression cassette may further comprise a sequence providing or encoding a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) at the 3' end of the construct, as digrammatically represented in the top construct of Figure 1. The WPRE sequence is routinely used to increase expression of genes delivered by viral vectors. Without wishing to be bound by theory, the inclusion of this sequence in the expression cassette may increase mRNA stability and therefore protein yield.

[0049] Alternatively, the expression cassette may include a sequence encoding a self-cleaving peptide. The self-cleaving peptide may be used in an expression cassette that includes more than one gene encoding an antibody or antibody fragment. For example, the expression cassette includes a single promoter operably linked to a gene encoding a light chain of an antibody or antibody fragment and a gene encoding a heavy chain of an antibody or antibody fragment. In such a case, the sequence encoding the self-cleaving peptide is located after the first gene (e.g., the gene encoding the light chain of the antibody or antibody fragment) and before the second gene (e.g., the gene encoding the heavy chain of the antibody or antibody fragment). Such a system may allow the self-cleavage of the peptide to occur co-translationally via ribosomal skipping (e.g., resulting in the separation of the heavy and light chain polypeptides of the antibody or antibody fragment from a single mRNA transcript). One particular class of self-cleaving peptides is the 2A peptide family (including the F2A peptide from foot and mouth disease virus), which shares the core sequence motif of DxExNPGP (SEQ ID NO: 16). Thus, in one embodiment, the expression cassette includes a sequence encoding a self-cleaving peptide from the 2A family. In a further embodiment, the expression cassette further comprises a sequence encoding a furin cleavage site upstream of the self-cleavage site. In other words, the expression cassette comprises a single promoter operably linked to a gene encoding the light chain of an antibody or antibody fragment and a gene encoding the heavy chain of an antibody or antibody fragment, and the sequence encoding the furin cleavage peptide and the self-cleavage peptide are located after the first gene and before the second gene. The addition of the furin cleavage site can be performed to remove additional amino acids of the self-cleavage peptide that remain attached to the upstream protein (e.g., the light chain of the antibody or antibody fragment) after self-cleavage. However, it is worth noting that even if there is a furin cleavage site upstream of the self-cleavage peptide, additional amino acids may remain in some upstream proteins (e.g., the light chain of the antibody or antibody fragment), which may lead to an immune response (e.g., immunogenicity) against the upstream protein.Furthermore, the inventors have surprisingly observed that constructs containing Furin and 2A increase aggregation to allow for self-cleavage. Thus, in a preferred embodiment, the expression cassette does not include a sequence encoding an auto-cleaving peptide after the first gene and before the second gene.

[0050] The expression cassette may further comprise a secretory peptide at the 5' end of the polynucleotide encoding the antibody or antibody fragment. In the context of the present invention, the secretory peptide assists in the delivery of the antibody or antibody fragment into the CNS, preferably into the brain parenchyma. When there are multiple genes encoding antibodies or antibody fragments, all genes may further comprise a sequence encoding a secretory peptide.

[0051] The present invention is not limited to a specific polynucleotide that encodes a specific antibody or antibody fragment.However, typically, the antibody or antibody fragment is a therapeutic antibody or antibody fragment.The therapeutic antibody or antibody fragment is one that effectively exerts its activity in the CNS, particularly in the brain.Therefore, the therapeutic antibody or antibody fragment can bind to the target antigen that is expressed in the CNS, particularly in the brain or spinal cord.

[0052] In general, the term "antibody" is used herein in the broadest sense and includes various antibody structures, such as, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. The antibody may also be a chimeric antibody (especially a mouse VH and VL region fused with a human constant domain), a recombinant antibody, an antigen-binding fragment of a recombinant antibody, or a humanized antibody.

[0053] An "antibody fragment" of an antibody refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to an antigen to which the intact antibody binds. In one embodiment, the antibody fragment is an Fv, Fab, Fab', Fab'-SH, F(ab')2; a diabody; a linear antibody; a single chain antibody molecule (e.g., scFv, preferably scFv); and a multispecific antibody formed from an antibody fragment. The term also encompasses single domain antibodies (e.g., VHH, VNAR or human single domain antibodies). One preferred antibody fragment according to the invention is the scFv-Fc, in which an scFv (a fusion protein of VH and VL domains, typically linked with a short linker peptide of about 10-25 amino acids) is linked to a fragment crystallizable (Fc) region. The scFv-Fc lacks the CH1 and CL domains.

[0054] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modified "monoclonal" indicates the character of the antibody as being amongst a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies used in accordance with the present invention can be made by the hybridoma method described in Kohler, Nature 256 (1975), 495.

[0055] Thus, in the context of the present invention, the term "antibody" refers to complete immunoglobulin molecules as well as to parts of such immunoglobulin molecules (i.e., "antibody fragments"). Furthermore, the term relates to modified and / or altered antibody molecules, as discussed above. The term also relates to recombinantly or synthetically produced / synthesized antibodies. The term also relates to intact antibodies as well as antibody fragments thereof, such as separated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term "antibody" also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs such as single chain Fvs (scFv), scFv-Fcs, or antibody fusion proteins.

[0056] Humanized antibody is a modified antibody, also called reshaped human antibody.Humanized antibody is constructed by transferring the CDR of the antibody from immunized animal to the complementarity determining region of human antibody.The conventional recombinant gene technology for such purpose is known (see EP239400; WO96 / 02576; Sato K. et al., Cancer Research 1993, 53: 851-856; WO99 / 51743).

[0057] The term "CDR" as used herein refers to "complementarity determining region" as is well known in the art. CDR is the part of an immunoglobulin that determines the specificity of said molecule and contacts a specific ligand. CDR is the most variable part of the molecule and contributes to the diversity of these molecules. There are three CDR regions CDR1, CDR2, CDR3 in each V domain. VH-CDR, or CDR-H, refers to the CDR region of the variable heavy chain, and VL-CDR, or CDR-L, refers to the CDR region of the variable light chain. VH means variable heavy chain and VL means variable light chain. The CDR regions of the Ig-derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest", 5th edit. NIH Publication no. 91-3242 US Department of Health and Human Services (1991); Chothia J., Mol. Biol. 196 (1987), 901-917 or Chothia, Nature 342 (1989), 877-883.

[0058] The "Fc" region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody, the two heavy chain fragments being held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0059] A "Fab' fragment" contains one light chain and a portion of one heavy chain including the VH and CH1 domains, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between the two heavy chains of the two Fab' fragments to form an F(ab')2 molecule.

[0060] An "F(ab')2 fragment" contains two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, resulting in the formation of an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0061] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0062] An "scFv-Fc" comprises the "Fv" variable regions from both the heavy and light chains fused to an "Fc" region comprising two heavy chain fragments containing the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0063] Thus, in the context of the present invention, there is provided an antibody molecule or antibody fragment thereof which has been humanized and which can be used in a pharmaceutical composition comprising a mixture of at least two antibody molecules or antibody fragments thereof.

[0064] An "antibody that binds to an epitope" within a defined region of a protein is an antibody that requires the presence of one or more amino acids within that region in order to bind to the protein.

[0065] In certain embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, amino acids of the protein are mutated, and binding of the antibody to the resulting modified protein (e.g., a modified protein containing an epitope) is determined to be at least 20% of binding to the unmodified protein. In some embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutation analysis, amino acids of the protein are mutated, and binding of the antibody to the resulting modified protein (e.g., a modified protein containing an epitope) is determined to be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of binding to the unmodified protein. In certain embodiments, antibody binding is determined by a suitable binding assay, such as FACS, WB, or ELISA.

[0066] The antibody or antibody fragment for use in accordance with the present invention is preferably an antibody or antibody fragment that binds to an epitope of the CNS, more specifically, the antibody or antibody fragment binds to an epitope of the CNS that is associated with a disease or disorder of the CNS. In preferred embodiments, the antibody or antibody fragment is selected from the group consisting of anti-ErbB2, anti-TDP-43 (NI-205), anti-Abeta (e.g. bapinezumab, solanezumab, lecanemab, azcanumab, donanemab, gantenerumab or crenezumab), anti-ApoE4 (apolipoprotein E4) and anti-DDX3X (ATP-dependent RNA helicase), anti-Tau (tirabonemab, goslanemab, zagotenemab, semolinemab, bepranemab, BIIB076, JNJ-63733657, Lu AF87908, PNT001, E-2814), anti-LINGO-1 (e.g. opisinumab), anti-alpha-synuclein (simpanemab, prasinezumab, MEDI-1341, Lu AF82422, BAN0805), anti-ASC (IC-100), anti-NLRP3, anti-C5 (ravulizumab, eculizumab), anti-C1q (ANX-005), anti-C3, anti-huntingtin (C-617, NI-302), anti-prion, anti-CD20 (e.g. ofatumumab, ocrelizumab, rituximab, BCD-132, ublituximab, BAT-4406F, AL-014), anti-PD-1 (IBC-Ab002) or anti-VEGF-A (bevacizumab, ranibizumab, brolucizumab, faricimab, vanucizumab).

[0067] As described in the experimental section herein, the human brain endothelial cell line hCMEC / D3 was transduced with AAV2, AAV8, AAV9 and AAVrh.10 (AAV rhesus isolate 10) delivering genes encoding one of the following: anti-TDP-43 antibody MAB1 (chimeric human IgG1), anti-ErbB2 antibody (Herceptin) MAB2 or eGFP (enhanced green fluorescent protein). This resulted in high quality and long-lasting expression of antibodies. We also confirmed those observations in mouse cell lines and human primary brain endothelial cells. The delivery systems resulted in the production of correctly folded antibodies in a variety of antibody formats (including full-length antibodies and antibody fragments). Importantly, in these experiments, secreted antibodies were detected on both the apical and basolateral sides, confirming delivery to the brain parenchymal side of the BBB in this model. Thus, the data contained in the Examples demonstrate that the methods and vectors described herein can be used to deliver correctly folded antibodies or antibody fragments (eg, therapeutic antibodies) to the CNS.

[0068] The object of the present invention is to provide, for the first time, a method for increasing antibody concentration in the CNS for treating CNS-related diseases or disorders by delivering polynucleotide(s) encoding an antibody or antibody fragment to cells in the CNS and / or BBB. This novel approach is useful for treating a variety of diseases or disorders that originate in the CNS.

[0069] For the avoidance of doubt, the term "treatment" as used herein includes therapeutic treatment, as well as symptomatic treatment and prevention of a condition. Use of the terms "treatment", "treat" or "treatment of" (and grammatical variations thereof) means that the severity of a subject's condition is reduced, at least partially ameliorated or alleviated, and / or some degree of alleviation, reduction or decrease in at least one clinical symptom is achieved, and / or the progression of a disease or disorder is delayed.

[0070] As used herein, the term "subject" refers to an individual, e.g., a mammal, such as a human, who has or is at risk of having a particular condition, disorder or symptom present in the CNS. The subject may be a subject in need of treatment according to the present invention. The subject may have been treated for a condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment according to the present invention.

[0071] The present invention relates to an innovative strategy that can be used in any mammal, including human subjects, to treat diseases or disorders associated with subjects suffering from diseases or disorders of the CNS, by delivering therapeutic antibody genes to brain endothelial cells, which act as reservoirs to provide high quality, long-term expression of antibodies into the CNS.

[0072] In one embodiment, the disease or disorder is a neurodegenerative disorder.

[0073] In further embodiments, the disease or disorder is associated with the CNS including, but not limited to, amyloid-beta protein, TDP-43-proteinopathy, alpha-synucleinopathy, tauopathy, trinucleotide repeat disorders including polyglutamine disorders such as Huntington's disease, brain-related cancers and tumors, epilepsy, psychiatric disorders, neuroinflammatory diseases, neuromuscular diseases, viral-induced encephalitis, and diseases characterized by microglial dysfunction.

