Antibodies targeting trans-active response DNA-binding protein-43 (TDP-43)

Abstract

Inventors followed an untargeted approach by screening a single chain variable fragment (scFv) library via phage display against recombinant human full-length wild-type (wt)TDP-43. They identified four wtTDP-43-specific scFv, two of which were retained following cellular expression and colocalization with TDP-43 in vitro. In silico binding site prediction on TDP-43 suggested the pathologically-relevant C-terminal and RRM1 domains as potential targets. One scFv diminished the amount of the insoluble 35-kDa C-terminal fragment of TDP-43 when the wildtype protein was overexpressed. Another scFv inhibited NF-κB activation associated with TDP-43 overexpression. Both scFv seemed to reverse some metabolic alterations caused by TDP-43 overexpression. Their findings offer two scFv molecules that bind wtTDP-43, alter its aggregation, and modify cellular pathways associated with TDP-43 proteinopathies. Accordingly, the present invention relates to scFv intrabodies targeting TDP-43 and their uses in diagnosis and treatment methods.

Description

[0001] ANTIBODIES TARGETING TRANS-ACTIVE RESPONSE DNA-BINDING

[0002] PROTEIN-43 (TDP-43)

[0003] FIELD OF THE INVENTION:

[0004] The invention is in the field of neurology. More particularly, the invention relates to scFv intrabodies targeting TDP-43 and their uses in diagnosis and treatment methods.

[0005] BACKGROUND OF THE INVENTION:

[0006] Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease caused by the rapid deterioration of the motor neurons of patients, leading to death 2-5 years on average following symptom onset.1Despite the availability of riluzole, edaravone, and recently AMX0035 as FDA-approved pharmaceutical treatments, their beneficial effects are fairly moderate.2,3,4The lack of effective treatment is in part a reflection of the difficulty researchers have met in comprehending the neurodegenerative mechanisms behind ALS. Developments in ALS research, however, suggest that Trans-Active Response DNA Binding Protein-43 (TDP-43) is a key player in the pathogenesis, as it has been shown to play roles in many of the pathological pathways driving cell death.5

[0007] TDP-43 is an RNA-binding protein implicated in the regulation of the metabolism, modification, and transport of approximately 6,000 mRNA targets, and is an essential protein for cell homeostasis.6,7Under physiological conditions, TDP-43 is mostly localized in the nucleus. In the majority of post-mortem neural tissue of ALS patients, TDP-43 in its wildtype form is found mislocalized to and aggregated in the cytoplasm. Indeed, TDP-43 aggregation is considered the hallmark of ALS.8However, it has been also observed in other neurodegenerative diseases including frontotemporal lobar dementia (FTLD) (10.1126 / science.l 134108) Alzheimer’s disease (10.1007 / s00401-008-0480-l) Parkinson’s disease (10.1159 / 000273591) Huntington disease (10.1097 / NEN.0b013e31818e8951) and limbic-predominant age-related TDP-43 Encephalopathy (LATE) (10.1093 / jnen / nlzl26). These diseases involving the pathologic features of TDP-43 have been termed TDP-43 proteinopathies.

[0008] TDP-43 aggregates mainly include post-translationally modified full-length and 35- kDa and 25-kDa C-terminal fragments (CTF) of TDP-43.9’10The C-terminal fragments are a result of proteolytic cleavage or alternative splicing, and have been widely implicated in toxic gain- or loss-of function mechanisms.11The regions of TDP-43 involved in its propensity to aggregate are its two RNA recognition motif (RRM) domains and C-terminus, especially the prion-like domain within this region.12It was shown that the dysregulation of TDP-43 in ALS triggers the activation of NF-KB, which plays a key role in inducing inflammatory responses.13In addition, various TDP -43 -induced metabolic alterations have been reported, particularly in pathways involving energy metabolism and neurotransmission.14’15Moreover, there seems to be a bidirectional interplay between the function of the degradation pathways of the cell, particularly the ubiquitin-proteasome system and TDP -43 proteinopathy, suggesting a key role of these pathways in ALS (PMID: 36158183; PMID: 35101542).

[0009] Previous studies have employed intrabodies or single-chain variable fragments (scFv) to target pathological aspects of TDP -43 in ALS models. In one study, an intrabody engineered against the RRM2 domain of TDP -43 to specifically target cytoplasmic, aggregated TDP -43 decreased cytoplasmic aggregates of TDP -43 and improved cell viability in cellular models of ALS.18Similarly, a scFv derived from a monoclonal antibody raised against the RRM1 region of TDP -43 reduced TDP -43 -mediated activation of NF-KB and lowered TDP -43 aggregation in cell models of ALS, and led to a decrease in motor decline in a mouse model of ALS.19

[0010] SUMMARY OF THE INVENTION:

[0011] The invention relates to intrabodies targeting wildtype Trans-active response DNA- binding protein-43 (TDP -43). In particular, the invention is defined by claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Inventors followed an untargeted approach by screening a single chain variable fragment (scFv) library via phage display against recombinant human full-length wild-type (wt)TDP-43. They identified four wtTDP -43 -specific scFv, two of which were retained following cellular expression and colocalization with TDP -43 in vitro. In silico binding site prediction on TDP -43 suggested the pathologically-relevant C-terminal and RRM1 domains as potential targets. One scFv diminished the amount of the insoluble 35-kDa C-terminal fragment of TDP -43 when the wildtype protein was overexpressed. Another scFv inhibited NF-KB activation associated with TDP -43 overexpression. Both scFv seemed to reverse some metabolic alterations caused by TDP -43 overexpression. Their findings offer two scFv molecules that bind wtTDP -43, alter its aggregation, and modify cellular pathways associated with TDP -43 proteinopathies.

[0014] Intrabodies targeting TDP-43

[0015] Accordingly, in a first aspect, the invention relates to antibodies targeting wildtype Trans-active response DNA-binding protein-43 (TDP-43).

[0016] As used herein, the term “TDP-43” refers to Trans-Active Response DNA Binding Protein-43, is an RNA-binding protein implicated in the regulation of the metabolism, modification, and transport of approximately 6,000 mRNA targets, and is an essential protein for cell homeostasis.6,7Under physiological conditions, TDP-43 is mostly localized in the nucleus. In the majority of post-mortem neural tissue of ALS patients, TDP-43 in its wildtype form is found mislocalized to and aggregated in the cytoplasm. The naturally occurring human TDP-43 gene has a nucleotide sequence as shown in Genbank Accession number NM 007375 and the naturally occurring human TDP-43 protein has an aminoacid sequence as shown in Genbank Accession number NP_031401 and NP_031401.1. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_001003898, NM_001003899, NM_001008545, NM_001008546, NMJ45556 and NP_001003898, NP_001003899, NP_001008545, NP_001008546, NP_001292354).

[0017] The sequence of said protein can be found under the Uniprot accession number Q13148. An exemplary amino acid sequence is represented by SEQ ID NO: 33:

[0018] SEQ ID NO: 33:

[0019] MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGACGLRYRNPVSQCMRGVRLVE GILHAPDAGWGNLVYVVNYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGLPW KTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGFGFVRFTEYETQVKVMSQRHMID GRWCDCKLPNSKQSQDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDVFIPKPF RAFAFVTFADDQIAQSLCGEDLIIKGISVHISNAEPKHNSNRQLERSGRFGGNPGGFGN QGGFGNSRGGGAGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQSSWGMMG MLASQQNQSGPSGNNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGS GSGFNGGFGSSMDSKSSGWGM.

[0020] In a particular embodiment, the invention relates to intrabodies scFv Bl, scFv D7, scFv A2 and scFv B6 targeting wildtype Trans-active response DNA-binding protein-43 (TDP-43) wherein: scFv Bl has:

[0021] (a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2; a H-CDR2 having a sequence set forth as SEQ ID NO: 3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;

[0022] (b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8; scFv D7 has: (a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 10; a H-CDR2 having a sequence set forth as SEQ ID NO: 11; a H-CDR3 having a sequence set forth as SEQ ID NO: 12;

[0023] (b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 14; a L-CDR2 having a sequence set forth as SEQ ID NO: 15; a L-CDR3 having a sequence set forth as SEQ ID NO: 16; scFv A2 has:

[0024] (a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 18; a H-CDR2 having a sequence set forth as SEQ ID NO: 19; a H-CDR3 having a sequence set forth as SEQ ID NO: 20;

[0025] (b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 22; a L-CDR2 having a sequence set forth as SEQ ID NO: 23; a L-CDR3 having a sequence set forth as SEQ ID NO: 24; scFV B6 has:

[0026] (a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 26; a H-CDR2 having a sequence set forth as SEQ ID NO: 27; a H-CDR3 having a sequence set forth as SEQ ID NO: 28;

[0027] (b) a light chain wherein the variable domain comprises: a L-CDR1 having a sequence set forth as SEQ ID NO: 30; a L-CDR2 having a sequence set forth as SEQ ID NO: 31; a L-CDR3 having a sequence set forth as SEQ ID NO: 32.

[0028] The inventors selected these antibodies for their properties to reverse some metabolic alterations caused by TDP-43 overexpression. Their findings offer two scFv molecules that bind wtTDP-43, alter its aggregation, and modify cellular pathways associated with TDP-43 proteinopathies.

[0029] As used herein the term "antibody" or "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. In one embodiment, the antibodies of the invention are monoclonal antibodies.

[0030] In the context of the invention, the amino acid residues of the antibodies of the invention are numbered according to the IMGT numbering system. The IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species (Lefranc M.-P., "Unique database numbering system for immunogenetic analysis" Immunology Today, 18, 509 (1997) ; Lefranc M.-P., "The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains" The Immunologist, 7, 132-136 (1999).; Lefranc, M.-P., Pommie, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, G., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains" Dev. Comp. Immunol., 27, 55-77 (2003).). In the IMGT unique numbering, the conserved amino acids always have the same position, for instance cysteine 23, tryptophan 41, hydrophobic amino acid 89, cysteine 104, phenylalanine or tryptophan 118. The IMGT unique numbering provides a standardized delimitation of the framework regions (FR1- IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. If the CDR3-IMGT length is less than 13 amino acids, gaps are created from the top of the loop, in the following order 111, 112, 110, 113, 109, 114, etc. If the CDR3-IMGT length is more than 13 amino acids, additional positions are created between positions 111 and 112 at the top of the CDR3-IMGT loop in the following order 112.1,111.1, 112.2, 111.2, 112.3, 111.3, etc.

[0031] (http: / / www.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefmition.html)

[0032] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind an epitope presented on an antigen, such as TDP-43. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 10: 1, about 20: 1, about 50: 1, about 100: 1, 10.000: 1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules (in this case the specific antigen is TDP-43). The term “affinity”, as used herein, means the strength of the binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of mAbs is the use of Biacore instruments. The present invention provides for anti-TDP-43 antibodies, particularly in a purified form or in an isolated form.

[0033] In some embodiments, the antibodies of the present invention are antibodies having a heavy chain comprising i) H-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) the H- CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 iii) the H-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6.

[0034] In some embodiments, the antibodies of the present invention are antibodies having a light chain comprising i) L-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) L-CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 and iii) L-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6.

[0035] In some embodiments, the antibodies of the present invention are antibodies having a heavy chain comprising i) H-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) the H- CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 iii) the H-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6 and a light chain comprising i) L-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) L-CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 and iii) L-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6.

[0036] More particularly, the antibodies of the present invention are antibodies having a heavy chain having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0037] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25.

[0038] In some embodiments, the antibodies of the present invention are antibodies having a light chain having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

[0039] 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

[0040] In some embodiments, the antibodies of the present invention are antibodies having a heavy chain having at least 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25 and a light chain having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

[0041] In some embodiments, the antibodies of the present invention are antibodies having a heavy chain which is identical to SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25. In some embodiments, the antibodies of the present invention are antibodies having a light chain identical to SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

[0042] In some embodiments, the antibodies of the present invention are antibodies having a heavy chain identical to SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25 and a light chain identical to SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

[0043] In another embodiment, the antibodies of the present invention are antibodies comprising: a heavy chain comprising i) a H-CDR1 having at least 90% of identity with SEQ ID NO: 2, SEQ ID NO: 10, SEQ ID NO: 18 or SEQ ID NO: 26, ii) a H-CDR2 having at least 90% of identity with SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 19 or SEQ ID NO: 27 and iii) a H-CDR3 having at least 90% of identity with SEQ ID NO: 4, SEQ ID NO: 12, SEQ ID NO: 20 or SEQ ID NO: 28 and a light chain comprising i) a L-CDR1 having at least 90% of identity with SEQ ID NO: 6, SEQ ID NO: 14, SEQ ID NO: 22 or SEQ ID NO: 30, ii) a L-CDR2 having at least 90% of identity with SEQ ID NO: 7, SEQ ID NO: 15, SEQ ID NO: 23 or SEQ ID NO: 31 and iii) a L-CDR3 having at least 90% of identity with SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 24 or SEQ ID NO: 32.

[0044] Table 1 : Sequences of scFv Bl, D7, A2 and B6

[0045] The terms "monoclonal antibody", "monoclonal Ab", "monoclonal antibody composition", "mAb", or the like, as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0046] As used herein, the term “epitope” refers to a specific arrangement of amino acids located on a protein or proteins to which an antibody binds. Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes can be linear or conformational, i.e., involving two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous.

[0047] The antibodies of the present invention are produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Typically, knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, antibodies of the present invention can be synthesized by recombinant DNA techniques well-known in the art. For example, antibodies can be obtained as DNA expression products after incorporation of DNA sequences encoding the antibodies into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired antibodies, from which they can be later isolated using well-known techniques.

[0048] In one embodiment, the monoclonal antibodies of the invention is chimeric antibodies, particularly chimeric mouse / human antibodies.

[0049] According to the invention, the term "chimeric antibody" refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.

[0050] In some embodiments, the human chimeric antibodies of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. As the CH domain of a human chimeric antibody, it may be any region which belongs to human immunoglobulin, but those of IgG class are suitable and any one of subclasses belonging to IgG class, such as IgGl, IgG2, IgG3 and IgG4, can also be used. Also, as the CL of a human chimeric antibody, it may be any region which belongs to Ig, and those of kappa class or lambda class can be used. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art (See Morrison SL. et al. (1984) and patent documents US5,202,238; and US5,204, 244). In another embodiment, the monoclonal antibodies of the invention is humanized antibodies. In particular, in said humanized antibodies, the variable domain comprises human acceptor frameworks regions, and optionally human constant domain where present, and nonhuman donor CDRs, such as mouse CDRs.

[0051] In one embodiment, the humanized antibodies can be derived from chimeric antibodies (obtained from the antibodies of the invention).

[0052] In another embodiment, the monoclonal antibodies of the invention is a caninized or primatized based on the same methods of humanization.

[0053] According to the invention, the term "humanized antibodies" refers to antibodies having variable region framework and constant regions from human antibodies but retains the CDRs of previous non-human antibodies.

