Anti-TDP-43 binding molecules and uses thereof

Abstract

The present invention is in the field of trans-activation responsive DNA binding proteins (TARDB or also referred to as TDP-43) having a molecular weight of 43 kDa. The present invention relates to TDP-43 specific binding molecules, in particular to anti-TDP-43 antibodies or antigen binding fragments or derivatives thereof, and uses thereof. The present invention provides means and methods for diagnosing, preventing, ameliorating, and / or treating diseases, disorders, and / or abnormalities associated with TDP-43 aggregates, including, but not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and edge dominant age-related TDP-43 encephalopathy (LATE).

Description

TECHNICAL FIELD

[0001] The present invention is in the field of trans-activation response DNA binding protein (TARDB or also TDP-43) having a molecular weight of 43 kDa. The present invention relates to TDP-43 specific binding molecules, in particular anti-TDP-43 antibodies or antigen-binding fragments or derivatives thereof and uses thereof. The present invention provides means and methods for diagnosing, preventing, alleviating and / or treating diseases, disorders and / or abnormalities associated with TDP-43, in particular with TDP-43 aggregates, or TDP-43 proteinopathies, including but not limited to Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), Chronic Traumatic Encephalopathy (CTE) and limbic-predominant age-related TDP-43 encephalopathy (LATE). BACKGROUND

[0002] Age-related encephalopathies characterized by pathological aggregation of proteins in the central nervous system (CNS) (proteinopathies) and peripheral organs are among the leading causes of disability and mortality worldwide. The best characterized protein forming aggregates is beta amyloid in Alzheimer’s disease and related disorders. Other disease-associated proteins that tend to aggregate include but are not limited to tau, alpha-synuclein (aSyn, a-syn), huntingtin, fused in sarcoma (FUS), dipeptide repeat proteins (DPRs) produced by non-canonical translation of C9orf72 repeat expansions, superoxide dismutase 1 (SOD1) and TDP-43. Diseases involving TDP-43 aggregates are generally listed as TDP-43 proteinopathies, including but not limited to ALS and FTD.

[0003] I. Introduction of TDP-43

[0004] The transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) is a protein of 414 amino acids encoded by the TARDBP gene on chromosome 1 p36.2 (ALS10). TARDBP is composed of six exons (exon 1 is non-coding; exons 2 to 6 are protein-coding). TDP-43 belongs to the heterogeneous ribonucleoprotein (hnRNP) RNA-binding protein family (Wang et al., Trends in Molecular Medicine Vol. 14 No. 11, 2008, 479-485; Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64). TDP-43 comprises five functional domains (Warraich et al., The International Journal of Biochemistry & Cell Biology 42 (2010) 1606-1609, Fig. 1): two RNA recognition motifs (RRM1 and RRM2) with two highly conserved hexanucleotide-binding regions (RNP2 and RNP1); a nuclear export signal (NES) and a nuclear localization signal (NLS), enabling it to shuttle between the nucleus and the cytoplasm, transporting bound mRNA; and a glycine-rich domain at the C-terminus, which mediates protein-protein interactions. TDP-43 is involved in multiple aspects of RNA processing, including transcription, splicing, transport and stabilization (Buratti and Baralle, FEBS Journal 277 (2010) 2268-2281). TDP-43 is a highly conserved, ubiquitously expressed and tightly autoregulated protein, which constantly shuttles between the nucleus and the cytoplasm, but is mainly localized to the nucleus.In 2006, TDP-43 was identified as the protein accumulating in the vast majority of cases of frontotemporal lobar degeneration (FTLD) with tau-negative, ubiquitin-positive inclusions (hereafter FTLD-TDP), as well as in most cases of amyotrophic lateral sclerosis (ALS) (Arai et al., Biochemical and Biophysical Research Communications 351 (2006) 602-611; Neumann et al., Science 314, (2006), 130-133).

[0005] Thirty-eight TDP-43 negative dominant mutations have been identified in sporadic and familial ALS patients as well as in patients with hereditary FTD, which are mainly located in the glycine-rich domain (Lagier-Tourenne and Cleveland, Cell 136, 2009, 1001-1004, Figure 1). TDP-43 is intrinsically aggregation-prone, as shown by sedimentation assays, and this propensity is further increased by some ALS-associated TARDBP mutations (Ticozzi et al., CNS Neurol. Disord. Drug Targets. 2010, 9(3), 285-296.) linking TDP-43 aggregation to clinical disease manifestation.