[0074] In another embodiment, the patient's disease or disorder or condition associated with amyloid-beta protein, TDP-43-proteinopathy, alpha-synucleinopathy, tauopathy, trinucleotide repeat disorders including polyglutamine disorders such as Huntington's disease, brain-related cancers and tumors, epilepsy, psychiatric disorders, neuroinflammatory diseases, neuromuscular diseases, viral-induced encephalitis, and diseases characterized by microglial dysfunction.

[0075] In some embodiments, the amyloid-beta related disease, disorder or condition according to the present invention includes mild cognitive impairment (MCI), Down's syndrome (DS), Alzheimer's disease associated with Down's syndrome, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease (PD), Parkinson's disease with dementia (PDD), dementia with Lewy bodies, and ALS (amyotrophic lateral sclerosis). Many of these pathologies are characterized by or are associated with loss of cognitive memory capacity. Thus, the conditions associated with or associated with loss of cognitive memory capacity according to the present invention include AD, mild cognitive impairment (MCI), Down's syndrome (DS), Alzheimer's disease associated with Down's syndrome, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease, Parkinson's disease with dementia (PDD), dementia with Lewy bodies, and ALS (amyotrophic lateral sclerosis).

[0076] In particular, the amyloid-beta related disease, disorder or condition may be selected from Alzheimer's disease (AD), Down's syndrome (DS), Alzheimer's disease associated with Down's syndrome, cerebral amyloid angiopathy (CAA), or dementia with Lewy bodies.

[0077] In some embodiments, the TDP-43-proteinopathy includes frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with progranulin (GRN) mutation, with C9orf72 mutation, with TARDBP mutation, with valosin-containing protein (VCP) mutation, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions, argyrophilic grain disease, Pick's disease, semantic primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), non-fluent primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, TARDBP, These include: angiogenin (ANG) mutations, with TDP-43 P mutations, Alexander disease (AxD), limbic dominant age-related encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathy with mutations in valosin-containing protein (VCP; also Paget's disease of bone and frontotemporal dementia)), traumatic brain injury (TBI), dementia with Lewy bodies (DLB) or Parkinson's disease (PD).

[0078] In particular, the TDP-43-proteinopathy, disease or condition may be selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE) and multiple sclerosis (MS).

[0079] In some embodiments, the synucleinopathy is Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with non-alpha-synuclein mutations, pure autonomic failure and Lewy body dysphagia), Lewy body dementia (LBD; Lewy body dementia (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), or diffuse Lewy body disease, sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2 or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration and olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, whiplash dementia, tau These conditions include: opiates (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia with parkinsonism linked to chromosome 17, and Niemann-Pick type C1 disease), Down's syndrome, Creutzfeldt-Jakob disease, Huntington's disease, motor neuron diseases, amyotrophic lateral sclerosis (sporadic, familial, and Guam ALS-dementia complex), neuroaxonal dystrophies, neurodegeneration type 1 with brain iron accumulation (Hallervorden-Spatz syndrome), prion diseases, Gerstmann-Straussler-Scheinker disease, ataxia-telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gaucher disease, Krabbe disease, and other lysosomal storage diseases (including Kufour-Rakeb syndrome and Sanfilippo syndrome), or rapid eye movement (REM) sleep behavior disorder.

[0080] In particular, the synucleinopathy may be selected from Parkinson's disease, multiple system atrophy, dementia with Lewy bodies (LBD; including dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD) or diffuse Lewy body dementia).

[0081] In some embodiments, the tauopathy is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down's syndrome, Gerstmann-Straussler-Scheinker disease, prion protein cerebral amyloid angiopathy, traumatic brain injury, Guam amyotrophic lateral sclerosis / Parkinson's disease-dementia complex, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal ganglia dementia, and cerebrovascular disease. degeneration, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, frontotemporal dementia with parkinsonism linked to chromosome 17, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, pallidopontonigral degeneration, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, senile dementia with neurofibrillary tangles, postencephalitic parkinsonism, and myotonic dystrophy.

[0082] In particular, the tauopathy may be selected from Alzheimer's disease or progressive supranuclear palsy.

[0083] In some embodiments, the neuroinflammatory disease, disorder or abnormality is Alzheimer's disease, Parkinson's disease, frontotemporal dementia, limbic predominant age-related TDP-43 encephalopathy, amyotrophic lateral sclerosis, motor neuron disease, trinucleotide repeat disorders including polyglutamine disorders, e.g., Huntington's disease, multiple sclerosis, demyelination, viral encephalitis, epilepsy, ischemic stroke, hemorrhagic stroke, traumatic brain injury, chronic traumatic encephalopathy, cryopyrin-associated periodic syndrome (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammation. The conditions selected are: chronic inflammatory syndrome (FCAS), neonatal onset multisystem inflammatory disease (NOMID), periodic fever syndrome (HIDS), sideroblastic anemia with B cell immunodeficiency, periodic fever with developmental delay (SIFD), Behçet's disease, Sjögren's syndrome, cerebral malaria, brain damage due to pneumococcal meningitis, chikungunya virus, Ross River virus, influenza, HIV, coronavirus, dengue fever, Zika virus, helminth infection, bacterial infection, depression, psychological stress, HIV-associated neurocognitive disorder, and coronavirus-associated inflammatory pathology.

[0084] In some embodiments, the neuroinflammatory disease, disorder or abnormality is preferably selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, demyelination, viral encephalitis, epilepsy, stroke, cryopyrin-associated periodic syndromes (CAPS), antineutrophil cytoplasmic antibody-associated vasculitis, lupus, psoriatic arthritis, and hereditary relapsing fever (HRF).

[0085] In some embodiments, the neuroinflammatory disease, disorder or abnormality is more preferably selected from Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, multiple sclerosis, demyelination, viral encephalitis, stroke, and cryopyrin-associated periodic syndrome (CAPS).

[0086] In some embodiments, the neuromuscular disease may include cerebrovascular accidents, Parkinson's disease, multiple sclerosis, Huntington's disease and Creutzfeldt-Jakob disease, spinal muscular atrophy and amyotrophic lateral sclerosis. In another embodiment, the disease or disorder of the CNS is a neurodegenerative disorder.

[0087] In preferred embodiments, the CNS disease or disorder is selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE) and multiple sclerosis (MS).

[0088] Brain and CNS cancers and tumors that may also be treated according to the present invention include astrocytoma (including cerebellar and cerebral), brain stem glioma, brain tumor, malignant glioma, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumor, medulloblastoma, myelodysplastic syndrome, oligodendroglioma, myelodysplastic / myeloproliferative disease, myeloid leukemia, myelocytic leukemia, multiple myeloma, myeloproliferative disease, neuroblastoma, plasma cell neoplasm / multiple myeloma, central nervous system lymphoma, intrinsic brain tumor, astrocytic brain tumor, glioma, and metastatic tumor cell infiltration in the central nervous system.

[0089] The vectors described herein can be administered to a subject by any conventional route, including injection, or by gradual infusion over time. Administration can be via parenteral administration. Administration can be, for example, by injection or intrathecal, intracisternal, intracerebroventricular, intraparenchymal, intranasal, intravitreal, subcutaneous, or intramuscular route. In one embodiment, the vector is administered by intravenous injection or infusion. As a further example, forms suitable for parenteral injection (including subcutaneous, intramuscular, intravascular, or infusion) include sterile solutions, suspensions, or emulsions. Intravenous injection is preferred.

[0090] Identifying the appropriate dosage of the composition of the present invention is within the ordinary skill of one of ordinary skill in the art. For example, the appropriate dosage for a given subject is determined by considering various factors known to modify the action of the vector for use according to the present invention. For example, the severity and type of CNS disease or disorder, body weight, sex, diet, time and route of administration, other drugs, and other relevant clinical factors. Dosage and schedule can vary depending on the specific condition, disorder or symptom, depending on the overall condition of the subject. Also, there may be no single dose that is accepted for the treatment of a given disease, but a range of doses may be considered appropriate. Effective dosages can be determined by either in vitro or in vivo methods.

[0091] Also provided herein is a method for treating a disease or disorder of the CNS in a subject. The method comprises administering to the subject a vector comprising a polynucleotide encoding an antibody or an antibody fragment. The method results in transduction or transfection of cells of the BBB, advantageously the transduced or transfected cells produce (e.g., express) the antibody or antibody fragment, and the antibody or antibody fragment is delivered into the CNS, preferably into the brain parenchyma.

[0092] In another aspect, the invention provides a method of delivering an antibody or antibody fragment to the BBB in a subject comprising administering to the subject a vector comprising a polynucleotide encoding the antibody or antibody fragment, the method resulting in transduction or transfection of cells of the BBB, wherein the transduced or transfected cells express the antibody or antibody fragment.

[0093] In a further aspect, the invention provides a method for delivering an antibody or antibody fragment to the CNS in a subject, the method comprising administering to the subject a vector comprising a polynucleotide encoding the antibody or antibody fragment, the method resulting in transduction or transfection of cells of the BBB, wherein the transduced or transfected cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

[0094] In yet a further aspect, the invention provides a method of treating a disease or disorder of the CNS in a subject, the method comprising administering to the subject a vector comprising a polynucleotide encoding an antibody or antibody fragment, the method resulting in transduction or transfection of cells of the BBB, wherein the transduced or transfected cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

[0095] In another aspect, the invention provides the use of a vector comprising a polynucleotide encoding an antibody or antibody fragment for the manufacture of a medicament for treating a disease or disorder of the CNS in a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB) ​​and the transduced or transfected cells express the antibody or antibody fragment resulting in delivery of the antibody or antibody fragment into the CNS.

[0096] In a further aspect, the invention provides the use of a vector comprising a polynucleotide encoding an antibody or antibody fragment to deliver the polynucleotide encoding the antibody or antibody fragment to the BBB of a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB) ​​and the transduced or transfected cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

[0097] All embodiments described herein in relation to vectors for use according to the preceding aspects of the invention are equally applicable to these further aspects of the invention.

[0098] The inventors have discovered that certain expression cassette constructs improve the quality of the antibodies produced. This is important when delivering antibodies in vivo. Thus, by using the expression cassettes of the invention (described in detail in various aspects and embodiments herein), advantageous delivery of antibodies and antibody fragments to the CNS can be obtained, either by direct delivery to the CNS or by delivery via BBB cells (in accordance with the invention as defined herein). Thus, the following aspects can be performed in vivo, where the cells are BBB cells, particularly brain endothelial cells, or more generally cells of the CNS. Thus, the antibody or antibody fragment is generally a therapeutic antibody or antibody fragment, as described herein. The following aspects can also be performed in some embodiments ex vivo or in vitro for antibody production. In these embodiments, any suitable cell type can be utilized. Again, the antibody or antibody fragment is generally a therapeutic antibody or antibody fragment.

[0099] Thus, the present invention provides a vector comprising an expression cassette comprising at least one promoter operably linked from 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment for use in a method of treating a disease or disorder of the central nervous system (CNS) in a subject, the vector transducing or transfecting cells of the blood-brain barrier (BBB) ​​or CNS, the transduced or transfected cells expressing the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS, preferably to the brain parenchyma. The inventors have found that ordering the light chain before the heavy chain in such a construct is highly advantageous. Preferably, an IRES is placed between the first and second genes.

[0100] In a further aspect, the present invention provides a vector comprising an expression cassette comprising, 5' to 3', a first promoter operably linked to a first gene encoding a light chain of an antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of the antibody or antibody fragment, for use in a method of treating a disease or disorder of the central nervous system (CNS) in a subject, the vector transducing or transfecting cells of the blood-brain barrier (BBB) ​​or CNS, the transduced or transfected cells expressing the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment within the CNS, preferably to the brain parenchyma. In such a construct, an IRES is not required.