[0054] The humanized antibodies of the present invention may be produced by obtaining nucleic acid sequences encoding CDR domains, as previously described, constructing humanized antibodies expression vector by inserting them into an expression vector for animal cell having genes encoding (i) a heavy chain constant region identical to that of a human antibody and (ii) a light chain constant region identical to that of a human antibody, and expressing the genes by introducing the expression vector into an animal cell. The humanized antibody expression vector may be either of a type in which a gene encoding an antibody heavy chain and a gene encoding an antibody light chain exists on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a humanized antibody expression vector, easiness of introduction into animal cells, and balance between the expression levels of antibody H and L chains in animal cells, humanized antibody expression vector of the tandem type is preferred. Examples of tandem type humanized antibody expression vector include pKANTEX93 (WO 97 / 10354), pEE18 and the like. Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (See, e. g., Riechmann L. et al. 1988; Neuberger MS. et al. 1985). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska MA. et al. (1994)), and chain shuffling (U.S. Pat. No.5, 565, 332). The general recombinant DNA technology for preparation of such antibodies is also known (see European Patent Application EP 125023 and International Patent Application WO 96 / 02576). In one embodiment, the antibodies of the invention are antigen biding fragment selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, an intrabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementary determining region (CDR), and fragments which comprise or consist of the VH or VL chains as well as amino acid sequence having at least 70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99 or 100% of identity with SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO: 25 or SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 29.

[0055] In the context of the invention, the antibodies are intrabodies.

[0056] As used herein, the term “intrabody” or “intrabodies” refers to an antibody that is expressed or can be expressed in a cell and that binds to an intracellular protein, for example an intrabody is an antibody that has been modified or adapted for intracellular localization and intracellular function. An intrabody comprises a heavy chain variable domain and a light chain variable domain and linker optionally in either variable domain orientation, e.g. heavy chain variable domain- linker - light chain variable domain or light chain variable domainlinker - heavy chain variable domain. Depending on the context, the term intrabody may refer to a nucleic acid molecule or a polypeptide molecule.

[0057] The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically binds to a given antigen (e.g., [TDP-43). Antigen biding functions of an antibody can be performed by fragments of an intact antibody. Examples of biding fragments encompassed within the term antigen biding fragment of an antibody include a Fab fragment, a monovalent fragment consisting of the VL,VH,CL and CHI domains; a Fab’ fragment, a monovalent fragment consisting of the VL,VH,CL,CH1 domains and hinge region; a F(ab’)2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; an Fd fragment consisting of VH domains of a single arm of an antibody; a single domain antibody (sdAb) fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain or a VL domain; and an isolated complementary determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by an artificial peptide linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (ScFv); see, e.g., Bird et al., 1989 Science 242:423-426; and Huston et al., 1988 proc. Natl. Acad. Sci. 85:5879-5883). "dsFv" is a VH::VL heterodimer stabilised by a disulfide bond. Divalent and multivalent antibody fragments can form either spontaneously by association of monovalent scFvs, or can be generated by coupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)2. Such single chain antibodies include one or more antigen biding portions or fragments of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies. A unibody is another type of antibody fragment lacking the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent biding region of IgG4 antibodies. Antigen binding fragments can be incorporated into single domain antibodies, SMIP, maxibodies, minibodies, intrabodies, diabodies, triabodies and tetrabodies (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126-1136). The term "diabodies" “tribodies” or “tetrabodies” refers to small antibody fragments with multivalent antigen-binding sites (2, 3 or four), which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Antigen biding fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH- CH1-VH-CH1) Which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al., 1995 Protein Eng. 8(10); 1057-1062 and U.S. Pat. No. 5,641,870).

[0058] The Fab of the present invention can be obtained by treating antibodies which specifically reacts with TDP-43 with a protease, papaine. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fab.

[0059] The F(ab')2 of the present invention can be obtained treating antibodies which specifically reacts with TDP-43with a protease, pepsin. Also, the F(ab')2 can be produced by binding Fab' described below via a thioether bond or a disulfide bond.

[0060] The Fab' of the present invention can be obtained treating F(ab')2 which specifically reacts with TDP-43 with a reducing agent, dithiothreitol. Also, the Fab' can be produced by inserting DNA encoding Fab' fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

[0061] The scFv of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv. To generate a humanized scFv fragment, a well known technology called CDR grafting may be used, which involves selecting the complementary determining regions (CDRs) from a donor scFv fragment, and grafting them onto a human scFv fragment framework of known three dimensional structure (see, e. g., W098 / 45322; WO 87 / 02671; US5,859,205; US5,585,089; US4,816,567; EP0173494).

[0062] Domain Antibodies (dAbs) are the smallest functional binding units of antibodies - molecular weight approximately 13 kDa - and correspond to the variable regions of either the heavy (VH) or light (VL) chains of antibodies. Further details on domain antibodies and methods of their production are found in US 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245; US 2004 / 0110941; EP 1433846, 0368684 and 0616640; WO 2005 / 035572, 2004 / 101790, 2004 / 081026, 2004 / 058821, 2004 / 003019 and 2003 / 002609, each of which is herein incorporated by reference in its entirety.

[0063] UniBodies are another antibody fragment technology, based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of a traditional IgG4 antibody and has a univalent binding region rather than a bivalent binding region. Furthermore, because UniBodies are about smaller, they may show better distribution over larger solid tumors with potentially advantageous efficacy. Further details on UniBodies may be obtained by reference to WO 2007 / 059782, which is incorporated by reference in its entirety.

[0064] In a particular embodiment, the antibodies according to the invention are bispecific antibody.

[0065] As used herein, the term “bispecific antibody” should be understood as antibodies that recognize two different epitopes either on the same or on different antigens.

[0066] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al, EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multi- specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan, M. et al, Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S.A. et al, J. Immunol. 148 (1992) 1547- 1553; using "diabody" technology for making bispecific antibody fragments (see, e.g., HoUiger, P. et al, Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g. Gruber, M et al, J. Immunol. 152 (1994) 5368-5374); and preparing trispecific antibodies as described, e.g., in Tutt, A. et al, J. Immunol. 147 (1991) 60-69).

[0067] Brinkmann and al., in “The making of bispecific antibodies”, (MABS; 2017, VOL.9, NO.2, 182-212) focuses on the various formats and strategies available to generate recombinant bispecific antibodies. Recombinant bispecific antibodies can be classified according to format and composition. A main discrimination is the presence or absence of an Fc region. Bispecific antibodies with no Fc will lack Fc-mediated effector functions and could be classified as “Fc-less bispecific antibody formats”.

[0068] Bispecific antibodies that include an Fc region can be further divided into those that exhibit a structure resembling that of an IgG molecule and those that contain additional binding sites, i.e., those with an appended or modified Ig-like structure. The different bispecific antibodies will have either a symmetric or an asymmetric architecture.

[0069] For example, the majority of bispecific IgG molecules are asymmetric, while IgG fusion proteins often are symmetric in their molecular composition.

[0070] Hence, Brinkamann and al. described in the previous cited article (“the making of bispecific antibodies”) various formats of bispecific antibodies classified in three categories: i) Fc-less bispecific antibody formats; ii) Bispecific IgGs with asymmetric architecture; iii) Bispecific antibodies with a symmetric architecture.

[0071] Format and strategies to generate recombinant bispecific antibodies for each of these three categories are detailed in Brinkmann and al., which is incorporated herein by reference.

[0072] Fc-less bispecific antibody formats

[0073] Fc-less bispecific antibody formats comprise:

[0074] - Tandem single-chain variable fragments (scFv2, taFv) and triplebodies;

[0075] - Bispecific single-domain antibodyfusion proteins;

[0076] - Diabodies and diabody derivatives;

[0077] - Fab fusion protein

[0078] - Other Fc-less fusion proteins; - Additional antigen-binding sites grafted onto scFv.

[0079] Bispecific IgGs with asymmetric architecture

[0080] Bispecific IgGs with asymmetric architecture comprise:

[0081] - Asymmetric IgGs with heavy chain and light chains from two different antibodies;

[0082] - Bispecific IgGs with an asymmetric Fc region ;

[0083] - Asymmetric Fc and CH3 fusion proteins.

[0084] Post-assembly approaches to purify bispecific antibodies, genetic engineering for solving the light chain problem in asymmetric antibodies and post-production assembly from half-antibodies for solving the light chain problem are also described and incorporated by reference.

[0085] Bispecific antibodies with a symmetric architecture

[0086] Bispecific antibodies with a symmetric architecture comprise:

[0087] - Appended IgGs: fusion of scFv;

[0088] - Appended IgGs: fusion of domain antibodies and scaffold proteins;

[0089] - Appended IgGs: fusion of Fab arms;

[0090] - Appended IgGs: fusion of additional variable heavy and light chain domains;

[0091] - Modified IgG molecules;

[0092] - Symmetric Fc- and CH3-based bispecific antibodies;

[0093] - Bispecific antibodies using immunoglobulin-derived homodimerization domains.

[0094] Hence, the one skilled in the art knows the best format for generating bispecific antibodies and knows how to generate by biochemical or genetic means bispecific antibodies.

[0095] Typically, the one skilled in the art can selected bispecific antibodies among:

[0096] - Bispecific antibody conjugates such as IgG2, F(ab’)2, CovX-Body;

[0097] - Hybrid bispecific IgGs such as IgG, mouse / rat chimeric IgG, K / k-body common HC;

[0098] - “variable domain only” bispecific antibody molecules such as tandem scFv (taFv), triplebodies, diabody (Db), DsDb, Db(kih), DART, scDb, dsFv-dsFv’, tandAbs, tripleheads, tandem dAb / VHH, triple dAb / VHH, tetravalent dAb / VHH;

[0099] - Chl / CL fusion proteins such as scFv2-CHl / CL, VHH2-CH1 / CL;

[0100] - Fab fusion proteins such as Fab-scFv (bibody), Fab-scFv2 (tribody), Fab-Fv, Fab- dsFv, Fab-VHH, orthogonal Fab-Fab;

[0101] Non-immunoglobulin fusion proteins such as scFv2-albumin, scDb-albumin, taFv-albumin, taFv-toxin, miniantibody, DNL-Fab3, DNL-Fab2-scFv, DNL-Fab2-IgG- cytokine2, ImmTAC(TCR-scFv); - Fc-modified IgGs such as IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab (IgG-kih), scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1 / CL charge pairs, hinge / ch3 charge pairs, duobody, four- in-one-CrossMab (kih), LUZ-Y common LC, LUZ-Y scFab-IgG, LUZ-Y scFab-IgG, FcFc;

[0102] - Appended & Fc-modified IgGs such as IgG(kih)-Fv, IgG(HA-TF-Fv), IgG(kih)- scFab, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, half DVD-Ig, DV Ig (four-in-one), CrossMab-Fab,

[0103] - Modified Fc and CH3 fusion proteins such as scFv-Fc(kih), scFv-Fc(CH3 charge pairs), ScFv-Fc(EW-RVT), scFv-Fc(HA-TF), scFv-Fc(SEEDbody), taFv-Fc(kih), scFv- Fc(kih)-Fv, Fab-Fc(kih)-scFv, Fab-scFv-Fc(kih), Fab-scFv-Fc(BEAT), Fab-scFv- Fc(SEEDbody), DART-Fc, scFv-C3(kih), TriFabs;

[0104] - Appended IgGs - HC fusions such as IgG-HC-scFv, IgG-dAb, IgG-taFv, IgG- CrossFab, IgG-orthogonal Fab, IgG-(CaCP)Fab, scFv-HC-IgG, tandem Fab-IgG(orthogonal Fab);

[0105] - Fab-IgG(CaCpFab), Fab-IgG(CR3), Fab-hinge-IgG(CR3);

[0106] - Appended IgGs - LC fusions such as IgG-scFv(LC), scFv(LC)-IgG, dAb-IgG;

[0107] - Appended IgGs - HC&LC fusions such as DVD-Ig, TVD-Ig, CODV-Ig, scFv4-IgG, zybody;

[0108] - Fc fusions such as Di-diabody, scDb-Fc, taFv-Fc, scFv-Fc-scFv, HCAb-VHH, Fab- scFv-Fc, scFv4-Ig, scFv2-Fcab;

[0109] - CH3 fusions such as di-diabody, scDb-CH3;

[0110] - IgE / IgM CH2 fusions such as scFv-EHD2-scFv, scFv-MHD2-scFv;

[0111] - F(ab’)2 fusions such as F(ab’)2-scFv2;

[0112] - CH1 / CL fusion proteins such as scFv2-CHl-hinge / CL;

[0113] - Modified IgGs such as DAF(two -in-one-IgG), DutaMab, mAb2;

[0114] - Non-immunoglobulin fusions such as DNL-Fab4-IgG.

[0115] In one embodiment, Fc-modified IgGs such as IgG(kih), IgG(kih) common LC, ZW1 IgG common LC, Biclonics common LC, CrossMab (IgG-kih), scFab-IgG(kih), orthogonal Fab IgG(kih), DuetMab, CH3 charge pairs + CH1 / CL charge pairs, hinge / ch3 charge pairs, duobody, four-in-one-CrossMab (kih), LUZ-Y common LC, LUZ-Y scFab-IgG, LUZ-Y scFab-IgG, FcFc will be used.

[0116] In one embodiment, the bispecific antibody is a tetraval ent IgGl-like chimeric bispecific antibody, a Fab-scFv, a Fab-scFv2, a scFv-Fc or a knob into hole antibody bispecific antibody, and preferably a knob into hole antibody or scFv bispecific antibody. Tetravalent IgGl-like chimeric bispecific antibody format is described in Golay et al, (Journal of immunology; 2016). and scFv-Fab ou (scFv)2 -Fab formats are described in Panke C et al. (Protein Engineering, Design & Selection vol. 26 no. 10 pp. 645-654, 2013).

[0117] In a particular embodiment, the bispecific antibody according to the invention, wherein the antibody recognizes a second different epitope which is transferrin receptor 1 (TfRl).

[0118] As used herein, the term “Transferrin” (Tf) refers to a serum protein of 80 kDa, the role of which is to fix soluble iron. Iron charged transferin (holo-Tf) is endocyted in cells due to its binding with the transferrin receptor 1 (TfRl) at physiological extracellular pH. Acidification of the endosome causes a conformational change which salts out the iron in the cytosol. Upon its natural ligand holo-Tf binding, TfRl is rapidly internalized and recycled after holo-Tf has released iron in the endosomes. Because apo-Tf (apo-Tf corresponds to Tf not saturated by iron) is still tightly bound to TfRl under acidic conditions of the endosome, the apo-Tf / TfR complex is then re-exported to the membrane, where the return to a physiological pH causes a dissociation of the apo-Tf / TfR complex.

[0119] Transferrin receptors (TfRs) act as the most important receptor mediated controls. TfRl and TfR2 are two subtypes of TfRs that bind with iron-transferrin complex to facilitate iron into cells. TfRl is ubiquitously expressed on the surfaces of generic cells, whereas TfR2 is specially expressed in liver cells.

[0120] Nucleic acid sequence, vectors and host cells:

[0121] Accordingly, a further object of the invention relates to a nucleic acid molecule encoding antibodies according to the invention. More particularly the nucleic acid molecule encodes a heavy chain and / or a light chain of an antibody of the present invention.