[0006] II. TDP-43 in neurodegeneration

[0007] TDP-43 aggregates have been identified in an increasing number of neurodegenerative disorders (Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64), including but not limited to: frontotemporal dementia (FTD, e.g. sporadic or familial, with or without motor-neuron disease (MND), with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosine-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), Argyrophilic grain disease, Pick’s disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), Nonfluent Variant Primary Progressive Aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g. sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer’s disease (AD, including sporadic and familial forms of AD), Down syndrome, Familial British dementia, polyglutamine disease (Huntington’s disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado Joseph Disease)), hippocampal sclerosis dementia and myopathies (sporadic inclusion body myositis, with valosine-containing protein (VCP);inclusion body myopathy associated with Paget bone disease and frontotemporal dementia), oculopharyngeal dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES)), Traumatic Brain Injury (TBI), Dementia with Lewy Body (DLB) or Parkinson’s Disease (PD). The term LATE is intended to encompass several previously used names associated with TDP-43 proteinopathy that can be associated with cognitive impairment, including hippocampal sclerosis, age-related hippocampal sclerosis, hippocampal sclerosis dementia, cerebral age-related TDP-43 and sclerosis (CARTS), and TDP-43 pathology in the elderly (for review see Kuslansky et al., 2004; Lippa and Dickson, 2004; Nelson et al., 2013, 2016b; Dutra et al., 2015).

[0008] Aggregated TDP-43 from patient brains shows a number of abnormal modifications, including hyperphosphorylation, ubiquitination, acetylation, and C-terminal fragments cleaved by proteolysis (Arai et al., Biochemical and Biophysical Research Communications 351 (2006) 602-611; Neumann et al., Science 314, (2006), 130-133; Neumann et al., Acta Neuropathol. (2009) 117: 137-149; Hasegawa et al., (2008) Annals of Neurology Vol 64 No 1, 60-70; Cohen et al., Nat Commun. 6:5845, 2015). The TDP-43 aggregation mechanism and the TDP-43 species involved in aggregation are not fully understood. However, there is evidence for the importance of the C-terminal region of TDP-43 in the pathological mechanism. The C-terminal domain, known as the low complexity domain (LCD), is intrinsically disordered and contains regions rich in glycine, hydrophobic residues, glutamine, and asparagine (Afroz et al., 2019). While this region of TDP-43 has intrinsic properties to form higher order physiological assemblies (e.g. stress granules) (Gasset-Rosa et al., 2019), in disease, irreversible inter- and intra-molecular interactions within this region can lead to pathological aggregates (Gasset-Rosa et al., 2019). Indeed, recent structural and biochemical studies have shown that, in the brains of ALS and FTD patients, a region within the C-terminal region, composed of amino acids 272-360 of TDP-43, adopts a stable protease-resistant amyloid core structure (Arseni et al., 2021; Arseni et al., 2023). Furthermore, disease-specific proteolytic cleavage that exposes this amyloid core shows a further enhanced seeding activity that is important for templated aggregation (Kumar et al., 2023). In addition to the post-translational modifications described above, such proteolytic processing of TDP-43 and its enrichment in patient brains is a disease-specific pathological feature.

[0009] Another characteristic feature of TDP-43 pathology is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. The hallmark lesions of FTLD-TDP are neuronal cytoplasmic inclusions and glial cytoplasmic inclusions (NCI and GCI, respectively) and dystrophic neurites (DN), which are immunoreactive for TDP-43 as well as ubiquitin and p62, but negative for other neurodegenerative disease-associated proteins. Differences in the morphology of the inclusions and their organizational distribution are associated with specific mutations and / or clinical presentation. To date, four types of TDP-43 pathology have been described by histological classification (Mackenzie and Neumann, J. Neurochem. (2016) 138 (Supplement 1), 54-70). FTLD-TDP type A cases are characterized by a large number of short dystrophic neurites (DN) and compact, oval or crescent-shaped NCI, mainly in the neocortical layer II (Mackenzie et al., 2016 J. Neurochem. 138 (Supplement 1), 54-70, Figure 2 f). This pathological profile is typically clinically seen in cases of behavioral variant frontotemporal dementia (bvFTD) or nonfluent / agrammatic primary progressive aphasia (nfvPPA) and is associated with progranulin (GRN) mutations. Type B cases show a moderate number of compact or granular NCI in both superficial and deep cortical layers with relatively fewer DN and NII (neuronal intranuclear inclusion; Mackenzie et al., 2016 J. Neurochem. 138 (Supplement 1), 54-70, Figure 2 g). Most cases with concurrent FTD and ALS symptoms are found to have FTLD-TDP type B pathology. Type C cases have a large number of long and curved neurites, mainly in the superficial cortical layers, with few or no NCI (Mackenzie et al., 2016 J. Neurochem. 138 (Supplement 1), 54-70, Figure 2 j). This pathology is specifically found in cases presenting with semantic variant primary progressive aphasia (svPPA). Type D FTLD-TDP shows abundant dystrophic neurites (DN) and neuronal intranuclear inclusions (NII) in the neocortical layers with only sparse NCI (Mackenzie et al., 2016 J. Neurochem. 138 (Supplement 1), 54-70, Figure 2k) Type E is characterized by granulofilamentous neuronal inclusion (GFNI) and very fine punctate neuropil aggregates in addition to curvilinear oligodendroglial inclusions in white matter (Edward B. Lee et al., Acta Neuropathol. 2017 July; 134(1): 65-78.). This pattern of pathology is only found in cases of VCP associated with inclusion body myositis.