[0101] The CNS, like the BBB, comprises multiple cell types, and in one embodiment, the vector transduces or transfects the polynucleotide(s) encoding the antibody or antibody fragment into at least one (up to all) cell type in the CNS. For example, the vector can transduce or transfect cells of the CNS selected from brain endothelial cells, neurons, pericytes, astrocytes, oligodendrocytes, microglia, and ependymal cells. Thus, the vector can transduce or transfect neuronal and non-neuronal (glial) cells of the CNS. Alternatively, the vector can transduce or transfect specific cell types of the CNS. For example, the vector can transduce or transfect brain endothelial cells of the CNS. In another example, the vector can transduce or transfect neurons of the CNS. In a further example, the vector can transduce or transfect astrocytes of the CNS. In yet another example, the vector can transduce or transfect oligodendrocytes. In yet a further example, the vector can be transduced or transfected into microglia of the CNS. Alternatively, the vector can be transduced or transfected into ependymal cells of the CNS.

[0102] In a further embodiment, the vector transduces or transfects multiple cell types from both the BBB and the CNS.

[0103] As mentioned above, vectors containing polynucleotide(s) encoding antibodies or antibody fragments can be transduced or transfected specifically into cells of the BBB and / or CNS. The same techniques described for targeting to the BBB described elsewhere are equally applicable to vectors that directly target to the CNS. In one embodiment, the vector expresses a peptide on the vector surface that targets the vector to cells of the BBB or CNS. In other words, the peptide expressed on the vector surface confers specificity of targeting to the BBB and / or CNS. In another embodiment, the peptide expressed on the vector surface targets the vector to a specific cell type(s) of the BBB and / or CNS. Examples of suitable peptides, as well as methods for generating and testing such peptides, including phage display methods, are known in the art.

[0104] Alternatively or additionally, a neurotropic vector comprising a polynucleotide(s) encoding an antibody or antibody fragment can be used to transduce or transfect cells of the BB and / or CNS, such as HSV, among others. Thus, in one embodiment, the vector comprises a neurotropic vector. In another embodiment, the vector comprises a modified HSV.

[0105] All embodiments described herein in relation to vectors for use according to the preceding aspects of the invention are equally applicable to these further aspects of the invention.

[0106] In one embodiment, the expression cassette comprises at least one promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second gene encoding the heavy chain of the antibody or antibody fragment. The expression cassette may further comprise an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody. In other words, the expression cassette comprises at least one promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment, an IRES, and a second gene encoding the heavy chain of the antibody or antibody fragment. In a further embodiment, the expression cassette comprises a first promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment. The position of each element within the expression cassette is relative to other elements and is conventionally represented in order moving from the 5' to the 3' end.

[0107] The present invention also describes unexpected high antibody expression efficiency and improved protein quality by using different secretory peptides, alternating chain positions, and customizing middle and downstream regulatory elements with respect to strategies previously reported in the prior art.

[0108] Thus, the present invention provides a method for reducing antibody or antibody fragment aggregation, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; Includes.

[0109] The same constructs also result in improved antibody quality compared to methods in which other constructs are used (particularly those having a heavy chain before the light chain in the cassette and / or incorporating a self-cleaving peptide such as the Furin / 2A peptide, as discussed herein). Thus, the present invention also provides a method for improving the maturation and / or quality of an antibody or antibody fragment, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; Includes.

[0110] In other words, one method according to the present invention results in an increase in the percentage of antibodies or antibody fragments that have the same three-dimensional structure as the native configuration of the antibody or antibody fragment. This can be measured, for example, using electrophoresis such as SDS-PAGE. Samples can also be reduced (e.g., with DTT) and then run on a gel to confirm correct disulfide bond formation and that both light and heavy chains migrate at the expected molecular weight.

[0111] The inventors have surprisingly observed that the position of the gene encoding the light chain of an antibody or antibody fragment relative to the gene encoding the heavy chain of the antibody or antibody fragment influences the observed rate of aggregation. Indeed, there is reduced aggregation of the expressed antibody when the gene encoding the light chain is the first gene in the expression cassette followed by the gene encoding the heavy chain, compared to the reverse orientation (e.g. when the gene encoding the heavy chain is the first gene in the expression cassette followed by the gene encoding the light chain).

[0112] In one embodiment, the cell is transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment, and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment, and antibody aggregation is reduced compared to a cell transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding the heavy chain of the antibody or antibody fragment, and the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment. As described in more detail elsewhere herein, the expression cassette may optionally include a sequence providing or encoding a WPRE at the 3' end of the construct, as represented diagrammatically in the top construct of Figure 1. Thus, in one embodiment, the expression cassette includes a sequence providing or encoding a WPRE followed by a second gene encoding the heavy chain of an antibody or antibody fragment. This applies to all relevant constructs and methods of the invention.

[0113] In a further embodiment, the cells are transformed with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of the antibody or antibody fragment, and exhibit reduced aggregation of the antibody or antibody fragment compared to a cell transformed with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a heavy chain of the antibody or antibody fragment, and a second promoter operably linked 5' to 3' to a first gene encoding a heavy chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a light chain of the antibody or antibody fragment.

[0114] For example, there is at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% reduction in antibody or antibody fragment aggregation.

[0115] The inventors have also surprisingly observed increased aggregation of constructs containing Furin and 2A (e.g., the 2A peptide from foot and mouth disease virus, F2A) to allow for self-cleavage. Therefore, the expression cassette preferably does not contain a self-cleaving peptide, and in particular does not contain Furin / 2A.

[0116] The inventors have also observed that the position of the gene encoding the light chain of an antibody or antibody fragment relative to the gene encoding the heavy chain of the antibody or antibody fragment affects the antibody titer. Indeed, when the gene encoding the light chain is the first gene in the expression cassette followed by the gene encoding the heavy chain, the titer of the expressed antibody or antibody fragment is increased compared to the reverse orientation (e.g. when the gene encoding the heavy chain is the first gene in the expression cassette followed by the gene encoding the light chain).

[0117] In a further aspect, the invention provides a method of increasing the titer of an antibody or antibody fragment, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising either an internal ribosome entry site (IRES) or a self (e.g., furin-2A) cleavage site after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising, 5' to 3', a first promoter operably linked to a first gene encoding the light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; Includes.

[0118] This observation applies to autocleavage sites, particularly Furin-2A containing constructs, although these are preferably not included due to the aggregation and immunogenicity issues discussed herein.

[0119] In one embodiment, the cell is transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second gene encoding the heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment. Thus, the antibody titer is increased compared to a cell transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding the heavy chain of the antibody or antibody fragment and a second gene encoding the light chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment. As described in more detail elsewhere herein, the expression cassette may optionally include a sequence providing or encoding a WPRE at the 3' end of the construct, as represented diagrammatically in the top construct in Figure 1. Thus, in one embodiment, the expression cassette includes a sequence providing or encoding a WPRE followed by a second gene encoding the heavy chain of an antibody or antibody fragment.

[0120] In another (less preferred) embodiment, the cell is transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second gene encoding the heavy chain of the antibody or antibody fragment, the expression cassette further comprising a self (e.g., furin-2A) cleavage site after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment, and the antibody titer is increased compared to a cell transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to the first gene encoding the heavy chain of the antibody or antibody fragment and the second gene encoding the light chain of the antibody or antibody fragment, the expression cassette further comprising a self (e.g., furin-2A) cleavage site after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment. As described in more detail elsewhere herein, the expression cassette may optionally include a sequence providing or encoding a WPRE at the 3' end of the construct, as represented diagrammatically in the top construct in Figure 1. Thus, in one embodiment, the expression cassette includes a sequence providing or encoding a WPRE followed by a second gene encoding the heavy chain of an antibody or antibody fragment.

[0121] In a further embodiment, the cells are transformed with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of the antibody or antibody fragment, resulting in increased antibody titer compared to a cell transformed with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a heavy chain of the antibody or antibody fragment, and a second promoter operably linked 5' to 3' to a first gene encoding a heavy chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a light chain of the antibody or antibody fragment.

[0122] For example, there is at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% increase in antibody or antibody fragment titer.

[0123] Without wishing to be bound by theory, the inventors also believe that antibodies produced using expression cassettes that do not utilize an autocleavage site such as Furin-2A have reduced immunogenicity compared to antibodies produced using expression cassettes that utilize an autocleavage site such as Furin-2A. This is because the antibodies lack any residues of an autocleavage site such as Furin-2A peptide, which is known to promote neutralizing antibody responses and / or cellular immunity against antibodies. As a result, antibodies produced using expression cassettes that do not utilize an autocleavage site (e.g., Furin-2A) are believed to result in reduced undesired immunogenicity. As used herein, the term "undesired immunogenicity" refers to an immune response by an animal (e.g., human) against an antibody (especially one that is directed against a peptide remnant of the autocleavage peptide contained in the expression cassette) that results in the production of neutralizing antibodies and / or cellular immunity, reducing the activity of therapeutic antibodies in vivo. Furthermore, the undesired immunogenicity may be the cause of adverse events associated with antibody or antibody fragment therapy in vivo.

[0124] Thus, in a further aspect, the invention provides a method for reducing the immunogenicity of an undesired antibody or antibody fragment, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked 5' to 3' to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; Includes.

[0125] Another aspect of the invention provides a method for reducing adverse events associated with antibody or antibody fragment therapy, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising, 5' to 3' to a first promoter operably linked to a first gene encoding the light chain of the antibody or antibody fragment and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; Includes.

[0126] In one embodiment according to these aspects, the cell is transformed or transduced with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment, and exhibits reduced undesirable immunogenicity compared to a cell transformed with an expression cassette comprising at least one promoter operably linked 5' to 3' to the first gene encoding the light chain of the antibody or antibody fragment and the second gene encoding the heavy chain of the antibody or antibody fragment, and the expression cassette further comprising a Furin-2A cleavage site after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment.

[0127] For example, there is at least a 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% reduction in immunogenicity of the antibody or antibody fragment, and / or a corresponding reduction in adverse events associated with antibody or antibody fragment therapy.

[0128] In the context of a comparator, one skilled in the art will understand that the same cells and the same conditions are used.

[0129] In a further embodiment, the antibody or antibody fragment does not comprise an autocleaving element, hi another embodiment, the antibody or antibody fragment does not comprise a Furin-2A peptide or a fragment thereof.

[0130] In another aspect, the present invention provides an antibody or antibody fragment obtained by the method according to the present invention. The antibody or antibody fragment obtained by the method according to the present invention can be used to reduce undesired immunogenicity (which can include inflammation) in a subject, compared to an antibody or antibody fragment produced by a method using an expression cassette that includes a self-cleavage site between the genes encoding the heavy and light chains of the antibody or antibody fragment. In a further embodiment, the antibody or antibody fragment produced by the method of the present invention can also reduce any toxicity associated with the antibody produced by a method using an expression cassette that includes a self-cleavage site between the genes encoding the heavy and light chains of the antibody or antibody fragment.

[0131] The present invention also provides a corresponding expression cassette that can be used in such a method. Thus, an expression cassette is provided that includes at least one promoter operably linked 5' to 3' to a first gene encoding the light chain of an antibody or antibody fragment and a second gene encoding the heavy chain of an antibody or antibody fragment, and the expression cassette further includes an IRES after the first gene encoding the light chain of an antibody or antibody fragment and before the second gene encoding the heavy chain of an antibody or antibody fragment. Introduction of the expression cassette into a cell generates a higher antibody titer compared to the antibody titer generated when an expression cassette including at least one promoter operably linked 5' to 3' to a first gene encoding the heavy chain of an antibody or antibody fragment and a second gene encoding the light chain of an antibody or antibody fragment, and an IRES after the first gene encoding the light chain of an antibody or antibody fragment and before the second gene encoding the heavy chain of an antibody or antibody fragment is introduced into the same cell. As described in more detail elsewhere herein, the expression cassette may optionally include a sequence providing or encoding a WPRE at the 3' end of the construct, as represented diagrammatically in the top construct in Figure 1. Thus, in one embodiment, the expression cassette includes a sequence providing or encoding a WPRE followed by a second gene encoding the heavy chain of an antibody or antibody fragment.