[0122] Typically, said nucleic acid is a DNA or RNA molecule, which may be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or a viral vector.As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. So, a further aspect of the invention relates to a vector comprising a nucleic acid of the invention. Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said antibody upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987k LTR orometer and enhancer of Molonev mouse leukemia virus tKuwana Y et al. 1987), promoter (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983) of immunoglobulin H chain and the like. Any expression vector for animal cell can be used, so long as a gene encoding the human antibody C region can be inserted and expressed. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSGl beta d2- 4-(Miyaji H et al. 1990) and the like. Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Other examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors ((PMID: 36158183; PMID: 35101542). Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0123] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0124] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.

[0125] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of ML Vs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0126] In another embodiment, the use of spion is contemplated for the introduction of antibodies into host cells.

[0127] As used herein, the term “spion” refers to superparamagnetic iron oxide nanoparticle.

[0128] SPIONs are composed of magnetite or iron oxide which is degradable in the body and non- toxic compared to other magnetic materials such as cobalt and nickel. The main forms of magnetite are Fe3O4 and its oxidized form maghemite or y-Fe2O3.

[0129] SPIONs may be produced by methods known in the art, for example, as described by Sun et al., J, American Chemical Society, 2002, 124, 8204. SPIONs may comprise one or more coatings or may be incorporated into micelles or liposomes to enhance desirably pharmacokinetic properties including biological half-life, biocompatibility, and targeting. The compositions of the invention contain SPIONs of a size compatible with in vivo administration and desired targeting functionality. Some representative SPION particle sizes range from about 1, 2, 5, 10, 20, 30, 40, 50, or 60 nm. A composition of the invention may contain a single size or single size distribution of SPIONs or may contain two or more sizes or size distributions. For example, various mixtures of large SPIONs ranging from about 10 to 60 nm in average size and small SPIONs ranging in size from about 2 to 22 nm may be used as described in Table 1-2. Mixtures of SPIONs of different sizes permit tuning of a biological responses or imaging functions. In some embodiments, a coprecipitation technique can be followed to form metal oxide composite with Ni or Cu or Mn and. Co nanoparticle to form respective MFe2O4 to enhance imaging capacity by increasing magnetization property.

[0130] In some embodiments the core of the SPIONs may be magnetite which is covered with one or more shells, for example, a polymer shell or a gold or metal shell. SPIONs may also be incorporated into, or coated with, one or more polymers including smart, pH-sensitive, or temperature-sensitive polymers.

[0131] Functionalized super paramagnetic iron oxide nanoparticles (SPIONs) may be used in accordance with one or more embodiments of the invention, for example, a SPION (or other components of a composition of the invention) may be functionalized with one or more curcuminoids, or with a combination of one or more curcuminoids and a targeting ligand such as an antibody that binds to a tumor-associated antigen. In some embodiments SPIONs may be conjugated to targeting moieties such as ligands that bind to, or agents that are internalized by, neural cells or by other target molecules, receptors, cells or tissues.

[0132] In another embodiment, the intrabodies according to the invention are delivered alone or in association with a viral vector.

[0133] In a particular embodiment, the method according to the invention, wherein the vector comprises a nucleic acid molecule as described above.

[0134] In a particular embodiment the nucleic acid molecule comprised in a vector encodes for an acid nucleic (such as siRNA, shRNA, miRNA, antisense oligonucleotide, ribozyme, or an endonuclease) specific to TDP-43 In another embodiment the nucleic acid molecule comprised in a vector encodes for an intrabody against TDP-43.

[0135] In a particular embodiment, the method according to the invention wherein the nucleic acid molecule is operatively linked to a promoter sequence (such as myocilin or det promoters).

[0136] In a particular embodiment, the method according to the invention, wherein the vector is a viral vector.

[0137] In a particular embodiment, the method according to the invention, wherein the viral vector is lentivirus (LV).

[0138] As used herein, the term “lentivirus” refers to enveloped RNA particles measuring approximately 120 nm in size are efficient drug delivery tools and more particularly gene delivery tools. The LV binds to, and enters into target cells through its envelope proteins which confer its pseudotype. Once the LV has entered into the cells, it releases its capsid components and undergoes reverse transcription of the lentiviral RNA before integrating the proviral DNA into the genome of target cells. Non-integrative lentiviral vectors have been generated by modifying the properties of the vector integration machinery and can be used for transient gene expression. Virus-like particles lacking a provirus have also been generated and can be used to deliver proteins or messenger RNA. LV can be used for example, for gene addition, RNA interference, exon skipping or gene editing. All of these approaches can be facilitated by tissue or cell targeting of the LV via its pseudotype.

[0139] Lentivirus-like particles are described for example in (Aoki et al., 2011; Kaczmarczyk et al., 2011; McBumey et al., 2006; Muratori et al., 2010). Examples of lentivirus-like particles are VLPs generated by co-expressing in producer cells, a syncytin protein with a gag fusion protein (Gag fused with the gene of interest). The drug and / or syncytin may be, either displayed on the surface of the particles, or enclosed (packaged) into the particles. The syncytin protein is advantageously displayed on the surface of the particles, such as coupled to the particles or incorporated into the envelope of (enveloped) virus particles or virus-like particles to form pseudotyped enveloped virus particles or virus-like particles. The drug is coupled to the particles or packaged into the particles. For example, the drug is coupled to viral capsids or packaged into viral capsids, wherein said viral capsids may further comprise an envelope, preferably pseudotyped with syncytin. In some preferred embodiments, the drug is packaged into the particles pseudotyped with syncytin protein. The drug which is packaged into particles is advantageously a heterologous gene of interest which is packaged into viral vector particles, preferably retroviral vector particles, more preferably lentiviral vector particles.

[0140] In a particular embodiment, the method according to the invention, wherein the viral vector is adenovirus.

[0141] As used herein, the term “adenovirus” refers to medium-sized (90-100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome.

[0142] In a particular embodiment, the method according to the invention, wherein the viral vector is an adeno-associated virus (AAV) vector.

[0143] As used herein the term "AAV" has its general meaning in the art and is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all serotypes and variants both naturally occurring and engineered forms. According to the invention the term "AAV" refers to AAV type 1 (AAV- 1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV- 5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), and AAV type 8 (AAV-8) and AAV type 9 (AAV9). The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank. See, e.g., GenBank Accession Numbers NC_001401 (AAV-2), AF043303 (AAV_,2), and NC_006152 (AAV-5). As used herein, a "rAAV vector" refers to an AAV vector comprising the antibody of interest (i.e the intrabodies as described above). The rAAV vectors contain 5' and 3' adeno-associated virus inverted terminal repeats (ITRs), and the polynucleotide of interest operatively linked to sequences, which regulate its expression in a target cell.

[0144] The AAV vector of the present invention typically comprises regulatory sequences allowing expression and, secretion of the encoded molecule polypeptide (i.e. the intrabody according to the invention), such as e.g., a promoter, enhancer, polyadenylation signal, internal ribosome entry sites (IRES), sequences encoding protein transduction domains (PTD), and the like. In this regard, the vector comprises a promoter region, operably linked to the polynucleotide of interest, to cause or improve expression of the protein in infected cells. Such a promoter may be ubiquitous, tissue-specific, strong, weak, regulated, chimeric, inducible, etc., to allow efficient and suitable production of the protein in the infected tissue. The promoter may be homologous to the encoded protein, or heterologous, including cellular, viral, fungal, plant or synthetic promoters. Examples of such regulated promoters include, without limitation, Tet on / off element-containing promoters, rapamycin-inducible promoters and metallothionein promoters. Examples of ubiquitous promoters include viral promoters, particularly the CMV promoter, CAG promoter (chicken beta actin promoter with CMV enhancer), the RSV promoter, the SV40 promoter, etc. and cellular promoters such as the PGK (phosphoglycerate kinase) promoter. The promoters may also be neurospecific promoters such as the Synapsin or the NSE (Neuron Specific Enolase) promoters (or NRSE (Neuron restrictive silencer element) sequences placed upstream from the ubiquitous PGK promoter), or promoters specific for iris cell types such as DCT or the trabeculum meshwork such as MYOC, or retinal cell types such as the RPE65, the BEST1, the Rhodopsin or the cone arrestin promoters. The vector may also comprise target sequences for miRNAs achieving suppression of transgene expression in non- desired cells. In some embodiments, the vector comprises a leader sequence allowing secretion of the encoded protein. Fusion of the polynucleotide of interest with a sequence encoding a secretion signal peptide (usually located at the N-terminal end of secreted polypeptides) will allow the production of the therapeutic protein in a form that can be secreted from the transduced cells. Examples of such signal peptides include the albumin, the P-glucuronidase, the alkaline protease or the fibronectin secretory signal peptides.

[0145] The recombinant AAV vector of the present invention is produced using methods well known in the art. In short, the methods generally involve (a) the introduction of the rAAV vector into a host cell, (b) the introduction of an AAV helper construct into the host cell, wherein the helper construct comprises the viral functions missing from the rAAV vector and (c) introducing a helper virus into the host cell. All functions for rAAV virion replication and packaging need to be present, to achieve replication and packaging of the rAAV vector into rAAV virions. The introduction into the host cell can be carried out using standard virological techniques simultaneously or sequentially. Finally, the host cells are cultured to produce rAAV virions and are purified using standard techniques such as CsCl gradients. Residual helper virus activity can be inactivated using known methods, such as for example heat inactivation. The purified rAAV vector is then ready for use in the method of the present invention.

[0146] In a particular embodiment, the method according to the invention, wherein the AAV vector is selected from vectors derived from AAV serotypes having tropism for and high transduction efficiencies in ocular cells.

[0147] In a particular embodiment, the method according to the invention, wherein the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV 5, AAV 6, AAV7, AAV 8 or AAV9. In a particular embodiment, the method according to the invention, wherein the AAV vector is an AAV1, AAV 2, AAV 5, AAV 7, 8 or AAV 9.

[0148] In a particular embodiment, the method according to the invention, wherein the AAV vector is AAV 9.

[0149] A further aspect of the invention relates to a host cell which has been transfected, infected or transformed by a nucleic acid and / or a vector according to the invention.

[0150] The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA bas been "transformed".

[0151] The nucleic acids of the invention may be used to produce an antibody of the present invention in a suitable expression system. The term "expression system" means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E.coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Agl4 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as "DHFR gene") is defective (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.2O cell (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cell"), and the like. The present invention also relates to a method of producing a recombinant host cell expressing an antibody according to the invention, said method comprising the steps of: (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described above into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and / or secrete said antibody. Such recombinant host cells can be used for the production of antibodies of the present invention.

[0152] Antibodies of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0153] Antibody engineering:

[0154] Engineered antibodies of the invention include those in which modifications have been made to framework residues within VH and / or VL, e.g. to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived. To return the framework region sequences to their germline configuration, the somatic mutations can be "backmutated" to the germline sequence by, for example, site- directed mutagenesis or PCR-mediated mutagenesis. Such "backmutated" antibodies are also intended to be encompassed by the invention. Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0155] In some embodiments, the glycosylation of an antibody is modified. Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0156] In some embodiments, some mutations are made to the amino acids localized in aggregation “hotspots” within and near the first CDR (CDR1) to decrease the antibodies susceptibility to aggregation (see Joseph M. Perchiacca et al., Proteins 2011; 79:2637-2647).

[0157] The antibodies of the present invention may be of any isotype. The choice of isotype typically will be guided by the desired effector functions. IgGl and IgG3 are isotypes that mediate such effectors functions as ADCC or CDC, when IgG2 and IgG4 don’t or in a lower manner. Either of the human light chain constant regions, kappa or lambda, may be used. If desired, the class of a monoclonal antibody of the present invention may be switched by known methods. Typical, class switching techniques may be used to convert one IgG subclass to another, for instance from IgGl to IgG2. Thus, the effector function of the monoclonal antibodies of the present invention may be changed by isotype switching to, e.g., an IgGl, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses.

[0158] In some embodiments, the antibodies of the present invention are full-length antibodies.

[0159] In some embodiments, the full-length antibodies are IgGl antibodies.

[0160] In some embodiments, the full-length antibodies are IgG2 antibodies.

[0161] In some embodiments, the full-length antibodies are an IgG3 antibodies.

[0162] In some embodiments, the antibodies of the present invention are antibodies which has been mutated such that the ability to mediate effector functions, such as ADCC, has been reduced or even eliminated. Such mutations have e.g. been described in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol. 75(24): 12161-12168 (2001).

[0163] In some embodiments, the hinge region of CHI is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0164] In some embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0165] In some embodiments, one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[0166] In some embodiments, one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94 / 29351 by Bodmer et al. In some embodiments, the Fc region is modified to increase the ability of the antibodies to mediate antibody dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibodies for an Fc receptor by modifying one or more amino acids. This approach is described further in PCT Publication WO 00 / 42072 by Presta. Moreover, the binding sites on human IgGI for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604, W02010106180).

[0167] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art, and refers to a cell-mediated reaction in which non- specific cytotoxic cells that express Fc receptors (FcRs) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

[0168] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

[0169] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation. For example, EPl, 176, 195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non- fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransf erase. PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyl transferases (e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues

[0170] (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).

[0171] In another embodiment, the antibodies are modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations can be introduced: T252L, T254S, T256F, as described in U.S. Patent No. 6,277,375 by Ward. Alternatively, to increase the biological half life, the antibodies can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121 ,022 by Presta et al. Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the foetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311,312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitutions of Fc region residue 434 (US Patent No. 7,371,826).

[0172] Another modification of the antibodies herein that are contemplated by the invention is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. The pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl- CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP0154316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0173] Another modification of the antibodies that is contemplated by the invention is a conjugate or a protein fusion of at least the antigen-binding region of the antibody of the invention to serum protein, such as human serum albumin or a fragment thereof to increase half-life of the resulting molecule. Such approach is for example described in Ballance et al. EP0322094. Another possibility is a fusion of at least the antigen-binding region of the antibody of the invention to proteins capable of binding to serum proteins, such human serum albumin to increase half-life of the resulting molecule. Such approach is for example described in Nygren et al., EP 0 486 525.

[0174] Polysialytion is another technology, which uses the natural polymer polysialic acid (PSA) to prolong the active life and improve the stability of therapeutic peptides and proteins. PSA is a polymer of sialic acid (a sugar). When used for protein and therapeutic peptide drug delivery, polysialic acid provides a protective microenvironment on conjugation. This increases the active life of the therapeutic protein in the circulation and prevents it from being recognized by the immune system. The PSA polymer is naturally found in the human body. It was adopted by certain bacteria which evolved over millions of years to coat their walls with it. These naturally polysialylated bacteria were then able, by virtue of molecular mimicry, to foil the body's defense system. PSA, nature's ultimate stealth technology, can be easily produced from such bacteria in large quantities and with predetermined physical characteristics. Bacterial PSA is completely non-immunogenic, even when coupled to proteins, as it is chemically identical to PSA in the human body.