[0132] Similarly, the invention provides an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of the antibody or antibody fragment. Introduction of the expression cassette into a cell produces a higher antibody titer compared to the antibody titer produced when an expression cassette comprising a first promoter operably linked 5' to 3' to a first gene encoding a light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of the antibody or antibody fragment is introduced into the same cell.

[0133] These expression cassettes preferably do not contain a self-cleavage site, such as a Furin / 2A cleavage site, as such constructs result in higher aggregation (i.e., lower quality) and further increased immunogenicity of the expressed antibodies.

[0134] The present invention also relates to a vector for transducing or transfecting cells of the blood-brain barrier (BBB) ​​or CNS, comprising an expression cassette of the invention as described herein. Such vectors are useful in a method for producing an antibody or antibody fragment, comprising transforming a cell with the vector and maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment.

[0135] The expression cassettes described herein were designed with delivery via viral vectors in mind. Such vectors are limited in the amount of genetic material that can be packaged. Thus, the present invention provides viral vectors, particularly neurotropic viral vectors, or viral vectors capable of transducing or transfecting cells of the BBB or CNS that contain the expression cassettes of the present invention. The viral vectors may include engineered AAV2 vectors, preferably AAV-BR1, or engineered AAV9 vectors, such as AAV-S, AAV-AF, AAV-PHP.eB, or AAV9-PHP-V1, or engineered AAV1 vectors, such as AAV1RX, AAV1R6, or AAV1R7. The viral vectors include an expression cassette that includes at least one promoter operably linked from 5' to 3' to a first gene encoding the light chain of an antibody or antibody fragment, and a second gene encoding the heavy chain of an antibody or antibody fragment, and further includes an IRES after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment. The viral vector may contain an expression cassette comprising, 5' to 3', a first promoter operably linked to a first gene encoding a light chain of an antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of an antibody or antibody fragment.

[0136] These vectors and expression cassettes can be used in any of the methods according to the invention. [Brief description of the drawings]

[0137] [Figure 1]Representation of different constructs investigated to produce pAAV-derived antibodies, linked derivatives and other proteins. The curved arrows indicate alternating positions of light and heavy chains in the expression cassette. SP: secretory peptide, IRES: internal ribosome entry site, FSG2A: Furin / 2A self-cleaving peptide, WPRE: Woodchuck Hepatitis Virus post-transcriptional regulatory element, pA: polyA. The heavy or light chain can be any antibody sequence. The promoter is preferably CMV (human cytomegalovirus) or other promoters, such as but not limited to EF1A (human eukaryotic translation elongation factor 1 alpha 1), CAG (CMV early enhancer fused to chicken β-actin promoter), CBh (CMV early enhancer fused to modified chicken β-actin promoter), SV40 (simian virus 40 enhancer / early promoter), GFAP (human glial fibrillary acidic protein promoter)*: 3' regulatory element not included. [Diagram 2] Figure 2A. CHO cell supernatant titers of MAB1 IgG and scFv-Fc after pAAV transfection. BLI Octet system, Protein A coated biosensor. The arrows indicate a clear drop in expression when the heavy chain genes are placed in front of the light chains in the construct (HC / LC) compared to their respective LC / HC counterparts. The clonal constructs follow the options in Figure 1 detailed in the figure. Figure 2B. CHO cell supernatant titers of MAB1 Fab after pAAV transfection. BLI Octet system, Anti-His coated biosensor. The arrows indicate again a clear down-expression when the heavy chain genes are placed in front of the light chains in the construct (HC / LC) compared to their respective LC / HC counterparts. [Diagram 3] SDS-PAGE separation of purified MAB1 IgG with Coomassie Blue staining. Samples were reduced in the presence of 5 mM dithiothreitol (DTT) where indicated. Arrows indicate a shift to higher molecular weight. [Figure 4]SDS-PAGE separation of purified MAB1 scFv-Fc with Coomassie Blue staining. Samples were reduced in the presence of 5 mM dithiothreitol (DTT) where indicated. [Diagram 5] MAB1 IgG binding to TP-62 peptide. Octet-Kinetics starts with 100 nM binder protein candidate and serially dilutes 2-fold. Top panels cover two-promoter MAB1 IgG with different promoters and Furin HC / LC IgG constructs. Bottom panels cover Furin HC / LC and LC / HC MAB1 IgG constructs, IRES LC / HC and HC / LC IgG constructs, and scFv-Fc IgG constructs. Details of each construct are found in the figures. Each panel starts with biosensor loading with 500 nM TP-62 peptide. [Figure 6] Size-exclusion chromatographic analysis of relevant purified pAAV-derived IgG using a Superdex 200 Increase 10 / 300GL column. Separation was performed in PBS buffer at 4°C. Details of individual constructs are shown in the figures. [Figure 7] Size-exclusion chromatogram overlay of relevant purified pAAV-derived IgG under the same conditions as described in Figure 6. [Figure 8] Analysis of the C-terminal portion of the Furin MAB2 IgG construct confirmed the Furin / F2A peptide left on the construct, thereby increasing the mass of the first antibody chain on the construct, after in-gel trypsin digestion, followed by LC / MS. [Figure 9] Purified MAB1 IgG and scFv-Fc per mL of culture obtained from CHO transduction of AAV2, 8, 9 and 10 were compared to transfection. The amount of purified protein per mL was quantified by OD280nm using their corresponding extinction coefficients. Details of each construct can be found in the figures. [Figure 10]SDS-PAGE separation of purified MAB1 IgG with Coomassie Blue staining. Samples were reduced in the presence of 5 mM dithiothreitol (DTT) where indicated. [Figure 11] Comparison of IgG titers after transduction of CHO, differentiated neuronal and BBB cells with AAV2, 8, 9 and 10 vectored antibody constructs. Left panel, CHO transduction; middle panel, differentiated human neuroblastoma cell line transduction; right panel, differentiated blood-brain barrier (BBB) ​​cell transduction. Details of each construct are found in the figure. [Figure 12] Comparison of IgG titers following transduction of rat brain primary cells with AAV2, 8, 9 and 10 vectored MAB1 IgG and scFv-Fc constructs. Details of each construct are found in the figure. [Figure 13] Detection of MAB1 (human IgG1 isotype) secreted by hCMEC / D3 cells in the apical supernatant of the OrganoPlate® 3D BBB model 3 days after transduction with different WT AAV serotypes. Expression of MAB1 was driven by the CMV promoter. An IRES element was used between the LC and HC to achieve bicistronic antibody production. In summary, the constructs tested had the following configuration: 5'-CMV promoter-gene encoding light chain and secretory peptide-IRES-gene encoding heavy chain and sequence encoding secretory peptide-3'. The presence of antibodies measured by ELISA binding to hTDP-43 full-length (FL) protein is expressed in OD (optical density). No binding was observed for the control AAV2-eGFP transduction. As expected, for the two controls, mIgG MAB1 and hIgG MAB2, anti-human secondary antibodies were used for detection and no antibody binding was detected since MAB2 cannot bind to the hTDP-43 FL protein. [Figure 14]Detection of MAB1 (human IgG1 isotype) secreted by hCMEC / D3 cells in both apical and basal compartments of the OrganoPlate® 3D BBB model 3 days after transduction with AAV2 constructs. Bicistronic expression of MAB1 driven by the CMV promoter was achieved using an IRES element between the LC and HC. In summary, the constructs tested had the following configuration: 5'-CMV promoter-gene encoding light chain and secreted peptide-IRES-gene encoding heavy chain and sequence encoding secreted peptide-3'. Presence of antibodies measured by ELISA binding to TDP-43 full-length protein. No binding was observed for the control AAV2-eGFP transduction. Data are expressed as OD. [Figure 15] Detection of MAB1 (human IgG1 isotype) secreted by hCMEC / D3 cells in the supernatant of 24-well culture plates 3 days after transduction with AAV2 constructs at different MOIs (multiplicity of infection). Bicistronic expression of MAB1 driven by the CMV promoter was achieved using an IRES element between the LC and HC. In summary, the constructs tested had the following configuration: 5'-CMV promoter-gene encoding light chain and secretory peptide-IRES-gene encoding heavy chain and sequence encoding secretory peptide-3'. Presence of antibodies measured by ELISA binding to TDP-43 full-length protein. Data are expressed as concentrations calculated from a standard curve. [Figure 16]Detection of MAB1 (human IgG1 isotype) secreted by hCMEC / D3 cells in both the apical and basal compartments of a transwell BBB model 3 days after transduction with AAV2 constructs. Bicistronic expression of MAB1 driven by the CMV promoter was achieved using an IRES element between the LC and HC. In summary, the constructs tested had the following configuration: 5'-CMV promoter-gene encoding light chain and secretory peptide-IRES-gene encoding heavy chain and sequence encoding secretory peptide-3'. Presence of antibodies measured by ELISA binding to hTDP-43 full-length protein. No binding was observed for the control AAV2-eGFP transduction (data not shown). Data are expressed as the relative percentage of antibodies present. [Figure 17] Detection of MAB1 hIgG1 and scFv-Fc in cell supernatants 6 days after transduction with AAV2, AAV-BR1 and AAVrh10 constructs in (A) b.End3 and b.End5 mouse brain endothelioma cell lines, and (B) hCMEC / D3 cells. Bicistronic expression of MAB1 driven by the CMV promoter was achieved using an IRES element between LC and HC. In summary, the constructs tested had the following configuration from 5' to 3': CMV promoter-secreted peptide-light chain-IRES-secreted peptide-heavy chain-WPRE-polyA. Presence of antibodies measured by HTRF (homogeneous time-resolved fluorescence) using an anti-hFc kit (PerkinElmer, Cisbio). Data expressed as concentrations interpolated from a standard curve generated from 3-6 biological replicates within a single experiment plotted as mean ± SD. [Figure 18]Detection of MAB1 hIgG1 or mIgG2a secreted by primary human brain microvascular endothelial cells from a commercial 3D human in vitro BBB model (Neuromics). Detection in both apical and basal compartments 7 days after transduction with AAV2 and AAV-BR1 vectors at an MOI of 100'000. Bicistronic expression of MAB1 driven by the CMV or CBh promoter was achieved using an IRES element between the LC and HC. In summary, the constructs tested had the following configuration from 5' to 3': CMV or CBh promoter-secreted peptide-light chain-IRES-secreted peptide-heavy chain-WPRE-polyA. Presence of antibodies measured by HTRF using an anti-hFc kit (PerkinElmer, Cisbio). Data expressed as (A) concentration or (B) amount interpolated from standard curves for the respective apical and basal compartments. To allow direct comparison between the conditions evaluated, two to three biological replicates were performed within a single experiment with mean values ​​± SD plotted. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0138] The invention is further described in the following examples, which serve only to illustrate the invention and are not intended to limit the scope of the invention in any way. EXAMPLES

[0139] [Example 1] Vectored antibody constructs: 1. Selection of optimal clones for vectorization Introduction One of the main challenges with the use of single-stranded DNA (ssDNA) vectorization in AAV capsids is capacity. The expression cassette size includes a) a promoter, b) an open reading frame (typically encoding an antibody or antibody fragment, including heavy and light chains), and c) downstream regulatory elements. Once the DNA is converted to single stranded and vectorized in the AAV capsid, the recommended size is 4.7 kb or less, including the 5'ITR and 3'ITR. Constructs of larger size will barely fit into the AAV viral capsid complex, thereby reducing production yields. Self-complementary constructs have even smaller capacity, i.e., 2.3 kb or less, within the 5'ITR and 3'ITR.

[0140] Commonly used antibody plasmid AAV (pAAV) designs are smaller in size and are generated using antibody fragments that fit into expression cassettes driven by a single promoter, such as a) antibody single-chain variable fragments (scFv) [44,55], b) scFv fused to an IgG Fc domain [56,57], or c) single-domain antibodies such as shark and / or camel antibodies [58-62].

[0141] In the case of complete antibodies, it is difficult to fit both light and heavy chain genes due to the aforementioned size limitations of the expression cassette. To date, most academic and industrial researchers rely on pAAV constructs consisting of an open reading frame translated with a self-processing protein fusion. In most cases, the respective coding sequence consists of 1) an antibody heavy chain, 2) a self-cleaving 2A peptide that is also known as PACE (paired basic amino acid cleaving enzyme

[63] , 3) a furin protease recognition site that promotes furin cleavage [47-49], which is expected to facilitate the cleaning process, i.e., E2A, F2A, P2A or T2A, and 4) an antibody light chain. Researchers significantly prefer this construct due to the high expression titers obtained and equimolar concentrations in antibody chain expression. However, in most cases, the remnants of furin and 2A fusion peptides included in the expressed protein

[64] do not allow the protein to mature properly, thereby exploiting the undesired immunogenicity of the expressed protein

[45] . Different 2A peptides have different efficiencies of self-cleavage, with T2A and P2A being the most efficient and F2A being the least efficient

[65] . Thus, up to 50% of F2A-bound proteins may remain in the cell as fusion proteins, potentially resulting in unexpected consequences such as gain-of-function

[66] . 2A sites cause ribosomes to leave the polynucleotide about 60% of the time. Together with about 10% ribosomal reads for P2A and T2A, this reduces the expression of downstream peptide chains by about 70%

[47] . Furthermore, it is important to note that 2A peptides are derived from viruses, i.e., F2A is derived from foot-and-mouth disease virus 18, E2A is derived from equine rhinitis virus A, P2A is derived from porcine teschovirus 1-2A, and T2A is derived from tosea signavirus 2A. Thus, 2A peptide residues left in either the heavy or light chains may be considered non-self by the mammalian and human immune systems.

[0142] Less common pAAV constructs that are alternatives to the Furin / 2A fusion for antibody expression are 1) the use of two promoters driving the heavy and light antibody chains, or 2) a single promoter with an internal ribosome entry site (IRES, a DNA sequence derived from the encephalomyocarditis virus) located between the two antibody gene chains. First, the constraints in pAAV design are increased because of the generally large size of the promoters, which necessitates the use of small promoters to control the heavy and light antibody genes and remove the 3'ITR regulatory elements that can greatly increase protein titers. Second, IRES constructs are considered to be poor protein expressors to date [56,67-69] and are therefore ignored for many pAAV constructs (where IRES constructs express much lower titers than 2A or Furin 2A constructs).

[0143] 1.2. Selection approach We screened over 55 pAAV constructs that were produced and investigated for expression, depending on the open reading frame component position, promoter strength, secretory peptide and fusion peptide. The different types of constructs are shown in Figure 1. To the best of our knowledge, the only complete IgG expression using AAV vectors reported in the literature is the Furin / 2A construct, where the heavy chain is placed before the light chain (see Figure 1, second construct). Our approach also investigated other light and heavy chain positions of this type of construct, as well as different promoter strengths of the bicistronic construct (Figure 1, fourth construct). Furthermore, we challenged the reported low expression of the IRES bicistronic construct by alternating chain positions with different secretory peptides and by customizing the middle and downstream regulatory elements (Figure 1, third construct). Finally, two full antibody clones and the corresponding antibody fragments, such as scFv-Fc, Fab and scFv, namely MAB1 (anti-TDP-43 antibody) and MAB2 (anti-ErbB2 antibody), were tested (Figure 1, first construct). Additionally, a clone expressing enhanced green fluorescent protein (eGFP) was included.

[0144] In our study, the selection criteria for identifying the best pAAV constructs were defined as follows: 1) high protein titer, 2) low aggregation levels, 3) correct maturation and folding, and 4) target binding.

[0145] 1.3. Chinese Hamster Ovary (CHO) Cell Transfection and Supernatant Titers Briefly, CHO cells (ExpiCHO-S™ (ThermoFisher, cat:A29127)) were transfected in triplicate with 1 μg / mL plasmid per construct according to the manufacturer's recommendations. Cells were grown in deep-well microplates for 24 hours at 37°C with optimized synthetic medium, then temperature shifted to 32°C and subsequently grown for 11 days in a final volume of approximately 3.5 mL medium. Each supernatant was then titered in triplicate using Bio-Layer Interferometry (BLI) with an Octet system and biosensor chips coated with either a) full-length antibodies and scFv-Fcs, or b) monoclonal antibodies conjugated His-tagged proteins (here Fabs, scFvs and EGFP). For all measurements, standard curves were performed with the corresponding, previously purified proteins. Results for antibodies and scFv-Fcs are shown in Figure 2A, and for Fabs in Figure 2B.

[0146] observation. In the case of MAB1 IgG, each of the two promoter constructs produced significant titers in the range of about 30-100 μg / mL. Surprisingly, about 2-10 times higher titers in the range of 150-350 μg / mL were obtained with the Furin and IRES constructs, but notably only when the light chain gene was placed in front of the heavy chain in the construct (LC / HC) compared to the two promoter IgG construct. The high protein levels in the IRES IgG construct LC / HC were comparable to the Furin IgG construct. This was unexpected and, to the best of our knowledge, the first report since the state of the art states the opposite [56,67-69]. A similar observation was made for the MAB1 Fab construct in Figure 2B, which shows similar titers to the Furin and two promoter constructs. Furthermore, positioning the heavy chain preceding the light chain (LC / HC) in IRES Fab constructs has significantly higher expression titers than their HC / LC construct counterparts. Data collected shows that in IgG or Fab constructs using Furin and an IRES, positioning the light chain before the heavy chain increases titers. Titers are significantly higher than their HC / LC counterparts. Finally, expression appears to be reduced when the medium-strength promoter SV40 is included as one of the two promoters in two promoter IgG constructs.

[0147] 1.4. Protein purification and analysis on SDS-PAGE Following this experiment, cells were harvested and supernatants were separated in triplicate and combined per clone. For full length antibodies and scFv-Fc clones, proteins were captured using a commercially available Protein A resin and eluted with 100 mM glycine pH 2.8 buffer supplemented with 100 mM NaCl. Proteins were then quantified spectrophotometrically at OD280nm using their corresponding extinction coefficients. For Fabs and scFvs constructs, a commercially available affinity column was used for His-tagged proteins.

[0148] Proteins were then separated by SDS-PAGE 12-4% and stained with Coomassie Blue. Independent of protein concentration, 13 μL per sample was loaded per gel well to a) obtain a second estimate of purification titer and b) confirm possible degradation products. Additionally, verification of interchain disulfide bond formation and proper complexation at 150 kDa was performed in the absence of dithiothreitol (DTT) and release of light and heavy chains in the presence of dithiothreitol. The resulting gel for the purified MAB1 IgG pAAV clone is shown in Figure 3.

[0149] Observation. All samples had the proper disulfide bond arrangement and the antibody migrated at the corresponding molecular weight of 150 kDa. Upon reduction, both constructs, 2 promoter and IRES IgG, were resolved at the expected light and heavy chain molecular weights of approximately 25 and 50 kDa, respectively. However, it was noted that the first chain of the Furin / 2A IgG construct was of a larger molecular weight, i.e., the heavy chain in the HC / LC construct arrangement and the light chain in the LC / HC arrangement. The size of each Furin / 2A IgG chain construct was further examined by LC / MS for peptide accuracy. This confirmed that all or part of the Furin / 2A peptide was added to the C-terminus of either the LC / HC construct or the heavy chain for the HC / LC construct (see Figures 3, 7 and 8). In contrast, the second antibody construct chain was properly processed and no peptide remnants were observed by LC / MS at the N-terminus. Overall, all clones showed low levels of degradation under the applied cell culture conditions. The 2 promoter and IRES protein appears to have the most accurate maturation quality. Finally, a similar observation was made for a different IgG antibody, clone MAB2. Again, the Furin / 2A construct had a higher molecular weight relative to the first construct chain, and the addition of the Furin / 2A peptide was confirmed by LC / MS, but the 2 promoter and IRES IgG showed the most accurate maturation. The purified MAB1 scFv-Fc sample was also separated under the same SDS-PAGE conditions. The results are shown in Figure 4.

[0150] Observations: Purified MAB1 samples show that the scFv-Fc has proper interchain disulfide bond formation and the protein migrates at its expected molecular weight of approximately 100 kDa. Reduction of the sample with 5 mM DTT releases a single chain that migrates at the expected molecular weight of 50 kDa. Overall, all clones showed low levels of degradation in the cell culture conditions used.

[0151] 1.5. Analysis of Binding Affinities of Transfection-Derived Proteins by BLI To demonstrate that the pAAV system-generated proteins were functional, we measured their binding affinity to the TAR DNA-binding protein 43 (TDP-43) C-terminal peptide by BLI using the Octet system. Briefly, a streptavidin biosensor chip was coated with 500 nM of the TDP-43 C-terminal peptide, called TP-62. Measurements were then performed using a reaction buffer consisting of PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween. Reactions were performed at 30°C. Samples were analyzed in two-fold serial dilutions starting from a 100 nM concentration. Protein molar concentrations were calculated using the corresponding extinction coefficients at OD280 nm. Sample association to the TP-62 peptide was left for 900 s in reaction buffer, followed by 600 s dissociation in the same buffer. The biosensor was regenerated with 10 mM glycine pH 2.0 and neutralized in reaction buffer before measurement of each sample. The collected data are shown in Figure 5 for the relevant purified samples.

[0152] Overall, all MAB1 clones were functional and had comparable K values ​​ranging from approximately 1 to 6 nM. D The 2 promoter and IRES constructs had similar Rmax values, ranging from about 2.5 to 3 nm. Importantly, the K DThe affinity did not vary depending on the promoter or secreted peptide (CMV / CMV, CMV / SV40 or SV40 / CMV promoter constructs, and Igk or GH1 secreted peptides, respectively). In contrast, the Furin IgG HC / LC construct produced a protein with approximately twice the Rmax than the 2 promoter and IRES construct, approximately 5-6 nm versus approximately 2.5-3 nm, respectively. Furthermore, the Furin IgG LC / HC construct had a less pronounced but larger Rmax than its 2 promoter and IRES counterpart, approximately 2.5 nm versus approximately 2.5-3 nm, respectively. According to the BLI system, the data show a larger molecular weight protein for the Furin / 2A construct at equimolar concentrations compared to its 2 promoter and IRES counterpart, e.g., Furin IgG HC / LC protein under the assay conditions. This observation was confirmed in the next analytical step using size exclusion chromatography separation (see 1.6). Finally, the data demonstrate that functional IgG and scFv proteins can be obtained in the pAAV expression system. Further Octet analysis is described below (see 1.6).

[0153] 1.6. Protein Analysis by Size Exclusion Chromatography and LC / MS The purified proteins were separated by size-exclusion chromatography to monitor for degradation, aggregation, and the presence of proper protein folding, separated as monomers at a molecular weight of 150 kDa. For this, 100 μL of purified samples were separated on a Superdex 200 Increase 10 / 300GL column from G&E Healthcare at 4 °C with PBS buffer. The separation obtained is shown in Figure 6.