[0175] Another technology includes the use of hydroxy ethyl starch ("HES") derivatives linked to antibodies. HES is a modified natural polymer derived from waxy maize starch and can be metabolized by the body's enzymes. HES solutions are usually administered to substitute deficient blood volume and to improve the rheological properties of the blood. Hesylation of an antibody enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity. By varying different parameters, such as the molecular weight of HES, a wide range of HES antibody conjugates can be customized. In another embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0176] In certain embodiments of the invention the antibodies have been engineered to remove sites of deamidation. Deamidation is known to cause structural and functional changes in a peptide or protein. Deamidation can result in decreased bioactivity, as well as alterations in pharmacokinetics and antigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar 1 ;77(5): 1432-9).

[0177] In certain embodiments of the invention the antibodies have been engineered to increase pl and improve their drug-like properties. The pl of a protein is a key determinant of the overall biophysical properties of a molecule. Antibodies that have low pls have been known to be less soluble, less stable, and prone to aggregation. Further, the purification of antibodies with low pl is challenging and can be problematic especially during scale-up for clinical use. Increasing the pl of the anti-TDP-43 antibodies of the invention or fragments thereof improved their solubility, enabling the antibodies to be formulated at higher concentrations (>100 mg / ml). Formulation of the antibodies at high concentrations (e.g. >100mg / ml) offers the advantage of being able to administer higher doses of the antibodies into eyes of patients via intravitreal injections, which in turn may enable reduced dosing frequency, a significant advantage for treatment of chronic diseases including cardiovascular disorders. Higher pls may also increase the FcRn- mediated recycling of the IgG version of the antibody thus enabling the drug to persist in the body for a longer duration, requiring fewer injections. Finally, the overall stability of the antibodies is significantly improved due to the higher pi resulting in longer shelf-life and bioactivity in vivo. Preferably, the pl is greater than or equal to 8.2.

[0178] Glycosylation modifications can also induce enhanced anti-inflammatory properties of the antibodies by addition of sialylated glycans. The addition of terminal sialic acid to the Fc glycan reduces FcyR binding and converts IgG antibodies to anti-inflammatory mediators through the acquisition of novel binding activities (see Robert M. Anthony et al., J Clin Immunol (2010) 30 (Suppl 1): S9— S 14; Kai-Ting C et al., Antibodies 2013, 2, 392-414).

[0179] Mimetics antibody: In some embodiments, the heavy and light chains, variable regions domains and CDRs that are disclosed can be used to prepare polypeptides that contain antigen binding region that can specifically bind to TDP-43. For example, one or more of the CDRs listed in Table 1 can be incorporated into a molecule (e.g., a polypeptide) covalently or noncovalently to make an immunoadhesion. An immunoadhesion may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDR(s) enable the immunoadhesion to bind specifically to a particular antigen of interest (e.g., anti-TDP43 or epitope thereof).

[0180] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well to naturally occurring amino acids polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0181] In some embodiments, the antigen biding fragment of the invention is grafted into non-immunoglobulin based antibodies also called antibody mimetics selected from the group consisting of an affibody, an affilin, an affitin, an adnectin, an atrimer, an evasin, a DARPin, an anticalin, an avimer, a fynomer, and a versabody.

[0182] The term “antibody mimetic” is intended to refer to molecules capables of mimicking an antibody’s ability to bind an antigen, but which are not limited to native antibody structures. Examples of such antibody mimetics include, but are not limited to, Adnectins, Affibodies, DARPins, Anticalins, Avimers, and versabodies, all of which employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms. Antigen biding fragments of antibodies can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). An affibody is well known in the art and refers to affinity proteins based on a 58 amino acid residue protein domain, derived from one of the IgG binding domain of staphylococcal protein A. DARPins (Designed Ankyrin Repeat Proteins) are well known in the art and refer to an antibody mimetic DRP (designed repeat protein) technology developed to exploit the binding abilities of non-antibody proteins. Anticalins are well known in the art and refer to another antibody mimetic technology, wherein the binding specificity is derived from lipocalins. Anticalins may also be formatted as dual targeting protein, called Duocalins. Avimers are well known in the art and refer to another antibody mimetic technology, Avimers are derived from natural A- domain containing protein. Versabodies are well known in the art and refer to another antibody mimetic technology, they are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core the typical proteins have. Such antibody mimetic can be comprised in a scaffold. The term “scaffold” refers to a polypeptide platform for the engineering of new products with tailored functions and characteristics.

[0183] In one aspect, the invention pertains to generating non-immunoglobulin based antibodies also called antibody mimetics using non-immunoglobulins scaffolds onto which CDRs of the invention can be grafted. Known or future non-immunoglobulin frameworks and scaffolds may be employed, as long as they comprise a binding region specific for the target anti-TDP-43 protein.

[0184] The fibronectin scaffolds are based on fibronectin type III domain (e.g., the tenth module of the fibronectin type III (10 Fn3 domain)). The fibronectin type III domain has 7 or 8 beta strands which are distributed between two beta sheets, which themselves pack against each other to form the core of the protein , and further containing loops (analogous to CDRs) which connect the beta strands to each other and are solvent exposed. There are at least three such loops at each edge of the beta sheet sandwich, where the edge is the boundary of the protein perpendicular to the direction of the beta strands (see US 6,818,418). These fibronectin-based scaffolds are not an immunoglobulin, although the overall fold is closely related to that of the smallest functional antibody fragment, the variable region of the heavy chain, which comprise the entire antigen recognition unit in camel and llama IgG. Because of this structure, the non-immunoglobulin antibody mimics antigen binding properties that are similar in nature and affinity to those of antibodies. These scaffolds can be used in a loop randomisation and shuffling strategy in vitro that is similar to the process of affinity maturation of antibodies in vivo. These fibronectin-based molecules can be used as scaffolds where the loop regions of the molecule can be replaced with CDRs of the invention using standard cloning techniques.

[0185] The Ankyrin technology is based on using proteins with Ankyrin derived repeat modules as scaffolds for bearing variable regions which can be used for binding to different targets. The Ankyrin repeat module is a 33 amino acid polypeptide consisting of two antiparallel a-helices and a P-turn. Binding of the variable regions is mostly optimized by using ribosome display.

[0186] Avimers are derived from natural A-domain containing protein such as LRP-1. These domains are used by nature for protein-protein interactions and in human over 250 proteins are structurally based on “A-domains” monomers (2-10) linked via amino acids linkers. Avimers can be created that can bind to the target antigen using the methodology described in, for example, U.S. patent Application publication Nos. 20040175756; 20050053973; 20050048512; and 20060008844.

[0187] Affibody affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of protein A. protein A is a surface protein form the bacterium Staphylococcus aureus. This scaffold domain consist of 58 amino acids, 13 of which are randomized to generate affibody librairies with a large number of ligand variants (See e.g., US 5,831,012). Affibody molecules mimic antibodies, they have a molecular weight of 6kDa. In spite of its small size, the binding site of affibody molecules is similar to that of an antibody.

[0188] Anticalins are products developed by the company Pieris ProteoLab AG. They are derived from lipocalins, a widespread group of small and robust proteins that are usually involved in the physiological transport or storage of chemically sensitive or insoluble compounds. Several natural lipocalins occur in human tissues or body liquids. The protein architecture is reminiscent of immunoglobulins, with hypervariable loops on top of a rigid framework. However, in contrast with antibodies or their recombinant fragments, lipocalins are composed of a single polypeptide chain with 160 to 180 amino acids residues, being just marginally bigger than a single immunoglobulin domain. The set of four loops, which makes up the binding pocket, shows pronounced structural plasticity and tolerates a variety of side chains. The binding site can can thus be reshaped in a proprietary process in order to recognize prescribed target molecules of different shape with high affinity and specificity. One protein of lipocalin family, the bilin-binding protein (BBP) of Pieris Brassicae has been used to develop anticalins by mutagenizing the set of four loops. One example of a patent application describing anticalins is in PCT Publication No. WO 199916873.

[0189] Affilin molecules are small non-immunoglobulin proteins which are designed for specific affinities towards proteins and small molecules. New affilin molecules can be very quickly selected from two libraries, each of which is based on a different human derived scaffold protein. Affilin molecules do not show any structural homology to immunoglobulin proteins. Currently, two affilin scaffolds are employed, one of which is gamma crystalline, a human structural eye lens protein and the other is “ubiquitin” superfamily proteins. Both human scaffolds are very small, show high temperature stability and are almost resistant to pH changes and denaturing agents. This high stability is mainly due to the expanded beta sheet structure of the proteins. Examples of “ubiquitin-like” proteins are described in W02004106368.

[0190] Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable and offer extended shelf-life. Additional information regarding Versabodies can be found in US 2007 / 0191272, which is hereby incorporated by reference in its entirety.

[0191] The above descriptions of antibody fragment and mimetic technologies is not intended to be comprehensive. A variety of additional technologies including alternative polypeptide- based technologies, such as fusions of complementarity determining regions as outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), as well as nucleic acid- based technologies, such as the RNA aptamer technologies described in US 5,789,157; 5,864,026; 5,712,375; 5,763,566; 6,013,443; 6,376,474; 6,613,526; 6,114,120; 6,261,774; and 6,387,620; all of which are hereby incorporated by reference, could be used in the context of the instant invention.

[0192] Diagnosis uses

[0193] A further aspect of the invention relates to the TDP-43 antibodies of the invention for diagnosing and / or monitoring and / or staging a TDP-43 proteinopathy and particularly a TDP- 43 proteinopathy in which TDP-43 is overexpressed in cytoplasm.

[0194] As used herein term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and / or prospects of recovery.

[0195] According to the invention, the TDP-43 proteinopathy in which TDP-43 overexpression found in cytoplasm is: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-predominant age-related TDP-43 encephalopathy (LATE), Alzheimer's disease, Parkinson's disease, multiple system atrophy, or progressive supranuclear palsy, post- traumatic diseases (chronic traumatic encephalopathy), neoplastic (pilocytic astrocytoma) or post-infectious (post-encephalitic parkinsonism).

[0196] In a particular embodiment, antibodies of the invention may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art as above described. For example, antibodies of the invention may be labelled with a radioactive molecule by any method known to the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, Ini 11, Rel86, Rel88. Antibodies of the invention may be also labelled with a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-i l l, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. Following administration of the antibody, the distribution of the antibody within the patient is detected. Methods for detecting distribution of any specific label are known to those skilled in the art and any appropriate method can be used. Some non-limiting examples include, computed tomography (CT), position emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence and sonography.

[0197] Typically, said diagnostic methods involve the use of a biological sample obtained from the subject.

[0198] As used herein the term "biological sample" encompasses a variety of sample types obtained from a subject and can be used in a diagnostic or monitoring assay. Biological samples include but are not limited to blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom, and the progeny thereof. For example, biological samples include cells obtained from a tissue sample collected from an individual suspected of having a TDP-43 proteinopathy associated with TDP-43 overexpression in the cytoplasm.

[0199] In a particular embodiment, the biological sample is fibroblastes obtained from a subject suffering from a TDP-43 proteinopathy.

[0200] In a particular embodiment, the biological sample is post-mortem neural tissue from a subject suffering from a TDP-43 proteinopathy such as ALS.

[0201] Therefore, biological samples encompass clinical samples, neural samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.

[0202] In another aspect, the antibodies according to the invention are useful for molecular imaging.

[0203] In a particular embodiment, such antibodies are able to detect TDP-43 as a molecular imaging target.

[0204] As used herein, the term “molecular imaging target” refers to a compound that can be detected by using molecular imaging techniques such as with an molecule imaging agent.

[0205] As used herein, the term “molecular imaging agent” refers to a compound that can be used to detect specific biological elements, in particular a molecular imaging target, by using molecular imaging techniques. The molecular imaging agent is typically an agent coupled to an imaging label, covalently or non-covalently. A chelating agent is a molecule covalently bound to the ligand, which allows complexing radiometal(s). Typically, chelating agents could be: 6-Hydrazinopyridine-3- carboxylic acid (HYNIC), chelating peptide such as Gly-Gly-Cys or His-based sequence (Francesconi 2004, Waibel 1999, Ali 2011), MAG3 en N-ter (Okarvi 2012, 2004), 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetracetic acid (DOTA), diethylene triamine penta-acetic 20 acid (DTPA), l,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine, 2,3-dimercaptosuccinic acid,2,3-dimercapto-l- propanesulfonic acid, 2, 3 -dimercaptopropanol (BAL), triethylenetetramine, ammonium tetrathiomolybdate anion, ethylenedi aminetetraacetic acid (EDTA), 2- (pisothiocyanatobenzyl)- 6-methyldiethylenetriaminepentaacetic acid (IB4M) or 25 hydroxypyridinone (HOPO).

[0206] The antibodies according to the invention may be linked to the chelating agent directly (for example the chelating agent being attached to a lateral amino acid long chain) or indirectly, for example via at least one spacer. A preferred chelating agent is 6- Hydrazinopyridine-3 -carboxylic acid (HYNIC). In such case, the antibodies according to the invention may be linked to HYNIC directly (for example HYNIC being attached to a lateral amino acid long chain) or indirectly, for example via at least one spacer. The spacer for example comprises or consists of at least one PEG (polyethylene glycol), for example one, two or at least three PEG, more preferably three PEG and / or at least one aliphatic spacer.

[0207] In the context of the invention, the molecular imaging agent is used to detect TDP-43 in vivo or in vitro, for example in a biological sample (blood sample, neural tissue...) of a subject suffering or susceptible to suffer from TDP-43 proteinopathies.

[0208] The present invention thus relates to antibodies anti-TD43 for use as a molecular imaging tool for imaging accumulation of TDP-43 in the cytoplasm of a subject susceptible of suffering from a TDP-43 proteinopathies.

[0209] The present invention particularly relates to antibodies anti-TD43 for use as a molecular imaging tool in a method for diagnosing TDP-43 proteinopathies particularly in vivo.

[0210] The present invention particularly relates to antibodies anti-TD43 for use as a molecular imaging tool in a method for monitoring the evolution of an TDP-43 proteinopathies particularly in vivo.

[0211] In a particular embodiment, the antibodies anti-TD43 according to the invention are labelled with a radiolabel for imaging. As used herein, the term “radiolabel” also known as radionuclide or radioactive tracer refers to a radioactive isotope which, when injected into a chemically similar substance, or artificially attached to a biological or physical system, can be traced by radiation detection devices.

[0212] Examples of radioactive tracer well known in the art are: tritium, carbon-11, carbon- 14, oxygen-15, fluorine-18, phosphorus-32, sulfur-35, technetium-99, iodine-123, gallium-67, and 89-zirconium.

[0213] In the context of the invention, the radioactive tracer is89Zr (zirconium).

[0214] 89Zr displays an adequate half-life (3.3 day) compatible with the antibodies anti-TDP- 43 of the invention biological half-life to monitor TDP-43 aggregates. Currently, the majority of 89Zr- immuno-PET studies use desferrioxamine B (DFO). DFO has been administered to thousands of patients, in free form for the treatment of iron overload or attached to slow kinetic drugs as chelator for 89 Zr labelling.

[0215] In another embodiment, the DFO is used with an octadentate chelator, named DFO*- NCS.

[0216] Typically, inventors start by anchoring these chelating agents to the antibodies of the present invention such as D7 using DFO-NCS as reagent toward the generation of strong thioamide bond. The objective is to target via this approach a lysine amino residue (11 available, only 2 in the paratope region) and quantify the number of substitutions by High Resolution Mass spectrometry. Several assays have been performed to modulate the number of anchoring. The preparation of D7-DFO* complexes with non-radioactive natZr salts have been performed to evaluate efficiency of the conjugation D7 substitution ratio (ICP). The radiolabelling of molecules with89Zr has been performed using available methods well know in the art. Purity as specific activity (GBq / pg) of the synthesized 89Zr-DFO*-D7 has been performed prior to any biological experiment.

[0217] In another embodiment, the radiolabel tracer is fluorine-18 (18F). 18F can be coupled with well-known chelator such as NOTA or RESCA.

[0218] In a particular embodiment, the radionuclide is detectable by nuclear medicine molecular imaging technique(s), such as, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid of SPECT and / or PET or their combinations. Single Photon Emission Computed Tomography (SPECT) herein includes planar scintigraphy (PS). An hybrid of SPECT and / or PET is for example SPECT / CT, PET / CT, PET / IRM or SPECT / IRM. In a particular embodiment, the antibodies anti-TD43 according to the invention are conjugated with a radiolabel for PET imaging.

[0219] As used herein, the term “PET” refers to Positron emission tomography (PET). PET is a technique that measures physiological function by looking at blood flow, metabolism, neurotransmitters, and radiolabelled drugs. PET offers quantitative analyses, allowing relative changes over time to be monitored as a disease process evolves or in response to a specific stimulus.

[0220] Typically, SPECT and PET acquire information on the concentration (or uptake) of radionuclides introduced into a subject’s body. PET generates images by detecting pairs of gamma rays emitted indirectly by a positron-emitting radionuclide. A PET analysis results in a series of thin slice images of the body over the region of interest (e.g., brain, breast, liver,. . .). These thin slice images can be assembled into a three dimensional representation of the examined area. SPECT is similar to PET, but the radioactive substances used in SPECT have longer decay times than those used in PET and emit single instead of double gamma rays. Although SPECT images exhibit less 5 sensitivity and are less detailed than PET images, the SPECT technique is much less expensive than PET and offers the advantage of not requiring the proximity of a particle accelerator. Actual clinical PET presents higher sensitivity and better spatial resolution than SPECT, and presents the advantage of an accurate attenuation correction due to the high energy of photons; so PET provides more accurate quantitative data than SPECT. Planar scintigraphy (PS) is similar to SPECT in that it uses the same radionuclides. However, PS only generates 2D-information.

[0221] SPECT produces computer-generated images of local radiotracer uptake, while CT produces 3-D anatomic images of X ray density of the human body. Combined SPECT / CT imaging provides sequentially functional information from SPECT and the anatomic information from CT, obtained during a single examination. CT data are also used for rapid and optimal attenuation correction of the single photon emission data. By precisely localizing areas of abnormal and / or physiological tracer uptake, SPECT / CT improves sensitivity and specificity, but can also aid in achieving accurate dosimetric estimates as well as in guiding interventional procedures or in better defining the target volume for external beam radiation therapy. Gamma camera imaging with single photon emitting radiotracers represents the majority of procedures.

[0222] In a particular embodiment, the presence, localization and / or amount of the labeled TDP-43 antibody of the invention may be determined by planar scintigraphy (PS), Positron Emission Tomography 5 (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid SPECT / CT or their combinations.

[0223] In a particular embodiment, the labeled TDP-43 antibody of the invention is used or administered by intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal administration.

[0224] In a particular embodiment, the labeled TDP-43 antibody of the invention is used or administered in an amount sufficient to obtain a detectable signal for an intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal administration.

[0225] In a particular embodiment, the labeled TDP-43 antibody of the invention is detected immediately after the administration of the labeled TDP-43 antibody.

[0226] In a particular embodiment, the signal may be detected in the entire body of the subject or in only one part of the subject (particularly in only one part of the body of the subject).

[0227] In a particular embodiment, the labeled TDP-43 antibody of the invention relates to a labeled TDP-43 antibody as defined above for use in a method for detecting TDP-43, wherein said method comprises:

[0228] - administering said labeled TDP-43 antibody to the subject, and

[0229] - imaging said subject or at least one part of the body of said subject, thereby detecting the binding of the labeled TDP-43 antibody to TDP-43 in said subject or in said part of the body of the subject.

[0230] The present invention thus particularly relates to a labeled TDP-43 antibody as defined above for use in a method for determining whether a subject suffers from a TDP-43 proteinopathy, is at risk of having TDP-43 proteinopathy condition or is at risk of recurrence of TDP-43 proteinopathy after a TDP-43 proteinopathy treatment or for monitoring the efficacy of a treatment of TDP-43 proteinopathy, wherein said method comprises:

[0231] - administering said labeled TDP-43 antibody to the subject, and

[0232] - imaging said subject or at least one part of the body of said subject, thereby detecting the binding of the labeled TDP-43 antibody to TDP-43 in said subject or in said part of the body of the subject.

[0233] In a particular embodiment, the the molecular imaging agent of the invention represents a powerful tool for diagnosing or assessing a TDP-43 proteinopathy condition associated with an accumulation of TDP-43. Therapeutic uses

[0234] Antibodies, fragments or immunoconjugates of the invention may be useful for treating any diseases associated with TDP-43 overexpression particularly, TDP-43 proteinopathies.

[0235] Accordingly, the invention relates to a method for treating TDP-43 proteinopathyies in a subject in need thereof comprising a step of administering to the subject a therapeutically effective amount of the antibody anti-TDP-43 according to the invention.

[0236] In a particular embodiment, the intrabodies targeting wildtype TDP-43 according to the invention for use as a medicament.

[0237] In a particular embodiment, the intrabodies targeting wildtype TDP-43 according to the invention for use as a medicament to treat TDP-43 proteinopathies.

[0238] In a particular embodiment, the intrabodies targeting wildtype TDP-43 according to the invention for use wherein the TDP-43 proteinopathy is selected from the group consisting of but not limited to: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic- predominant age-related TDP-43 encephalopathy (LATE), Alzheimer's disease, Parkinson's disease, multiple system atrophy, or progressive supranuclear palsy, post-traumatic diseases (chronic traumatic encephalopathy), neoplastic (pilocytic astrocytoma) or post-infectious (post-encephalitic parkinsonism).

[0239] Thus, the invention also relates to antibodies, fragments or immunoconjugates of the invention for use in the treatment of any diseases associated with TDP-43 overexpression particularly TDP-43 proteinopathy.

[0240] The antibodies of the invention may be used alone or in combination with any suitable agent.

[0241] The present invention also relates to a method of treatment of any diseases associated with TDP-43 overexpression particularly TDP-43 proteinopathy in a subject in need thereof comprising administering to the subject antibodies, fragments or immunoconjugates of the invention, in particular intrabodies targeting wildtype TDP-43 of the present invention.

[0242] In each of the embodiments of the treatment methods described herein, the anti-TDP- 43 antibody or anti-TDP-43 antibody-drug conjugate is delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody or antibody-drug conjugate is administered to a patient in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder. As used herein, the terms "treatment" and "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

[0243] As used herein, the term "therapeutically effective amount" or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the antibodies of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibodies of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibodies or antibodies portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the antibodies of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the antibodies of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to inhibit TDP-43 proteinopathie may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition may be evaluated by examining the ability of the compound to induce cytotoxicity by in vitro assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, for example about 0.1- 20 mg / kg, such as about 0.1-10 mg / kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example about 0.5-2 mg / kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using labeled antibodies of the present invention, fragment or mini-antibodies derived from the antibodies of the present invention. If desired, an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some embodiments, the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects. An effective dose of antibodies of the present invention may also be administered using a weekly, biweekly or triweekly dosing period. The dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established. As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of antibodies of the present invention in an amount of about 0.1-100 mg / kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0244] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., intrabodies of the invention) into the subject, such as by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraventricular, intrathecal, intraorbital, ophthalmic, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, oral, topical, intravitreal, mucosal, intradermal, delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0245] In some embodiments, the subject suffers from a TDP-43 proteinopathy.

[0246] Accordingly, a further object of the present invention relates to a method of treating TDP-43 proteinopathy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antibodies of the present invention.

[0247] As used herein, the term " TDP-43 proteinopathy" has its general meaning in the art and refers to all diseases in which TDP-43 is overexpressed in the cytoplasm. The TDP-43 proteinopathy is selected from the group consisting of but not limited to: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-predominant age-related TDP-43 encephalopathy (LATE), Alzheimer's disease, Parkinson's disease, multiple system atrophy, or progressive supranuclear palsy, post-traumatic diseases (chronic traumatic encephalopathy), neoplastic (pilocytic astrocytoma) or post-infectious (post-encephalitic parkinsonism).

[0248] In a particular embodiment, the TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

[0249] As used herein, the term "Amyotrophic lateral sclerosis” (ALS) refers to the most common motor neuron disorder in adults, with an incidence of 1-2 / 100,000 and a prevalence of 4-6 / 100,000 each year. The progressive degeneration of both upper and lower motor neurons typically leads to death for respiratory failure in three to five years after diagnosis. About 15% of ALS patients develop also signs of frontotemporal dementia (FTD). FTD represents the second most common cause of dementia after Alzheimer’s disease, leading to personality and behavioral changes and speech disabilities. It is characterized by a progressive neuronal loss in the frontal and anterior temporal lobes of the brain.

[0250] In a particular embodiment, the TDP-43 proteinopathy is Alzheimer's disease.

[0251] As used herein, the term "Alzheimer's disease” refers to a chronic neurodegenerative disease that usually starts slowly and gets worse over time. The most common early symptom is difficulty in remembering recent events (short- term memory loss). As the disease advances, symptoms can include: problems with language, disorientation (including easily getting lost), mood swings, loss of motivation, not managing self-care, and behavioral issues. As a person's condition declines, she or he often withdraws from family and society. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the average life expectancy following diagnosis is three to nine years.

[0252] In a particular embodiment, the TDP-43 proteinopathy is Parkinson 's disease.

[0253] As used herein, the term "Parkinson's disease” refers to a chronic neurodegenerative disease which is caused by a loss of nerve cells in part of the brain called the substantia nigra. This leads to a reduction in a chemical called dopamine in the brain. Dopamine plays a vital role in regulating the movement of the body. A reduction in dopamine is responsible for many of the symptoms of Parkinson's disease.

[0254] In another aspect the invention relates to the anti-TDP43 antibodies for use according to the invention, and a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of TDP-43 proteinopathies in a subject in need thereof.

[0255] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

[0256] As used herein, the term “administration simultaneously” refers to administration of at least two or three active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of at least two or three active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of at least two or three active ingredients at different times, the administration route being identical or different.

[0257] In the context of the invention, the term “simultaneous use” denotes the use of an anti-TDP43 antibody and at least one drug occurring at the same time. In the context of the invention, the term “separate use” denotes the use of an anti-TDP43 antibody and at least one drug not occurring at the same time. In the context of the invention, the term “sequential use” denotes the use of an anti-TDP43 antibody and at least one drug occurring by following an order.

[0258] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat TDP-43 proteinopathy.

[0259] In some embodiments, the anti-TDP43 antibodies for uses according to the invention wherein the classical treatment is selected from the group consisting of but not limited to: Riluzole (Rilutek or Tiglutik™); Edaravone (Radicava™), RELYVRIO TM (AMX0035), and Nuedexta™.

[0260] In certain embodiments, anti-TDP43 antibodies or antibodies-drug conjugates are used in combination with a second agent for treatment of a disease or disorder.

[0261] The present invention also provides for therapeutic applications where antibodies of the present invention is used in combination with at least one further therapeutic agent. Such administration may be simultaneous, separate or sequential. For simultaneous administration the agents may be administered as one composition or as separate compositions, as appropriate. The further therapeutic agent is typically relevant for the disorder to be treated.

[0262] In a further embodiment, the method according to the invention, wherein the intrabody as described above is delivered alone or in association with liposomes, nanoparticles, spion and / or a viral vector. Accordingly, in a particular embodiment, the invention relates to a method of treating TDP-43 proteinopathyies in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a vector as described above which comprises the intrabody according to the invention.

[0263] In a further embodiment, the invention relates to a method of treating TDP-43 proteinopathyies in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of a liposomes, nanoparticles or spion as described above which comprises the intrabody according to the invention.

[0264] Pharmaceutical compositions

[0265] In another aspect, the invention relates to a pharmaceutical composition comprising the antibodies according to the invention.

[0266] Typically, the antibodies of the present invention are administered to the subject in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, antibodies present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and Sterile Water for Injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg / m2and 500 mg / m2. However, it will be appreciated that these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials. A pharmaceutical composition of the invention for injection (e.g., intramuscular, i.v.) could be prepared to contain sterile buffered water (e.g. 1 ml for intramuscular), and between about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg or more preferably, about 5 mg to about 25 mg, of an anti -myosin 18A antibodies of the invention.

[0267] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0268] Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made.

[0269] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of ML Vs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. In another embodiment, the use of spion is contemplated for the introduction of antibodies into host cells.

[0270] As used herein, the term “spion” refers to superparamagnetic iron oxide nanoparticle. SPIONs are composed of magnetite or iron oxide which is degradable in the body and nontoxic compared to other magnetic materials such as cobalt and nickel. The main forms of magnetite are Fe3O4 and its oxidized form maghemite or y-Fe2O3.

[0271] SPIONs may be produced by methods known in the art, for example, as described by Sun et al., J, American Chemical Society, 2002, 124, 8204. SPIONs may comprise one or more coatings or may be incorporated into micelles or liposomes to enhance desirably pharmacokinetic properties including biological half-life, biocompatibility, and targeting. The compositions of the invention contain SPIONs of a size compatible with in vivo administration and desired targeting functionality. Some representative SPION particle sizes range from about 1, 2, 5, 10, 20, 30, 40, 50, or 60 nm. A composition of the invention may contain a single size or single size distribution of SPIONs or may contain two or more sizes or size distributions. For example, various mixtures of large SPIONs ranging from about 10 to 60 nm in average size and small SPIONs ranging in size from about 2 to 22 nm may be used as described in Table 1-2. Mixtures of SPIONs of different sizes permit tuning of a biological responses or imaging functions. In some embodiments, a coprecipitation technique can be followed to form metal oxide composite with Ni or Cu or Mn and. Co nanoparticle to form respective MFe2O4 to enhance imaging capacity by increasing magnetization property.

[0272] In some embodiments the core of the SPIONs may be magnetite which is covered with one or more shells, for example, a polymer shell or a gold or metal shell. SPIONs may also be incorporated into, or coated with, one or more polymers including smart, pH-sensitive, or temperature-sensitive polymers.

[0273] Functionalized super paramagnetic iron oxide nanoparticles (SPIONs) may be used in accordance with one or more embodiments of the invention, for example, a SPION (or other components of a composition of the invention) may be functionalized with one or more curcuminoids, or with a combination of one or more curcuminoids and a targeting ligand such as an antibody that binds to a tumor-associated antigen. In some embodiments SPIONs may be conjugated to targeting moieties such as ligands that bind to, or agents that are internalized by, neural cells or by other target molecules, receptors, cells or tissues.