[0154] Observations. For all constructs, IgG monomers were separated as expected with an elution volume of approximately 11.7 mL. In contrast, the Furin IgG construct had a higher level of aggregates, 26% for the LC / HC construct and 38% for the HC / LC construct. Furthermore, the monomer peak of the Furin construct does not align with the IRES and 2 promoter IgG constructs (see overlay Figure 7), has a slightly higher molecular weight in the separation, and elutes earlier. This is clearly the case for the Furin IgG LC / HC construct, which elutes before 0.25 mL. The higher aggregation level of the Furin / 2A construct (Figures 6, 7 and 8) may reflect the reported unexpected outcomes and toxicity of this type of protein

[66] . These data confirm the BLI measurements above showing the larger molecular weight of the Furin / 2A derived IgG. Conversely, the IRES IgG construct protein demonstrated a higher quality, with a very low 2% aggregation level and 98% abundant monomeric protein. The two-promoter IgG protein had a low aggregation level of 13%, although higher than the IRES construct. These results identified the IgG IRES LC / HC and the two-promoter LC / HC as constructs with better quality, expression yield, stability, and potentially less undesirable immunogenicity and / or toxicity.

[0155] 2. Vectorization of MAB1 and MAB2 lead candidates 2.1. Selection of AAV capsids for cell transduction As mentioned above, the best constructs of MAB1 and MAB2 that provided the highest quality protein were selected for vectorization, i.e., a) MAB1 IgG IRES LC / HC and two promoter IgG LC / HC constructs, b) MAB2 two promoter IgG LC / HC, and c) MAB1 scFv-Fc. A panel of different cells of interest was selected for transduced gene delivery, i.e., human neuroblastoma cell line differentiated with Chinese ovarian hamster cells (CHO), neuronal and brain endothelial cell line (hCMEC / D3). For this, the best pAAV constructs mentioned above were vectorized in AAV2, AAV8, AAV9 and AAV10 capsids. The resulting low endotoxin ultra-purified capsids were used to transduce cells.

[0156] 2.2. CHO cell transduction, protein purification and titer comparison with the same transfected clone Briefly, CHO cell cultures were transfected in triplicate with vectorized lead constructs at 100K gc (genome copies) / CHO cell, then grown in deep-well microplates for 24 hours at 37°C in synthetic medium optimized as above, then temperature shifted to 32°C, and then grown for 11 days in a final volume of 3.5 mL per well. Cell growth was not affected by the presence of AAV. Cells were then harvested and supernatants were separated in triplicate and combined per clone. Full-length antibodies and scFv-Fc clones were captured as above using protein A resin and eluted with 100 mM glycine pH 2.8 buffer supplemented with 100 mM NaCl. Proteins were then quantified spectrophotometrically at OD280nm using their corresponding extinction coefficients. The resulting titers are shown in Figure 9.

[0157] Observations: All transduced lead gene constructs were expressed with each tested AAV capsid. Expression levels vary as a function of the capsid used for vectorization, but are overall similar to the same constructs transfected with purified plasmid.

[0158] 2.3. Transduction of Rat Primary Brain Cells and Antibody Titers Using MAB1 Antigen ELISA Briefly, rat primary cells were dissected from rat pup brains and grown in 96-well microplates with 50K primary cells per well in 100 μL of neurobasal medium supplemented with B27™ (ThermoFisher, cat:17504044) at 37°C. Rat primary brain cells were transduced in triplicate with 100K gc / rat primary cell with vectorized lead constructs and then grown in neurobasal medium supplemented with B27 for 7 days at 37°C. Microscopic morphological evaluation showed that the cells were not affected by AAV presence. Triplicate transduced cell supernatants were then collected and antibody titers against hTDP-43 full-length protein were quantified by ELISA. Briefly, 96-well microplates were coated overnight with 1 μg / ml human full-length TDP-43 in PBS buffer at 4°C. The plates were then washed three times with PBS supplemented with 0.05% Tween and then blocked with PBS supplemented with 1% bovine serum albumin, 0.05% Tween for 1 h at 37°C. The collected antibody-containing supernatants were diluted in blocking buffer, diluted 20, 40, and 80 times, and 50 μL of the samples were added to the microplate and incubated for 1 h at 37°C. The plates were then washed as above and incubated with goat anti-human IgG Fc-HRP (ab cam, #98624) diluted 1 / 10000 in blocking buffer for 1 h at 37°C. The plates were washed as above and the wells were supplemented with 100 μL of TMB substrate and incubated for a few minutes at room temperature. The HRP reaction was then blocked with 50 μL of H2SO4 0.16 M. Finally, the resulting solutions were red-lighted at 450 nm using a microplate reader (BioTek). The corresponding antibody titers are shown in Figure 12. The data show that MAB1 IgG and scFv-Fc clones vectored in AAV2 have significantly lower expression titers ranging from 5-50 ng / mL of cell supernatant. In contrast, other AAV8, 9 and 10 vectored candidates have much greater IgG and scFv-Fc titers ranging from approximately 500-2000 ng / mL of cell supernatant regardless of expression drive system (IRES or 2 promoter), except for MAB1 vectored in AAV9, which is expressed under two CMV promoters at a titer of approximately 180 ng / mL.

[0159] Observation: All transduced lead gene constructs were expressed with each tested AAV capsid. As noted above, expression levels vary as a function of the capsid used for vectorization, but are overall similar to the same constructs transfected with purified plasmid.

[0160] 2.4. Analysis of Protein Maturation by SDS-PAGE The purified lead proteins were separated by SDS-PAGE 12-4% and stained with Coomassie Blue. As above, protein samples were loaded in equal amounts (13 μL / sample) without matching the loading amount to verify the amount measured by OD280nm. The resulting separation is shown in Figure 10 for the MAB1 pAAV clone.

[0161] Observation. All samples had the proper disulfide bond arrangement and the antibody migrated at the corresponding molecular weight of 150 kDa, as shown in Figure 10. When reduced, both constructs, the 2 promoter and IRES IgG, separated at the expected light and heavy chain molecular weights of approximately 25 and 50 kDa, respectively. The scFv-Fc formed the proper interchain disulfide bonds and the protein migrated at the expected molecular weight of approximately 100 kDa. When the samples were reduced with 5 mM DTT and migrated at the expected molecular weight of 50 kDa, a single chain was released. Overall, as expected from previous screening experiments, all clones showed low levels of degradation in the cell culture conditions employed.

[0162] 2.5. Analysis of Binding Affinities of Transfection- and Transduction-Derived Proteins by BLI To demonstrate that the proteins produced using the pAAV system were functional, we measured their binding affinity to the TAR DNA-binding protein 43 (TDP-43) C-terminal peptide by BLI using the Octet system under the same conditions as described above. Briefly, a streptavidin biosensor chip was coated with 500 nM of the TDP-43 C-terminal peptide, called TP-62. Measurements were then performed using a reaction buffer consisting of PBS supplemented with 0.1% bovine serum albumin and 0.02% Tween. Reactions were performed at 30°C. Samples were analyzed in two-fold serial dilutions starting from a 100 nM concentration. The collected data are shown in Table 1 below.

[0163] [Table 1] Table 1.

[0164] The data show that IgG clones from either the IRES or two-promoter pAAV constructs had K values ​​comparable to the standards used. D It has been shown that it has (Table 1). D are comparable between construct types, either transfected or vectorized, in the range of approximately 4-6 nM, thus demonstrating that the high-quality proteins produced by vectorization have the expected binding affinity for the target TDP-43 antigen. The same is true for the scFv-Fc proteins, although perhaps with minor variations.

[0165] 2.6 Transduction of human neuroblastoma cell lines and BBB cells and protein titers in cell supernatants The next step was to verify whether cell types other than CHO could be efficiently transduced with AAV2, 8, 9 and 10 vectored MAB1 IgG and scFv-Fc constructs as well as the negative control MAB2 IgG 2 promoter LC / HC. For this, human neuroblastoma cells differentiated into neurons were transduced at 37°C under 5% CO2 using concentrated synthetic medium supplemented with 10 μM retinoic acid and 2% fetal bovine serum. Approximately 200K cells were transduced in triplicate and incubated in 24-well microplates with 500 μL medium per well. Transduction was performed by adding approximately 100K genome copies / cell. Both vectored MAB1 IgG and scFv-FC titers were measured by ELISA. Similar experiments were performed with hCMEC / D3 cells differentiated in blood-brain barrier microvessels (see Example Procedure below). Here, blood-brain barrier cells were grown at 50K or 100K / well at 37°C with 5% CO2 and transduced with 50K genome copies (gc) per cell of AAV-vectored MAB1 IgG IRES LC / HC construct. For both cell types, the medium was changed every 3 days. Briefly, 96-well microplates were coated with full-length TDP-43 protein and then saturated with PBS buffer supplemented with 1% bovine serum albumin and 0.05% Tween. Samples were then added to the microplate wells and incubated for 1 h at 37°C. At the same time, the corresponding sample standards were serially diluted 2-fold starting at 2 μg / mL concentration with the same buffer as the samples (PBS buffer supplemented with 1% bovine serum albumin and 0.05% Tween). The plates were washed with PBS buffer supplemented with 0.05% Tween. After this, anti-human IgG Fc antibody labeled with horseradish peroxidase was added to the plate in PBS buffer supplemented with 1% bovine serum albumin and 0.05% Tween and incubated for 1 hour at 37°C. The plate was washed as above and horseradish peroxidase substrate (3,3',5,5'-tetramethylbenzidine, called TMB) was added to the wells, incubated at room temperature for approximately 5-10 minutes, and the reaction was stopped with 0.5M H2SO4.Samples were then read individually in a microplate reader at OD450nm. The collected data is shown in Figure 11 and is compared to the purified titers obtained with the same vectored CHO transduction using the MAB1 lead construct.

[0166] Observations. Transduced commercially available cell types, differentiated neurons derived from a human neuroblastoma cell line, and a brain endothelial cell line (hCMECD / 3) all produced functional MAB1 IgG and scFv-Fc capable of binding full-length TDP-43 protein (Figure 11). Overall, antibodies and derivatives are produced over time in large quantities (see middle panel at day 18 for differentiated human neuroblastoma cell line). They represent an average of about 0.2-1 pg antibody / cell / day, a robust cellular expression, similar to that reported for CHO cells grown at higher cell densities (~10 million / mL), which express about 0.5-4 pg / cell / day. In these experiments, the titers obtained are higher with AAV2 vectored MAB1 IgG and scFv-Fc. Furthermore, scFv-Fc titers are higher with each AAV8, 9 and 10 vectorization compared to their vectored IgG counterparts. The differentiated human neuroblastoma cell line titers of the IRES constructs were higher than the two-promoter construct counterparts as a function of the capsid used for vectorization. Finally, the vectorized MAB2 IgG two-promoter LC / HC was not detected due to its property of binding specific antigens other than TDP-43 (see middle panel). In conclusion, the collected data confirmed earlier observations showing that the IgG IRES construct reaches higher expression titers than the two-promoter using the LC / HC configuration.

[0167] Table 2: Nucleic acid sequences used in vectored antibody constructs: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Example 2]

[0168] cell culture Human brain microvascular endothelial hCMEC / D3 cells were cultured on 75 cm endothelial cells pre-coated with 100 μg / mL rat tail collagen type I (08-115, Merck) and 1 ng / mL bFGF (Merck, GF003) in EndoGRO-MV growth medium (Merck, SCME004) supplemented with all factors included in the kit. 2 The flasks were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0169] B.End3 and b.End5 mouse brain endothelioma cell lines were cultured in DMEM medium supplemented with Pen / strep and 10% FBS (growth medium). The cells were cultured in TC-treated 75 cm 2 The cells were cultured in flasks, detached with trypsin-EDTA solution, and subcultured at a ratio of 1:10. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. [Example 3]

[0170] OrganoPlate® 3-Lane In Vitro BBB Model The OrganoPlate® 3-lane system used for 3D in vitro BBB modeling encompasses 40 microfluidic cell culture structures embedded in a standard 384-well microtiter plate format. Each tissue chip is composed of three lanes connected to corresponding wells of the microtiter plate that serve as inlets and outlets to access the microfluidic cultures. First, an extracellular matrix (ECM) gel of 4 mg / mL collagen-I (rat tail, Merck) was prepared by mixing 1 M HEPES, 37 g / L NaHCO3, and 5 mg / mL collagen-I and introduced into the center lane. A phase guide was used to selectively pattern the ECM gel in the center lane by meniscus pinning. After ECM gelation (overnight or over the weekend at 37 °C, 5% CO2), hCMEC / D3 cells were seeded in EndoGRO-MV growth medium supplemented with 1 ng / mL bFGF at a density of 40,000 cells per chip in the top lane. Once cells were attached, plates were placed horizontally on an interval rocker inducing flow by reciprocal leveling between reservoirs and incubated at 37°C, 5% CO2 for at least 3 days to allow tubule formation. Medium changes were performed approximately every 3 days to maintain optimal barrier integrity, which was controlled before each transduction or transcytosis experiment by permeability assay. Barrier function was assessed by perfusing 0.5 mg / mL FITC-dextran (Sigma 46946, average 150 kDa; FD20S, average 20 kDa, and Sigma FD10S, average 10 kDa) in medium through the tube lumen and then determining the fluorescence level in the basal gel area normalized to the fluorescence in the lumen. Fluorescence measurements were performed every 5 min for 1 h using an Incucyte live cell reader. [Example 4]

[0171] Transwell in vitro BBB model In the in vitro BBB model based on transwells, the pore size was 0.4 μm (0.33 cm 2The apical side of a 24-well plate containing a Corning transwell membrane with a culture area of ​​100,000 mm (Sigma) was first coated with rat tail collagen-I (Merck) for 1 h in a humidified incubator. hCMEC / D3 cells were then plated at 100,000 cells / cm. 2 EndoGRO-MVs were seeded in EndoGRO-MV growth medium supplemented with 1 ng / mL bFGF at a density of 100 μL. Medium changes were performed approximately every 2–3 days, always maintaining an apical volume of 100 μL and a basal volume of 600 μL. Permeability and transendothelial electrical resistance (TEER) measurements were performed starting on day 4 after endothelial cell seeding, and we found that the cell monolayer maintained appropriate barrier properties up to day 15 after seeding. For permeability measurements, the endothelial cell medium was replaced with 600 μL of fresh medium in the basal juxtalateral compartment. Then, 100 μL of 0.25 mg / mL FITC (Sigma 46946, average 150 kDa; FD20S, average 20 kDa and Sigma FD10S, average 10 kDa) in the medium was added to the upper compartment, and the cells were incubated for 1 h in a humidified incubator. Then, 100 μL fractions were collected from the basolateral compartment and transferred to a Greiner black 96-well plate for fluorescence measurement using a Tecan Spark microplate reader. Apparent permeability was calculated according to the formula Papp = (ΔQ / Δt) × (1 / AC0), where Papp is the apparent permeability coefficient (cm / min), ΔQ / Δt is the permeation rate of dextran across the endothelial cell layer (μg / min), and A is the surface area of ​​the cell layer (cm 2 ), C0 is the initial dextran concentration (μg / ml) applied to the apical cell surface. For TEER measurements, the endothelial cell medium was replaced with 1050 μL of fresh medium in the lower compartment and 325 μL in the upper compartment. TEER was measured using an EVOM-3 epithelial voltmeter (WPI). [Example 5]

[0172] AAV transduction of hBMEC / hBMEC and antibody detection by target binding - antibody secretion by hCMEC / D3 cells using the OrganoPlate® BBB model Using the established OrganoPlate® hCMEC / D3 in vitro BBB model, we evaluated whether vectored antibodies in AVV WT capsids are efficiently secreted by hCMEC. MAB1 (human IgG1 isotype) antibody was vectored into AAV2, AAV8; AAV9, and AAVrh10 capsids and transduced into hCMEC / D3 monolayers 24 hours after seeding in OrganoPlate® 3 lanes at an MOI of 50'000. Supernatants in the top lane (apical) and bottom lane (basal) were harvested 3 days after transduction, and antibody concentrations in the apical and basolateral compartments were determined by binding to human full-length (FL) TDP-43 using indirect ELISA. Briefly, ELISA plate coating with 1 μg / ml human FL TDP-43 was performed in carbonate buffer at 4°C. Plates were washed with 0.05% Tween-20 / PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20 / PBS for 1 hour at 37°C, then the collected antibody-containing supernatants were added to the plates and incubated for 2 hours at 37°C, after which the plates were washed. AP-conjugated anti-mouse IgG secondary antibody (Jackson, 115-055-206) was added at 1 / 1000 dilution in 0.05% Tween-20 / PBS for 1 hour at 37°C. After the final wash, plates were incubated with pNPP solution and read at 405 nm after 1 hour using a plate reader (BioTek). MAB1 (human IgG1 isotype) was detected in the apical supernatants of all WT AV capsid-transduced hCMEC / D3 monolayers that were assessed and retained binding to TDP-43, as shown in Figure 13. MAB1 titers in the apical compartment were estimated to be approximately 40 ng / mL for AAV2, and approximately 4 ng / mL for AAV8, AAV9, and AAVrh10 serotypes. The relevant negative control (AAV2-eGFP) did not induce any signal in the TDP-43 ELISA assay.MAB1 (human IgG1 isotype) transduced by AAV2 was also detected in the basal compartment at 20-fold lower levels compared to the apical side (Figure 14), in accordance with the limited diffusion observed across the dense ECM layer of the OrganoPlate® (data not shown). The MAB1 (human IgG1 isotype) AAV2 construct produced antibody titers in a dose-dependent manner when transduced in hCMEC / D3 cells at MOIs between 5'000 and 160'000, as shown in Figure 15. The delivery of vectored antibodies was further validated using a transwell in vivo BBB model. hCMEC / D3 monolayers were transduced 24 hours after seeding in 24-well transwell inserts with the MAB1 AAV2 construct. Three days after transduction, supernatants were collected from both the apical and basolateral sides and antibody concentrations in the apical and basolateral compartments were determined by binding to human FL TDP-43 using an indirect ELISA as previously described. This preliminary assessment suggested a relatively uniform secretion of antibody to both the apical and basolateral sides, and thus a non-polarized secretion of the vectored MAB1 antibody, as shown in Figure 16. [Example 6]

[0173] Antibody secretion by mouse and human brain microvascular endothelial cell lines. The immortalized hCMEC / D3, b.End3 and b.End5 cell lines were used to evaluate whether brain endothelial cells can produce high-quality antibodies. Different AAV vectors, such as AAV2, AAV-BR1 and AAVrh10, were evaluated for their ability to deliver the MAB1 antibody transgene to human and mouse cell lines. Expression of all antibodies (such as hIgG1 and scFv-Fc) was driven by the CMV promoter; an IRES element was used between the genes encoding the LC and HC of hIgG1 to achieve bicistronic antibody production. Cells were cultured at 100'000 cells / cm in 96-well culture plates. 2The endothelial cell lines were then transduced 4 to 16 hours after seeding at an MOI of 100'000 in growth medium. The cells were incubated overnight at 37°C with 5% CO2 and the medium was changed the next day to remove the AAV particles. Seven days after transduction, cell culture supernatants were harvested and secreted antibody titers were measured by homogeneous time-resolved fluorescence (HTRF) using the hFc kit (PerkinElmer, Cisbio, 62HFCPEH) according to the manufacturer's instructions. This quantification method allowed the detection of secreted and correctly folded antibodies. Purified recombinant MAB1 hIgG1 was used as a standard for antibody quantification in the culture medium. The fluorescent signal was read using a Tecan Spark® microplate reader (Em: 317 nm; Ex: 620 nm and 665 nm; 75 flashes; 400 μs integration time; 100 μs delay time). The interpolated secreted antibody titers are shown in Figure 17. MAB1 hIgG1 delivered by AAV2 was quantified at (A) 20 ng / mL in b.End3 and b.End5 mouse endothelioma cell line supernatants, and (B) 200 ng / mL in hCMEC / D3 cell supernatants. MAB1 scFv-Fc constructs were generated and quantified at 50 / 100 ng / mL and 2500 ng / mL in the supernatants for b.End3 / b.End5 and hCMEC / D3, respectively. In all three cell lines, AAVrh10 and AAV-BR1 serotypes yielded MAB1 hIgG1 below 10 ng / mL. The antibody levels obtained indicate that brain endothelial cells produce correctly folded antibodies independent of the AAV capsid or antibody format used. Expression titers depended on the conditions evaluated, with higher titers observed in the human hCMEC / D3 cell line compared to both mouse cell lines (Figures 17A and B). Also, consistently higher antibody titers were observed for MAB1 scFv-Fc when delivered by AAV2 compared to the entire MAB1 IgG1 counterpart. In this experiment, AAV2 was more potent at delivering the transgene compared to AAV-BR1. [Example 7]

[0174] Antibody secretion by human primary brain microvascular endothelial cells in a 3D human BBB model Antibody production by BBB cells was evaluated using a commercially available Transwell-based model composed of human primary brain endothelial microvasculature cells, astrocytes and pericytes purchased from Neuromics (3D45002). The model was cultured according to the manufacturer's instructions. Briefly, 24-well plates were thawed on day 0 and the freezing medium was replaced with warm growth medium (medium 1). After 3 h of incubation in a humidified incubator, medium 1 was removed and replaced with a second maintenance medium (medium 2). No further medium changes were performed and cells were maintained in culture until day 11 post-thaw. AAV2 and AAV-BR1 vectors were evaluated to deliver human (hIgG1) and mouse (mIgG2a) versions of the MAB1 antibody transgene. Antibody expression was driven by either the CMV or CBh promoter; an IRES element was used between the LC and HC to achieve bicistronic antibody production. Endothelial cells present in cell culture inserts were transduced 4 days after thawing with an MOI of 100'000 in growth medium as previously described with AAV constructs. Supernatants of the apical and basal compartments were harvested 7 days after transduction and secreted antibody titers were measured by HTRF using human Fc and mouse Fc kits (PerkinElmer Cisbio, 62HFCPEH and 6FMIGPEH) according to the manufacturer's instructions. Purified recombinant MAB1 as hIgG1 or mIgG2a format, respectively, was used as standard for antibody quantification and titrated in the same medium used to maintain the models. Fluorescence signals were read using a Tecan Spark® microplate reader (Em: 317 nm; Ex: 620 nm and 665 nm; 75 flashes; 400 μs integration time; 100 μs delay time). Interpolated secreted antibody titers are shown in Figure 18. Similar MAB1 hIgG1 titers were obtained for AAV2 and AAV-BR1 serotypes, approximately 50 ng / mL in the apical compartment (cell culture insert) and 10-20 ng / mL in the basolateral. AAV-BR1 delivery of the MAB1 mIgG2a isotype expressed from either the CMV or CBh promoter demonstrated two- to three-fold lower titers compared to the hIgG1 counterpart.Differences in tropism were observed with AAV-BR1 in the hCMEC / D3 cell line (Figure 17A, MAB1 hIgG1 expression not detected) when compared to data from primary human brain microvascular endothelial cells (Figures 18A and B). Although in vitro cell line data predicts in vivo effects, primary cell data is a favorable predictor of AAV tropism in vivo. Figure 18B shows the relative amounts of antibody determined in the apical (200 μL) and basal (500 μL) compartments for each AAV vector. Redistribution of antibody suggests bilateral secretion of antibody from the endothelial cell layer.

[0175] In summary, the antibody titers obtained demonstrated that primary brain endothelial cells produce correctly folded antibodies, independent of the AAV capsid used and regardless of the antibody format. Importantly, antibodies were detected both apically and basolaterally, the latter observation mimicking brain parenchyma. The data generated validate the innovative method described herein and confirm that high quality IgG titers were achieved with this novel delivery strategy.