[0274] Kits

[0275] Finally, the invention also provides kits comprising at least one antibody of the invention. Kits containing antibodies of the invention find use in detecting anti-TDP43 expression (increase or decrease), or in therapeutic or diagnostic assays. Kits of the invention can contain antibodies coupled to a solid support, e.g., a tissue culture plate or beads (e.g., sepharose beads). Kits can be provided which contain antibodies for detection and quantification of anti-TDP-43 in vitro, e.g. in an ELISA or a Western blot or in vivo (for imaging such as PET imaging). Such antibodies useful for detection may be provided with a label such as a fluorescent or radiolabel.

[0276] In certain embodiments, the kit comprises at least one intrabody as described above and at least one imaging label, preferably at least one antibody anti-TDP43, and, optionally, instructions for associating said antibody anti-TDP43 and said imaging label, preferably radionuclide, to form a labeled antibody anti-TDP43 according to the invention.

[0277] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0278] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0279] FIGURES:

[0280] Figure 1: ELISA of purified intrabodies reveal the binding of Bl and D7 to wtTDP- 43. Wells of 96-well plate coated with commercially purchased wtTDP-43 diluted to 1 pg / mL were incubated with the control scFv, Bl, or D7 at increasing concentrations of 1 pg / mL, 5 pg / mL, and 10 pg / mL (x-axis). The control intrabody did not show signs of binding to TDP- 43 at any concentration, while Bl and D7 showed binding via increasing absorbances. Mann Whitney test: **p=0.004 or *p=0.03 versus 5 pg of control intrabody; ##p<0.005 versus 10 pg of control intrabody. N=6-7.

[0281] Figure 2: Intrabody overexpression in HEK293T cells. MTT reduction assay on HEK293T cells transfected with the scFv’s. Control = non-transfected cells (blue dashed line). Quantity of scFv reflects the amount of plasmid transfected (N=3).

[0282] Figure 3: scFv D7 decreases the amount of insoluble TDP-35 CTF through proteasomal degradation. A. Quantification of insoluble TDP-35 CTF normalized to the TDP43 condition (dotted line). * / ?=0.0286; Mann-Whitney test, N=4. B. Quantification of insoluble TDP-35 CTF normalized to TDP43 condition (dotted line) in the presence of autophagy and proteasome inhibitors. The pink histogram represents co-transfected cells; the blue histogram indicates co-transfected cells treated for 6 hours with 10 pg / mL cycloheximide and 300 nM Bafilomycin Al ; the red histogram shows co-transfected cells treated for 6 hours with 10 pg / mL cycloheximide and 0.5 pM MG-132 *p=0.0286 vs TDP43; #p=0.0286 vs TDP43 / D; Mann-Whitney test, N=4.

[0283] Figure 4: TDP-43 activates NF-KB and Bl reverses it in NSC-34 cells. Quantification of the active subunit of NF-KB, p65, in the total lysate normalized to stain-free lanes. Values were normalized to the mock condition (dotted line). *p = 0.02, Mann-Whitney test, N=4.

[0284] Figure 5: Metabolome profiles of HEK293T cells. Quantification of some of the metabolites of interest. Values were normalized to the vehicle condition (dotted line).

[0285] Figure 6: Metabolome profiles of NSC-34 cells. Quantification of some of the metabolites of interest. Values were normalized to the vehicle condition (dotted line).

[0286] EXAMPLE:

[0287] Material & Methods:

[0288] Plasmids and cloning

[0289] We had previously cloned full-length, human wtTDP-43 cDNA 40 into the pcDNA™3.3-TOPO™ TA vector (InvitrogenTM). Starting from this clone, we added the 6xHis tag at the 3' end by performing PCR on the plasmid using the following primers (Eurogentec): Forward (5’-TCTGAATATATTCGGGTAACCGAAG-3' SEQ ID NO: 34) and Reverse (5’-

[0290] CTAGTGATGGTGATGGTGATGAGAACCCCCCATTCCCCAGCCAGAAGACTTAG-3’ SEQ ID NO: 35) The resulting amplicon was then integrated into the Vivid Colors™ pcDNA™6.2 / C-EmGFP-GW / TOPO™ Mammalian Expression Vector (InvitrogenTM), which added GFP to the 5' end and yielded GFP-wtTDP-43-6xHis to permit mammalian cell expression and downstream purification for phage display. The amplicon was also integrated into the pcDNA™3.3-TOPO™ TA vector to create wtTDP-43 -6xHis for surface plasmon resonance analyses. To clone the sequence for the control antigen for phage display, GFP- 6xHis, a similar approach was taken by first performing PCR on the plasmid containing GFP- wtTDP-43-6xHis using the following primers (Eurogentec): Forward (5’- GCCACCATGGTGAGCAAGGGCGAGGA-3’ SEQ ID NO: 36) and Reverse (5’- CTAGTGGTGATGGTGATGATGAGAACCCCCCTTGTACAGCTCGTCCATGCC-3’ SEQ ID NO: 37) Then, the GFP-6xHis sequence was integrated into an empty pcDNA3.3- TOPO plasmid. As for scFv cloning, using the Ncol and Notl restriction sites surrounding the 5' and 3' ends of the scFv cDNA sequence, respectively, the pHEN-HuscI plasmid was digested to completion. The liberated scFv sequence was then integrated into previously digested empty pET-23NN vector using T4 DNA Ligase (Thermo Fisher ScientificTM). This vector added a c-myc and 6xHis tag at the 3' end of the clone, which permitted expression and purification from BL21(DE3)pLysS bacteria (New England BioLabs®). From the integrated pET-23NN vector, we cloned the scFv sequence with the 3' tags into pcDNA3.3-TOPO plasmid to permit mammalian cell expression according to the manufacturer's instructions (Thermo Fisher ScientificTM). For the PCR step, the following primers were utilized (Eurogentec): Forward (5 -GCCACCATGGCCGAGGTGCAGCTG-3’ SEQ ID NO: 38) and Reverse (5'- CTAATGGTGATGATGGTGATGTGCGG-3’ SEQ ID NO: 39).

[0291] Mammalian Cell Culture

[0292] HEK-293T cells (ATCC) were the main cell line of choice due to its robust transfection efficiency and common application in studies on TDP-43 proteinopathy.40, 60-62 The NSC-34 motor neuron-like mouse cell line (ATCC) was used in aggregation and metabolic analyses to serve as a more comparable cell model to ALS motor neurons (10.1007 / s00216-015-9047-x ). We maintained cells in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% (v / v) fetal bovine serum (FBS) at 37 °C and in an incubator maintaining an atmosphere of 5% CO2.

[0293] Purification of antigens from HEK-293T cells

[0294] The recombinant proteins GFP-6xHis and GFP-wtTDP-43-6xHis were overexpressed in cells plated at a density of 0.44 x 106 cells / T-150 flask, with a total of 4 flasks (CytoOne®) in 20 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 5% FBS / pyruvate / 1% non-essential amino acids (Gibco®). After 24 h in an incubator at 37°C and with 5% CO2, the cells were transfected with 10 mL of Opti-MEM (Gibco®) containing 60 pg of expression plasmid mixed with 120 pL of Lipofectamine-2000 (InvitrogenTM) following the manufacturer's protocol. Overexpression was permitted for 48 h in the incubator. The cells were harvested and centrifuged at 500 x g for 10 min at 4°C to remove the medium. The cells were resuspended in 40 mL of cold denaturing lysis buffer (500 mM NaCl, PBS pH = 8.0, 10% glycerol, 0.1% Brij-35, 6 M urea, 1 mM DTT) supplemented with IX Halt Protease Inhibitor Cocktail (Thermo Fisher ScientificTM). Four freeze-thaw cycles using liquid nitrogen and a 37°C water bath were applied to the cells. 4 pL of Pierce Universal Nuclease (250 U / pL, InvitrogenTM) and 10 mM MgC12 were added to the lysate. After 15 min of agitation at 4°C, the lysate was centrifuged at 15,000 x g for 20 min at 4°C. 1 mL of Ni-NTA resin (Thermo Fisher ScientificTM) equilibrated in lysis buffer with 10 mM imidazole was introduced to the supernatant. After 1 h of agitation at 4°C, the mixture was passed through a disposable column by gravity-flow (Thermo Fisher ScientificTM). The column was washed with 10 mL of cold lysis buffer supplemented with 25 mM imidazole. Finally, the recombinant protein was eluted by addition of cold lysis buffer supplemented with 250 mM imidazole. The fractions containing recombinant protein were identifed by SDS-PAGE and pooled.

[0295] The pooled eluate was transferred to dialysis tubing (CelluSep®Hl, Membrane Filtration Products, Inc) with a MWCO of 15 kDa and incubated in 500 mL of dialysis buffer (50 mM Tris-HCl pH = 7.4, 10 % glycerol, 150 mM NaCl, 0,1 mM EDTA, 2 mM 0- mercaptoethanol) at 4°C for 16 h to permit the refolding of purified protein. The buffer was then replaced with 500 mL of fresh buffer for another incubation for 2 h at 4°C. The dialyzed solution containing refolded protein was transferred to an Amicon® Ultra tube (Merck Millipore®) with a MWCO of 10 kDa for concentration by centrifugation at 10,000 x g at 4°C until reaching a volume of 500 pL. The concentration of the purified, refolded protein was determined by Bradford assay (Thermo Fisher ScientificTM) following the manufacturer's protocol.

[0296] To characterize the size of the purified recombinant proteins, a sample of 100 pL was injected into an FPLC AKTA PurifierTMIO (GE Healthcare) with a 25-mL SuperdexTM 200 Increase 10 / 300 GL column (GE Healthcare) equilibrated in dialysis buffer at 4°C. 500-pL fractions were collected at a flow rate of 0.5 mL / min. The identity of the purified recombinant protein was confirmed by Western blot. Briefly, samples representing all the steps of the purification were separated in 4-20% SDS-PAGE gel apparatus (Bio-Rad) and transferred to PVDF (polyvinylidine fluoride) membranes (Bio-Rad). After blocking with 5% (w / v) Regilait powdered milk in Tris-buffered saline supplemented with 0.2% Tween-20 (TBS-T), membranes were incubated for 1 h at room temperature with a mouse HRP-conjugated primary antibody against the 6xHis tag (HRP-66005, Proteintech). Visualization of bands was realized with Clarity MaxTM Western ECL Blotting (Bio-Rad) on a ChemiDocTM Touch Imaging System (Bio-Rad). The MW of GFP-6xHis was 28 kDa, while the MW of GFP- wtTDP-43-6xHis was 75 kDa.

[0297] HuscI phage library and control scFv

[0298] The HuscI (Human scFv Intrabody Library) library provided by Dr. Pierre Martineau from the Montpellier Cancer Research Institute63-65 is a human synthetic library with a diversity of 3.109 functional clones. The library is cloned in pHENl vector (pHEN-HuscI), resulting in Ml 3 phage clones expressing single-chain variable fragments (scFv) at the N- terminal extremity of pill protein. Each scFv consisted of a variable heavy (VH) and light (VL) chain connected by a linker sequence (GGGGSGGGGSGGGGS SEQ ID NO: 40). The molecular weight of the scFv ranged from 25 - 30 kDa (data not shown). The scFv employed as the control in this work was the anti-P-galactosidase scFv, termed 13R4, described by Martineau and others.63

[0299] Phage display - polyclonal selection

[0300] Before panning on GFP-wtTDP-43-6xHis (GTH), the HuscI scFv library was depleted on anti-GFP and GFP-His protein. 3 wells of a Nunc Maxisorp 96-well plate (Thermo Fisher ScientificTM) were coated with 10 pg / mL of anti-GFP antibody (Roche, 11814460001) diluted in 100 pL of PBS for 24 h at 4°C. After 5 washes in PBS-Tween 0.1% (PBST) and one wash in PBS, the wells were saturated in 200 pL of 4% powdered milk in PBS (MPBS) for 2 h at RT. After briefly washing, the first well was incubated with 50 pL of MPBS and 50 pL of the HuscI phage library. In parallel, the second well was incubated with 10 pg / mL of purified GFP-6xHis diluted in 100 pL of PBST. After 2 h, the supernatant from the first well was transferred to the second well for another 2 h incubation. During this time, the third well was incubated with 5 pg / mL of purified GFP-wtTDP-43-6xHis diluted in 100 pL of PBST. After 2 h, the supernatant from the second well was transferred to the third well to begin the first round of selection (data not shown).

[0301] Following 2 h of incubation in the third well at RT, the supernatant was discarded and the well was washed 20 times with PBST and 3 times with PBS. 100 pL of elution buffer (50 mM Tris pH = 8.0, 1 mM CaC12) containing 125 pg / mL of trypsin (TPCK-treated, Sigma- Aldrich®) was added to the well for 15 min at RT. The supernatant was transferred to 200 pL of 2xYT medium (Sigma-Aldrich®) containing TGI E. Coli at an optical density (OD) of 0.5. After 1 h at 37°C with shaking, the bacteria were centrifuged at 3,000 x g for 10 min at RT and resuspended in 500 pL of 2xYT / 2% glucose / 100 pg / mL ampicillin. The bacteria were allowed to grow at 30°C with shaking for 16 h. 20 pL of the culture were then added to 1 mL of fresh 2xYT / 2% glucose / 100 pg / mL ampicillin shaken at 37°C. After reaching an OD of 0.5, the culture was infected with 20 pL of KM13 helper phage66 comprised of approximately 1011 TU / mL. Following shaking at 37°C for 1 h, the bacteria were centrifuged at 3,000 x g for 10 min at RT and resuspended in 1 mL of 2xYT with 100 pg / mL ampicillin and 25 pg / mL kanamycin. The culture was shaken at 30°C for 16 h. 500 pL of this culture were centrifuged at 10,000 x g for 10 min at 4°C. 400 pL of the supernatant containing new phage were added to 100 pL of 20% polyethylene glycol-8000 (PEG) and sterile 2.5 M NaCl at 4°C to precipitate the phage. After 15 min on ice, the solution was centrifuged at 13,000 rpm for 20 min at 4°C.

[0302] The pellet was resuspended in 200 pL of 15% sterile glycerol diluted in PBS. The phage titer was calculated using the following formula: number of virions / mL = (A269 - A320) x 1013

[0303] 50 pL of the new phage stock containing approximately 1011- 1012phage were then added to 50 pL of MPBS in a new well coated with 5 pg / mL of GFP-wtTDP-43-6xHis to undergo the second round of selection. In total, 4 rounds of selection were performed.

[0304] Phage display - monoclonal selection

[0305] 200 pL of HB2151 cells provided by the group of Dr. Pierre Martineau (strain allowing the expression of soluble unfused scFv) at a optical density of 0.5 were infected with 106 phage from polyclonal selection round 4. After 30 min at 37°C without shaking, infected bacteria were plated on LB / agar with 1% glucose and 100 pg / mL ampicillin and incubated at 37°C for 16 h. 96 isolated HB2151 colonies were transferred to wells of a 96-well deep-well plate (Thermo Fisher ScientificTM) containing 300 pL of 2xYT, 100 pg / mL ampicillin, and 1% glucose. The plate was incubated at 37°C with agitation for 16 h. 2 to 5 pL of culture from each well were transferred to a new flat-bottom plate (Thermo Fisher ScientificTM) containing 200 pL of 2xYT, 100 pg / mL ampicillin, and 0.1% glucose in each well. At an optical density of 0.6, 25 pL of fresh medium supplemented with 9 mM IPTG (Sigma- Aldrich®) were added to each well for induction. Induction lasted 24 h at 30°C with shaking.

[0306] The plate was centrifuged at 1,800 x g for 10 min. The supernatant contained soluble scFv with a C-terminal myc tag. 50 pL of each supernatant were added to 50 pL of 4% MPBS in each well of a 96-well plate pre-coated with 1.0 pg / mL of GTH and GH antigen (2 plates total). After a 1-h incubation at RT, the wells were washed 3 times in PBS. 100 pL of anti-c- Myc antibody (9E10, Thermo Fisher ScientificTM) diluted 2,000-fold in saturation solution were added to each well and incubated for 1 h at RT. The wells were washed 3 times with PBST and 1 time with PBS. 100 pL of TMB-ELISA Substrate Solution (Thermo Fisher ScientificTM) were then added for an incubation of 5 - 15 min at RT in the dark. The reaction was halted with 50 pL of 1 M H2SO4. Absorbance was measured at 450 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad).

[0307] For the alternative monoclonal selection, the glycerol stock of TGI infected with phage from round 4 of selection (data not shown) was seeded in 2xYT / 2% glucose / 100 pg / mL ampicillin for overnight shaking at 37°C. A small volume of the culture was then plated on LB / agar / 100 pg / mL ampicillin. After 16 h at 37°C, 95 colonies were transferred to each well of a deep-well 96-well plate containing 300 pL of 2xYT / l% glucose / 100 pg / mL ampicillin. Following 16 h of shaking at 37°C, 5 pL of culture from each well were transferred to another flat-bottom 96-well plate with 200 pL of 2xYT / 2% glucose / 100 pg / mL ampicillin. At an OD of 0.5, the cultures were infected with KM13 helper phage, as described previously.

[0308] The cultures were then centrifuged at 3,000 x g for 10 min and resuspended in fresh medium supplemented with 25 pg / mL kanamycin. After 16 h at 30°C with shaking, the cultures were centrifuged again, and 50 pL of supernatant were added to 50 pL of MPBS and incubated in a 96-well plate pre-coated with 1.0 pg / mL GFP-6xHis and another plate precoated with 1.0 pg / mL GFP-wtTDP-43-6xHis. Following 1 h at RT, the wells were washed 3 times with PBST and 1 time with PBS. 100 pL of 4% MPBS containing HRP-conjugated anti -Ml 3 bacteriophage antibody (Sino Biological) at 1 / 2,000 dilution were added to the wells for an incubation at RT for 1 h. The same washing procedure was applied before adding 100 pL of TMB-ELISA Substrate Solution (Thermo Fisher ScientificTM) for an incubation of 5 - 15 min at RT in the dark. The reaction was halted with 50 pL of 1 M H2SO4. Absorbance was measured at 450 nm .

[0309] In silico scFv binding site prediction

[0310] The potential binding sites of each scFv of interest were predicted in silico for wtTDP- 43 by MabSilico (Nouzilly, France). The 3D structure of full-length TDP-43 is not available. Therefore, a pseudo-3D structure was generated by combining the resolved structures of the separate domains of TDP-43 and homologous proteins available on the RCSB PDB Protein Data Bank. Residus 1-88 were modeled by region 1-102 of TDP-43 (PDB file 5MRG)67 Residues 89-102 were modeled by the structure of nuclear ribonucleoprotein L (PDB file 2MQN.68 Residues 103-177 and 191-258 were modeled by both RRM domains of TDP-43 (PDB file 4BS2.69 Residues 259-306 were modeled by U1 nuclear ribonucleoprotein 70 (PDB file 5UZ5.70 Residues 307 - 346 were modeled by the hydrophobic, prion-like domain of TDP-43 (PDB file 2N2C). Finally, residues 347 - 413 were modeled by human splicing factor (PDB file 4WIJ).71

[0311] Briefly, the binding site prediction was based on a docking method.72 The probability of an interaction for each residue involved in the top 30 conformations of the scFv-TDP-43 complex was evaluated. Residues were attributed 1 of 4 ratings: very highly probable, highly probable, probable, possible.

[0312] Overexpression of recombinant proteins in mammalian cells 0.3 x 106 cells were seeded in 2 mL of DMEM / 5% FBS / pyruvate / 1% non-essential amino acids in 6-well plates (CytoOne®). After 24 h in an incubater at 37°C and with 5% CO2, the transfection solution was prepared: Plasmid was diluted in 100 pL of steril 150 mM NaCl (Polyplus transfection®), and a volume of jetPEI transfection reagent (Polyplus transfection®) equivalent to twice the amount of plasmid in pg was also diluted separately in 100 pL of 150 mM NaCl. The diluted jetPEI was added dropwise to the diluted plasmid. After gentle mixing and incubation for 20 min at RT, the mixture was added to the cells to commence transfection. Regarding wtTDP-43, 3.0 pg of plasmid were transfected. Unless noted otherwise, all TDP-43 transfections included the untagged, full-length, wild-type form. With respect to co-transfection, wtTDP-43 remained at 3.0 pg and was mixed with 1.5 pg of intrabody or empty plasmid. All types of overexpression were done in 6-well plates on 0.6 x 106 cells and lasted 48 h unless noted otherwise.

[0313] Immunofluorescence

[0314] 0.02 x 106 HEK293T cells were seeded in 250 pL of culture medium in an 8-well Nunc Lab-Tek II plate (No. 1.5 borosilicate glass, Thermo Fisher ScientificTM) that was precoated with 50 pg / mL of poly-D-lysine (Sigma-Aldrich®). After 24 h, transfection was performed as described previously with 0.2 pg of TDP-43 plasmid or empty plasmid and 0.1 pg of scFv plasmid. Following 48 h of overexpression, the cells were fixed in 250 pL of PBS with 4% paraformaldehyde and 2% sucrose for 20 min at RT. The cells were washed 3 times in PBS. The fixed cells were permeabilized by incubating them in 250 pL of 0.2% Triton-X 100 diluted in PBS for 15 min at RT. Then, the cells were saturated in 250 pL of permeabilization buffer supplemented with 10% donkey serum (ab7475, abeam®) for 1 h at RT. 250 pL of permeabilization buffer supplemented with 3% donkey serum, 1,000-fold diluted rabbit anti-TDP-43 primary antibody (12892-1-AP, Proteintech®), and 1,000-fold diluted mouse anti-6xHis primary antibody (66005-1-Ig, Proteintech®) were added to the cells for an incubation of 2 h at RT. Cells were washed 5 times in permeabilization buffer before adding 250 pL of buffer / 3% donkey serum with 500-fold diluted goat anti -rabbit Alexa Fluor® 488 (A-11034, Thermo Fisher ScientificTM) and 500-fold diluted donkey anti-mouse Alexa Fluor® 594 (A-11005, Thermo Fisher ScientificTM) for an incubation of 1 h at RT away from light. After 3 washes in permeabilization buffer and 1 wash in PBS, each well was covered with VECTASHIELD® Antifade Mounting Medium with DAPI (H-1200-10, Vector Laboratories). The wells were air-dried for 48 h at RT protected from light. The cells were visualized with a SP8 confocal microscope (Leica Microsystems®) using a magnification of 60X. Cell viability

[0315] Following 48 h of overexpression, the culture medium was withdrawn and replaced with 1 mL of 0.5 mg / mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich®) diluted in HBSS (Gibco®) that was pre-heated to 37°C. After 30 min of incubation at 37°C with 5% CO2, the medium was discarded and replaced with 500 pL of DMSO (Sigma-Aldrich®), in which the formazan crystals were dissolved. The absorbance was recorded at 570 nm.

[0316] Purification of scFv from BL21(DE3)pLysS

[0317] A pre-culture for a given clone was started in 5 mL of LB containing 1% glucose and 100 pg / mL ampicillin for which the incubation lasted 16 h at 37°C with shaking. 150 pL of pre-culture were added to 15 mL of fresh medium. At an OD of 0.6, 12 mL of culture were added to 500 mL of auto-inducible medium consisting of fresh LB containing 100 pg / mL ampicillin, 1.0 mL of 1 M MgSO4, 10 mL of solution 50x5052 (2.7 M glycerol, 0.14 M glucose, 0.28 M a-lactose monohydrate), and 10 mL of solution 50xM (1.3 M Na2HPO4, 1.3 M KH2PO4, 2.5 M NH4C1, 0.25 M Na2SO4). The culture was incubated at 37°C with shaking for 3 h then moved to 22°C for 16 h. The bacteria were pelleted by centrifugation at 5,000 x g for 10 min at 4°C and resuspended in 20 mL of lysis buffer (PBS pH = 8.0, 1% Brij- 35, 1.0 mM EDTA, 10 mM MgC12, 1.0 mM DTT). To this solution were added 200 pL of 100X Halt Protease Inhibitor Cocktail (Thermo Fisher ScientificTM), 2 pL of Pierce Universal Nuclease (Thermo Fisher ScientificTM), and 100 pL of 50 mg / mL lysosyme. After 15 min of agitation at 4°C, the bacteria were lysed by sonication applying the following parameters: 50% amplitude, 15-second pulse, 15-second rest for a total of 5 cycles. The lysate was centrifuged at 15,000 x g for 20 min at 4°C. The supernatant was combined with 1.0 mL of Ni-NTA resin (Thermo Fisher ScientificTM) equilibrated in lysis buffer with 10 mM imidazole for 1 h at 4°C. The resin was added back to a disposable column and was washed with 20 mL of cold lysis buffer supplemented with 25 mM imidazole. The scFv was eluted by adding cold lysis buffer supplemented with 250 mM imidazole. Fractions containing protein were identified by SDS-PAGE. The pooled eluate was dialyzed in 100 X its volume of MilliQ water supplemented with 1 mM DTT overnight at 4°C. The dialyzed product was quantified by Bradford assay and concentrated to 0.5 - 1.0 mg / mL if necessary. The final product was stored at 4°C. The identity of the scFv was confirmed by Western blot, as described previously.

[0318] Enzyme-linked immunosorbent assay (ELISA) on purified scFv Wells of a Nunc MaxiSorp 96-well plate (Thermo Fisher ScientificTM) were coated overnight at 4°C with 100 pL of commercial human wtTDP-43 (abeam, ab224788) diluted to 1 pg / mL in PBS. Wells were washed 3 times in 0.2% PBST. 200 pL of blocking solution consisting of 0.02% PBST and 5% powdered milk (Regilait) were added to wells and incubated for 2 h at RT. 3 washes were then done, as before. 100 pL of milliQ water containing purified scFv diluted to either 1, 5, or 10 pg / mL were added to corresponding wells and incubated for 1 h at RT. 3 washes were then done, as before. 100 pL of HRP- conjugated mouse anti-6xHis antibody (66005, Proteintech®) diluted 10,000-fold in 0.02% PBST were then added to wells and incubated for 1 h at RT. 5 washes were performed in 0.2% PBST followed by 1 wash in PBS. 100 pL of TMB-ELISA Substrate Solution (Thermo Fisher ScientificTM) were then added for an incubation of 10 - 15 min at RT in the dark. The reaction was halted with 50 pL of 2 M H2SO4. Absorbance was measured at 450 nm using an iMark™ Microplate Absorbance Reader (Bio-Rad).

[0319] TDP-43 solubility

[0320] Following 48 h of overexpression in HEK293T or NSC-34 cells, the medium was removed and cold PBS was added to the wells. The cells in PBS were centrifuged at 900 x g for 5 min at 4°C and resuspended in 200 pL of cold lysis buffer (PBS pH = 8.0, 1% Triton-X 100, 10 mM MgC12, 1 mM DTT) supplemented with IX Halt Protease Inhibitor Cocktail (Thermo Fisher ScientificTM) and Pierce Universal Nuclease (Thermo Fisher ScientificTM). After 30 min at 4°C with agitation, the lysates were centrifuged at 17,000 x g for 20 min at 4°C. The supernatant (soluble fraction) was transferred to a new tube, while the pellet (insoluble fraction) was resuspended in the same volume as for the supernatant of lysis buffer supplemented with 6 M urea. Protein concentration of the crude extract of the lysates was measured by the detergent-compatible Pierce® 660nm Protein Assay reagent (Thermo Fisher Scientific). 10 pg of crude extract, along with equivalent volumes of the insoluble fractions, were migrated by SDS-PAGE, transferred to a PVDF membrane, and prepared for HRP- conjugated chemiluminescence, as described previously. TDP-43 was stained overnight at 4°C by a rabbit primary antibody targeted against the C-terminus (12892-1 -AP, Proteintech®) diluted 5,000-fold. Then, a 1-h incubation at RT was performed with HRP-conjugated goat anti -rabbit secondary antibody (W401B, Promega®) diluted 10,000-fold. The 6xHis tag of the scFv was stained for 1-h at RT by the HRP-conjugated mouse anti-6xHis antibody (66005-1- Ig, Proteintech®) diluted 100,000-fold.

[0321] TDP-43 degradation Following wtTDP-43 and / or scFv overexpression in HEK293T cells, the medium was replaced with OptiMEM (Gibco, Thermo Fisher Scientific) and the cells were treated for 6 hours with 10 pg / mL cycloheximide (SigmaAldrich) with either 300 nM Bafilomycin Al (Merck, SigmaAldrich) or 0.5 pM MG-132 (SigmaAldrich). Lysate extraction and western blots were performed as described previously.

[0322] NF-KB activation

[0323] For the assessment of the NF-KB activation, 50 ug of proteins were loaded on the gel. The active subunit of NF-KB was stained overnight at 4°C by a mouse primary antibody targeting p65 (MAB3026, Sigma-Aldrich) diluted 1,000-fold, followed by 1-h incubation at RT with HRP-conjugated goat anti-mouse secondary antibody (W402B, Promega®). Detection of proteins was achieved using the Enhanced Chemiluminescence (ECL) reagent (Biorad). Loading control was done by normalizing the target protein to the stain-free images of total protein.73

[0324] Energy metabolism analysis

[0325] The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were determined using a Seahorse™ XF96 Flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). Experiments were performed according to the manufacturer’s instructions. Briefly, HEK293T or NSC-34 cells were plated in XF96 cell culture plates precoated with 50 pg / mL poly-D-lysine at 1.0 x 104 cells / well and transfected with 120 ng of TDP-43 plasmid, 120 ng of empty plasmid, and / or 60 ng of scFv plasmid, depending on the conditions. Overexpression was permitted for 48 h. 24 h after transfection, the XF96 sensor cartridges were hydrated with 200 pL of calibrant at pH 7.4 and maintained overnight at 37 °C without CO2. On the day of analysis, the protocol was followed as previously described.40

[0326] Metabolomics analysis

[0327] After 48 h of overexpression in HEK293T and NSC-34 cells, the cells were harvested and washed in ice-cold PBS two times while centrifuging at 200 x g for 2 min at RT to remove the supernatant. The dry cell pellet was frozen at -80°C until the day of analysis. Cells were thawed on ice and resuspended in 150 pL of Me0H:H20 (1 / 1) followed by centrifugation for 5 min at 4°C. Liquid chromatography in tandem with high-performance mass spectrometry (LC-HPMS) was performed as previously described by our group.74, 75

[0328] Data analyses

[0329] Unless otherwise stated, results are shown as mean ± standard deviation (SD). When relevant, the Mann-Whitney non-parametric t-test was performed using Prism v.7.0 (GraphPad Software, San Diego, CA, USA). Results were considered significant with p<0.05. For metabolomic analysis, each overexpression condition was normalized to the empty vector condition and the mean of the normalization ratios were calculated. We selected 10% of the metabolites that increased the most and 10% of the metabolites that decreased the most. A Venn diagram allowed us to identify the common and specific metabolites impacted by the scFv, compared to naive cells. We also normalized within each series to the “TDP condition” and conducted the same process to observe the metabolites impacted by the scFv on TDP -43 overexpression. Moreover, we built the Venn diagrams between the elevated metabolites on scFv-TDP-43 versus scFv-empty vector, and between the decreased metabolites. Finally, among the metabolites impacted by TDP -43 overexpression alone, we selected the ones also impacted by the scFv to see the most relevant ones in our anti-TDP-43 strategy.