[0176] Throughout the description and claims of this specification, the terms "comprise" and "contain" and variations thereof mean "including but not limited to" and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0177] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where indefinite articles are used, the specification should be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0178] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes related to the present invention.

[0179] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. Moreover, all aspects and embodiments of the present invention described herein are considered to be broadly applicable and can be combined with any and all other consistent embodiments, including those taken from other aspects of the present invention (alone or otherwise), as appropriate.

[0180] References: [Table 3] [Table 4] [Table 5] [Table 6]

Claims

1. A vector comprising a polynucleotide encoding an antibody or antibody fragment for use in a method for treating a disease or disorder of the central nervous system (CNS) in a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB) ​​and the transduced or transfected BBB cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

2. A vector for use in a method for delivering an antibody or antibody fragment to the BBB in a subject, the method comprising administering to the subject a vector comprising a polynucleotide encoding the antibody or antibody fragment, resulting in transduction or transfection of cells of the BBB, wherein the transduced or transfected cells express the antibody or antibody fragment.

3. A vector comprising a polynucleotide encoding an antibody or antibody fragment for use in the manufacture of a medicament for treating a disease or disorder of the CNS in a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB), and the transduced or transfected cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

4. A vector comprising a polynucleotide encoding an antibody or antibody fragment for use in delivering the polynucleotide encoding the antibody or antibody fragment to the BBB of a subject, wherein the vector transduces or transfects cells of the blood-brain barrier (BBB) ​​such that the transduced or transfected cells express the antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

5. For use in a method of treating a disease or disorder of the central nervous system (CNS) in a subject, a. at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second gene encoding a heavy chain of an antibody or antibody fragment; b. a first promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second promoter operably linked 5' to 3' to a second gene encoding a heavy chain of an antibody or antibody fragment; A vector comprising an expression cassette comprising: A vector that transduces or transfects cells of the blood-brain barrier (BBB) ​​or CNS such that the transduced or transfected cells express an antibody or antibody fragment, resulting in delivery of the antibody or antibody fragment into the CNS.

6. A vector for use according to any one of claims 1 to 5, wherein the antibody or antibody fragment is delivered into the brain parenchyma and, optionally, said antibody or antibody fragment is secreted into the brain parenchyma.

7. 7. The vector for use according to claim 5 or 6, wherein the vector transduces or transfects cells of the CNS, the cells being selected from brain endothelial cells, neurons, pericytes, astrocytes, oligodendrocytes, microglia and ependymal cells.

8. A vector for use according to any one of claims 1 to 7, for transducing or transfecting endothelial cells of the BBB.

9. A vector for use according to any one of claims 1 to 8, for transducing or transfecting pericytes or astrocytes of the BBB.

10. The vector for use according to any one of claims 5 to 9, wherein the antibody or antibody fragment is secreted into the CNS, preferably into the brain parenchyma.

11. a. comprising a modification on the vector surface that targets the vector to cells of the BBB; and / or b. expressing on the surface of the vector a peptide, a small molecule, an antibody or antibody fragment thereof, a protein, a nanoparticle, a lipid, an oligonucleotide, an aptamer, or a cationic molecule that targets the vector to cells of the BBB or CNS; and / or c. comprises a neurotropic vector; and / or d. Organic nanomaterials, such as liposomes, exosomes, dendrimers and micelles, or inorganic nanomaterials, such as gold nanoparticles, silica nanoparticles and carbon nanotubes; A vector for use according to any one of claims 1 to 10.

12. a. comprising a wild-type or engineered viral vector; and / or b. is selected from an adeno-associated virus (AAV), an adenovirus, a retrovirus, a rhinovirus, a lentivirus, a herpes simplex virus (HSV) or a virus-like particle, and / or c. An AAV selected from AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9) and AAV serotype 10 (AAV10); A vector for use according to any one of claims 1 to 11.

13. The vector is an engineered AAV vector, optionally (i) the engineered AAV vector is an engineered AAV2 vector, preferably AAV-BR1; or (ii) the engineered AAV vector is an engineered AAV9 vector, e.g., AAV-S, AAV-F, AAV-PHP.eB, AAV9-PHP-V1; or (iii) the engineered AAV vector is an engineered AAV1 vector, such as AAV1RX, AAV1R6 or AAV1R7; or (iv) the engineered AAV vector is an engineered AAV10 vector; A vector for use according to any one of claims 1 to 12.

14. A CNS disease or disorder, a. diseases associated with amyloid-beta protein, TDP-43-proteinopathy, alpha-synucleinopathy, tauopathy, trinucleotide repeat disorders including polyglutamine disorders, such as Huntington's disease, brain-related cancers and tumors, epilepsy, psychiatric disorders, neuroinflammatory disorders, neuromuscular disorders, viral-induced encephalitis, and diseases characterized by microglial dysfunction, and / or b. Frontotemporal Dementia (FTD), Amyotrophic Lateral Sclerosis (ALS), Alzheimer's Disease (AD), Parkinson's Disease (PD), Chronic Traumatic Encephalopathy (CTE), Limbic-Predominant Age-Related TDP-43 Encephalopathy (LATE), and Multiple Sclerosis A vector for use according to any one of claims 1, 3 or 5 to 13, selected from:

15. Antibodies or antibody fragments include, but are not limited to, anti-ErbB2, anti-TDP-43 (NI-205), anti-Abeta (e.g., bapinezumab, solanezumab, lecanemab, azcanumab, donanemab, gantenerumab or crenezumab), anti-ApoE4 (apolipoprotein E4) and anti-DDX3X (ATP-dependent RNA helicase), anti-Tau (tirabonemab, goslanemab, zagotenemab, semolinemab, bepranemab, BIIB076, JNJ-63733657, Lu AF87908, PNT001, E-2814), anti-LINGO-1 (e.g., opicinumab), anti-alpha-synuclein (simpanemab, prasinezumab, MEDI-1341, Lu AF82422, BAN0805), anti-ASC (IC-100), anti-NLRP3, anti-C5 (ravulizumab, eculizumab), anti-C1q (ANX-005), anti-C3, anti-huntingtin (C-617, NI-302), anti-prion, anti-CD20 (e.g. ofatumumab, ocrelizumab, rituximab, BCD-132, ublituximab, BAT-4406F, AL-014), anti-PD-1 (IBC-Ab002) or anti-VEGF-A (bevacizumab, ranibizumab, brolucizumab, faricimab, vanucizumab).

16. A vector for use according to any one of claims 1 to 15, which is administered to a subject parenterally, optionally by intravenous injection or infusion.

17. 17. The vector for use according to any one of claims 5 to 16, wherein the expression cassette further comprises either an internal ribosome entry site (IRES) or a Furin-2A cleavage site after a first gene encoding a light chain of the antibody or antibody fragment and before a second gene encoding a heavy chain of the antibody or antibody fragment, optionally the IRES is derived from an encephalomyocarditis virus and comprises SEQ ID NO:1 or SEQ ID NO:8 as appropriate.

18. 18. The vector for use according to any one of claims 5 to 17, wherein a first gene encoding a light chain of the antibody or antibody fragment further comprises a secretory peptide and / or a second gene encoding a heavy chain of the antibody or antibody fragment further comprises a secretory peptide.

19. The polynucleotide comprises at least one promoter, and the at least one promoter and / or the first promoter and / or the second promoter are selected from the group consisting of a cytomegalovirus (CMV) promoter, EF1A (human eukaryotic translation elongation factor 1 alpha 1), CAG (CMV early enhancer fused to a modified chicken β-actin promoter), CBh (CMV early enhancer fused to a modified chicken β-actin promoter), SV40 (simian virus 40 enhancer / early promoter), GFAP (human glial fibrillary acidic protein promoter), ATP1A2_1 (Na,K ATKase α2), CLDN_5 (claudin 5), ADRB2_1 (adrenergic receptor beta 2), TNFRSF6B_1 (TNF receptor superfamily member 6b), PDYN_1 (prodynorphin), GH1_1 (human growth hormone), OPALIN_1 (opalin), SYN1_1 (synapsin 1), CAMK2A_1 (calcium / calmodulin-dependent protein kinase II alpha), NEFH_1 (neurofilament heavy chain polypeptide), NEUROD6_1 (neuronal differentiation factor 6) or OLIG2_1 (oligodendrocyte transcription factor 2), GFAP, ATP1A2_1, CLDN_ 5, a CMV early enhancer fused to any of the ADRB2_1, TNFRSF6B_1, PDYN_1, GH1_1, OPALIN_1, SYN1_1, CAMK2A_1, NEFH_1, NEUROD6_1 or OLIG2_1 promoters, preferably selected from a cytomegalovirus (CMV) promoter or a CBh promoter, wherein at least one promoter is operably linked to a sequence encoding an antibody or antibody fragment, optionally at least one promoter and / or said first promoter and / or second promoter is selected from ATP1A2_1 (Na,K6. The vector for use according to any one of claims 1 to 5, wherein the promoter is selected from: CLDN_5 (Claudin 5), ADRB2_1 (Adrenergic receptor beta 2) and TNFRSF6B_1 (TNF receptor superfamily member 6b), and optionally wherein at least one promoter and / or the first promoter and / or the second promoter is operably linked to an enhancer, such as a CMV early enhancer.

20. 20. The vector for use according to any one of claims 5 to 19, wherein the expression cassette comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity thereto.

21. 1. A method for reducing antibody or antibody fragment aggregation and improving antibody or antibody fragment maturation and / or quality, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or an expression cassette comprising 5' to 3' to a first promoter operably linked to a first gene encoding the light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; The method includes:

22. 1. A method for increasing the titer of an antibody or antibody fragment, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising either an internal ribosome entry site (IRES) or a self (e.g., furin-2A) cleavage site after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or transforming a cell with an expression cassette comprising 5' to 3' to a first promoter operably linked to a first gene encoding the light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; The method includes:

23. 1. A method for reducing undesired antibody or antibody fragment immunogenicity and / or adverse effects associated with antibody or antibody fragment therapy, the method comprising: (i) transforming a cell with an expression cassette comprising at least one promoter operably linked 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment, and a second gene encoding a heavy chain of the antibody or antibody fragment, the expression cassette further comprising an internal ribosome entry site (IRES) after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or an expression cassette comprising 5' to 3' to a first promoter operably linked to a first gene encoding the light chain of the antibody or antibody fragment, and a second promoter operably linked to a second gene encoding the heavy chain of the antibody or antibody fragment; and (ii) maintaining the transformed cell under conditions suitable for the production of the antibody or antibody fragment; The method includes:

24. The method of any one of claims 21 to 23, wherein the antibody or antibody fragment does not contain a self-cleaving element.

25. The method according to any one of claims 21 to 24, wherein the expression cassette is comprised in a vector, the method being further defined according to the features according to any one of claims 6 to 20.

26. A viral vector comprising an engineered AAV2 vector, preferably AAV-BR1, or an engineered AAV9 vector, such as AAV-S, AAV-AF, AAV-PHP.eB or AAV9-PHP-V1, or an engineered AAV1 vector, such as AAV1RX, AAV1R6, AAV1R7, or an engineered AAV10 vector, wherein the engineered viral vector is a. comprising an expression cassette comprising at least one promoter operably linked from 5' to 3' to a first gene encoding a light chain of an antibody or antibody fragment and a second gene encoding a heavy chain of the antibody or antibody fragment, and further comprising an IRES after the first gene encoding the light chain of the antibody or antibody fragment and before the second gene encoding the heavy chain of the antibody or antibody fragment; or b. comprising an expression cassette comprising, from 5' to 3', a first promoter operably linked to a first gene encoding a light chain of an antibody or antibody fragment, and a second promoter operably linked to a second gene encoding a heavy chain of an antibody or antibody fragment; Optionally, the engineered viral vector is AAV-BR1 or AAV9-PHP-V1. Viral vector.

27. The viral vector according to claim 26, further defined in any one of claims 6 to 20.

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