[0330] Results:

[0331] Phage Display revealed four distinct anti-TDP-43 clones

[0332] Following four rounds of bio-panning, we observed the highest enrichment of bound phage at round 4. ELISA of retained phage from round 4 revealed 9 clones showing strong positive signal for TDP -43 and not for the control protein, suggesting specific binding to TDP -43. Sanger sequencing (data not shown) of the VH and VL domains from the nine phages revealed 4 distinct scFv clones, named Bl, A2, B6, and D7.

[0333] In silico binding site prediction suggested affinity to pathologically-relevant regions of TDP-43

[0334] The prediction of the binding sites of the scFv required us to generate a pseudo-3D model of TDP-43, which was done by MabSilico (data not shown). The in silico binding site prediction revealed several possible TDP-43 regions as targets for binding by scFv. Specifically, scFv A2 could target the N-terminus, the RRM2 domain, and the hinge 2 region (data not shown). The scFv B6 might bind two regions within the N-terminus, the RRM1, and the RRM2 region (data not shown). As for scFv Bl, it could target the RRM1 domain and the C-terminal end of the prion-like domain (data not shown). Finally, scFv D7 could bind the RRM1 domain and the N-terminal side of the prion-like domain (data not shown).

[0335] Intrabodies Bl and D7 bound wtTDP-43

[0336] Following the transfection of HEK293T cells by plasmids expressing the scFv, only Bl and D7 were successfully expressed. We therefore retained these 2 intrabodies in subsequent experiments. The overexpression of wtTDP-43 resulted in its cytoplasmic accumulation (data not shown). Additionally, the intrabodies were found throughout the cytoplasm, and seem to colocalize with cytoplasmic TDP-43, particularly with aggregated material (data not shown), contrary to the control intrabody 13R4, directed against E. coli 0- galactosidase.

[0337] To further verify whether intrabodies Bl and D7 interacted with human wtTDP-43, we conducted an indirect ELISA on commercial TDP-43 using purified Bl, D7, and the control intrabody, 13R4. The control wells containing increasing amounts of 13R4 showed no significant binding to TDP-43. However relative to equivalent quantity of the control scFv, the wells incubated with increasing amounts of Bl and D7 exhibited a significant dosedependent increase in absorbance (Fig. 1). Upon incubation with 5 pg of Bl and D7, there was a 5- and 2-fold increase in the signal, respectively (Bl, p = 0.004; D7, p = 0.03). Upon incubation with 10 pg of Bl and D7, there was 3- and 2-fold increase in the signal, respectively (Bl, p = 0.004; D7, p = 0.005). These results suggest that only Bl and D7 physically interact with TDP-43.

[0338] Intrabodies Bl and D7 had no effect on cellular viability

[0339] The MTT reduction assay revealed that the overexpression of the intrabodies alone in HEK293T cells did not result in any significant cellular toxicity (Fig. 2). On the other hand, wtTDP-43 overexpression decreased cellular viability to 30-40% of the control (empty vector transfection). The co-overexpression of the intrabodies with TDP-43 did not significantly change the TDP-43 -induced toxicity (data not shown).

[0340] D7 decreased the amount of the insoluble 35-kDa fragment

[0341] We quantified by Western blot the amount of full-length TDP-43 (TDP-43), the 35- kDa C-terminal fragment (TDP-35 CTF), and the 25-kDa C-terminal fragment (TDP-25 CTF) in the total lysate and insoluble fractions of HEK293T cell lysates. Upon transfection of cells with the plasmid overexpressing TDP-43, we observed a clear increase in the amount of TDP- 43 and TDP-35 CTF, as well as the appearance of TDP-25 CTF compared to control (empty vector; data not shown). The co-overexpression of the scFv had no effect on the quantity of each TDP-43 form in the total lysate compared to TDP-43 overexpression alone (data not shown). In the insoluble fraction, we observed TDP-43 and TDP-35 CTF but not TDP-25 CTF (data not shown). The quantity of insoluble TDP-43 did not change upon overexpression of D7 in HEK293T cells but the amount of insoluble TDP-35 CTF was significantly decreased (Fig. 3A p = 0.0286). Interestingly, there was a significant increase in the amount of insoluble TDP-35 CTF when the cells co-overexpressing TDP-43 and D7 were also treated with proteasome inhibitor MG-132 (Fig. 3B, p = 0.0286). This effect was not observed with the autophagy inhibitor Bafilomycin Al. In the motor neuron-like NSC-34 mouse cell line, analysis of the total lysate suggested that the transfection of TDP-43 plasmid did not yield a higher expression of its full-length form compared to cells transfected with the empty vector and TDP-25 CTF was undetected. Nevertheless, the TDP-35 CTF was overproduced (data not shown). Upon co-transfection with scFv plasmids, the total levels of TDP-43 and TDP-35 CTF remained unchanged (data not shown). As for the insoluble fraction of the lysates, the TDP-25 form appeared after TDP-43 transfection but was also lightly expressed in the vehicle condition (data not shown), which was not observed in the HEK293T cell line. Similar to the HEK293T cells, the insoluble form of TDP-35 CTF seemed to be lowered by D7, although this did not achieve significance (p = 0.1, data not shown).

[0342] Bl decreased TDP-43-induced activation of NF-KB in NSC-34 cells

[0343] Transfection by TDP-43 plasmid triggered the activation of NF-KB in NSC-34 cells (depicted as an increase in the active subunit of NF-KB, p65; Fig.4). Upon co-transfection with Bl, the level of p65 was significantly decreased and returned to baseline levels (Fig.4, p = 0.02). This decrease was not observed with the control intrabody or D7. These results suggest that Bl prevented NF-KB activation associated with TDP-43 overexpression.

[0344] Intrabodies altered cellular bioenergetics and the metabolome in different ways depending on cell type

[0345] As ALS alters many aspects of motor neuron metabolism, we turned to the metabolism of our cell models to evaluate the effects of Bl and D7. In HEK293T cells, while we observed a slight decrease in ATP production upon co-overexpression with intrabodies, including the 13R4 control (data not shown), in NSC-34 cells we noticed a slight increase in the mitochondrial ATP production rate over the glycolytic production rate (data not shown). Overall, we did not observe significant changes in ATP production upon intrabody overexpression in both cell lines.

[0346] We next analyzed the metabolome profiles of HEK293T and NSC-34 cells transfected either with TDP-43 plasmid alone or co-transfected with TDP-43 plasmid and each intrabody plasmid (Bl, D7, 13R4) to evaluate i) metabolic effect of scFv administration, ii) metabolic modification associated to TDP-43 overexpression, and iii) correction of TDP-43 -linked metabolic changes by scFv. We identified a total of 220 metabolites in HEK293T cells. 10% of the metabolites were upregulated and downregulated. Compared to cells transfected with the empty vector, the majority of the metabolites impacted seem to be common to all three intrabodies (data not shown). They represent the global effect of the overexpression of an scFv on naive cells with only a few metabolites specific to both or either Bl and D7. The same was observed when comparing the conditions of TDP-43 overexpression and TDP- 43 / scFv co-transfection, with a majority of metabolites being common to the three intrabodies (data not shown). The metabolic effects of Bl and D7 also seemed to vary depending on whether TDP-43 was overexpressed or not (data not shown), suggesting different effects when TDP-43 is pathological. We were able to identify three metabolites (L- palmitoylcamitine, PC(36:4), 4-hydroxy-phenylglycine) that were altered when TDP-43 was overexpressed and that returned to baseline levels (empty vector condition) when the scFv Bl or D7 were co-expressed (Fig.5).

[0347] In the analysis performed with NSC-34 cells, we detected and identified 192 metabolites This time, Bl and D7 seemed to exert more specific alterations, as few of the impacted metabolites were common with the control intrabody in both naive cells and cells overexpressing TDP-43 (data not shown). Distinct effects were also observed depending on whether TDP-43 was overexpressed or not, especially with D7 (data not shown). Among the metabolites that varied with TDP-43 overexpression (data not shown), six of them (hippuric acid, nicotinamide mononucleotide, PC(37:4), glyceraldehy dr-3 -phosphate diethyl acetal, O- phospho-L-serine, and caprylic acid) seemed to return to baseline levels when either Bl or D7 were co-expressed (Fig.6).

[0348] In conclusion, we present two scFv molecules, D7 and Bl, that alter different aspects of TDP-43 pathology. While they do not seem to alter cell viability when co-expressed with TDP-43, they could serve as important tools to help decipher more specifically the function of TDP-35 and the role of TDP-43 in inflammation, respectively. In addition, given their engineerability, the scFv could be modified or combined to enhance their therapeutic potential. Finally, targeting cellular alterations linked to TDP-43 proteinopathy for both therapeutic or diagnostic purposes will be essential for improving the quality of life of ALS patients in the future.

[0349] REFERENCES:

[0350] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. Intrabodies scFv Bl, scFv D7, scFv A2 and scFv B6 targeting wildtype Trans-active response DNA-binding protein-43 (TDP-43) wherein: scFv Bl has:(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 2; a H-CDR2 having a sequence set forth as SEQ ID NO: 3; a H-CDR3 having a sequence set forth as SEQ ID NO: 4;(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 6; a L-CDR2 having a sequence set forth as SEQ ID NO: 7; a L-CDR3 having a sequence set forth as SEQ ID NO: 8; scFv D7 has:(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 10; a H-CDR2 having a sequence set forth as SEQ ID NO: 11; a H-CDR3 having a sequence set forth as SEQ ID NO: 12;(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 14; a L-CDR2 having a sequence set forth as SEQ ID NO: 15; a L-CDR3 having a sequence set forth as SEQ ID NO: 16; scFv A2 has:(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 18; a H-CDR2 having a sequence set forth as SEQ ID NO: 19; a H-CDR3 having a sequence set forth as SEQ ID NO: 20;(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 22; a L-CDR2 having a sequence set forth as SEQ ID NO: 23;a L-CDR3 having a sequence set forth as SEQ ID NO: 24; scFV B6 has:(a) a heavy chain wherein the variable domain comprises: a H-CDR1 having a sequence set forth as SEQ ID NO: 26; a H-CDR2 having a sequence set forth as SEQ ID NO: 27; a H-CDR3 having a sequence set forth as SEQ ID NO: 28;(b) a light chain wherein the variable domain comprises : a L-CDR1 having a sequence set forth as SEQ ID NO: 30; a L-CDR2 having a sequence set forth as SEQ ID NO: 31; a L-CDR3 having a sequence set forth as SEQ ID NO: 32.

2. The intrabodies targeting wildtype TDP-43 of claim 1 having a heavy chain comprising i) H-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) the H-CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 iii) the H-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6 and a light chain comprising i) L-CDR1 of scFV Bl, scFV D7, scFV A2 or scFV B6, ii) L-CDR2 of scFV Bl, scFV D7, scFV A2 or scFV B6 and iii) L-CDR3 of scFV Bl, scFV D7, scFV A2 or scFV B6.

3. The intrabodies targeting wildtype TDP-43 of claim 1 having a heavy chain identical to SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25 and a light chain identical to SEQ ID NO: 5 SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

4. The intrabodies targeting wildtype TDP-43 of claim 1 having a heavy chain having at least 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 1, SEQ ID NO: 9, SEQ ID NO: 17 or SEQ ID NO: 25 and a light chain having at least 70, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% of identity with SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 29.

5. The intrabodies targeting wildtype TDP-43 of claim 1 is a chimeric antibody.

6. The intrabodies targeting wildtype TDP-43of claim 1 is a humanized antibody which comprises the CDRs of scFv Bl, scFv D7, scFv A2 and scFv B6 antibody.

7. The intrabodies targeting wildtype TDP-43 of claim 1 is selected from the group consisting of Fab, F(ab')2, Fab' and scFv.

8. A nucleic acid molecule encoding the intrabodies targeting wildtype TDP-43 of claim 1.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell which has been transfected, infected or transformed by the nucleic acid of claim 8 and / or the vector of claim 9.

11. The intrabodies targeting wildtype TDP-43 of claim 1 for use as a diagnostic tool.

12. The intrabodies targeting wildtype TDP-43 of claim 1 and claim 11 are conjugated with a radiolabel for PET imaging.

13. The intrabodies targeting wildtype TDP-43 of claim 1 for use as a medicament.

14. The intrabodies targeting wildtype TDP-43 of claim 1 for use as a medicament to treat TDP-43 proteinopathies.

15. The intrabodies targeting wildtype TDP-43 of claim 14 for use wherein the TDP-43 proteinopathy is selected from the group consisting of but not limited to: amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD-TDP), primary lateral sclerosis, progressive muscular atrophy, and limbic-predominant age-related TDP-43 encephalopathy (LATE), Alzheimer's disease, Parkinson's disease, multiple system atrophy, or progressive supranuclear palsy, post-traumatic diseases (chronic traumatic encephalopathy), neoplastic (pilocytic astrocytoma) or post-infectious (postencephalitic parkinsonism).

16. The intrabodies targeting wildtype TDP-43 of claim 1 for use is delivered alone or in association with liposomes, nanoparticles, spion and / or a viral vector.

17. The i) intrabodies targeting wildtype TDP-43 of claim 1 and ii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of TDP-43 proteinopathies.

18. The intrabodies targeting wildtype TDP-43 for use of claim 17 wherein the classical treatment is selected from the group consisting of but not limited to : Riluzole (Rilutek or Tiglutik™); Edaravone (Radicava™), RELYVRIO ™, and Nuedexta™.

19. A pharmaceutical composition comprising the intrabodies of claim 1.

20. A kit comprising at least one antibody according to claim 1 and at least one imaging label, preferably at least one antibody anti-TDP43, and, optionally, instructions for associating said antibody anti-TDP43 and said imaging label.

21. A method of treatment of TDP-43 proteinopathy in a subject in need thereof comprising administering to the subject intrabodies targeting wildtype TDP-43 according to claim 1.

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