[0010] III. TDP-43 in FTD

[0011] Frontotemporal dementia (FTD) is a clinical term encompassing a spectrum of disorders based on degeneration of the frontal and temporal lobes - a pathological feature called frontotemporal lobar degeneration (FTLD). FTD is the second most common cause of early onset dementia in the age group under 65 years (Le Ber, Revue Neurologique 169 (2013) 811-819). FTD manifests as several syndromes including bvFTD characterized by changes in personality and behavior; semantic dementia (SD) and progressive nonfluent aphasia (PNFA) characterized by changes in language function; corticobasal syndrome (CBS), progressive supranuclear palsy syndrome and motor neuron disease (FTD-MND) characterized by motor dysfunction. The clinical diagnosis of these syndromes is complex and the final conclusion can only be made by performing post-mortem histopathological analysis to detect aggregated proteins and determine the affected brain regions. In terms of pathological protein inclusions, about 45% of cases show pathological accumulation of misfolded tau, 45% of cases have pathological TDP-43 and a smaller subset have aggregates of FUS and other proteins.

[0012] IV. TDP-43 in ALS

[0013] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the premature loss of upper and lower motor neurons. The progression of ALS is characterized by fatal paralysis and respiratory failure, with a disease course from diagnosis to death of 1 to 5 years. In most sporadic ALS cases, this neuropathology is characterized by an abnormal cytoplasmic accumulation of TDP-43 in neurons and glial cells of the primary motor cortex, brainstem motor nuclei, spinal cord, and associated white matter tracts. ALS with dementia involves the accumulation of TDP-43 in the extramotor neocortex and hippocampus. The role of TDP-43 phosphorylation in ALS patients has been explored with the help of antibodies that specifically bind to phosphorylated TDP-43 in nuclear and cytoplasmic inclusions, with amino acids S379, S403, S404, S409, S410 being the main sites of TDP-43 phosphorylation (Hasegawa et al., Ann Neurol 2008; 64:60-70; Neumann et al., Acta Neuropathol (2009) 117: 137-149).

[0014] VI. TDP-43 in AD and other diseases

[0015] TDP-43 pathology occurs in up to 57% of patients with Alzheimer’s disease (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450; McAleese et al., Brain Pathol. 2017 Jul; 27(4):472-479). TDP-43 aggregation is associated with the age of the patient and is associated with cognitive decline, memory loss, and medial temporal lobe atrophy in AD. Apparently, in AD, TDP-43 represents a second or independent pathology with an overlapping brain distribution with beta-amyloid and tau pathology in the medial temporal lobe. Pathological TDP-43 follows a regular progressive deposition pattern, which has been described by the so-called staging scheme for TDP-43 in AD (TAD): TDP-43 is first deposited in the amygdala (stage I), then in the hippocampus, limbic, temporal, and finally frontostriatum (stage V) (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450).

[0016] VI. TDP-43 spreading

[0017] While ALS and FTD onset and initial symptoms vary significantly between patients, a common feature of disease progression is the spread of pathology from the initial lesioned area to anatomically connected brain regions. The progressive worsening of symptoms can be explained by the progressive spread of TDP-43 pathology. TDP-43 pathology in the brain of ALS patients shows a four-stage process of spread and is thought to occur through synaptically transmitted propagation using anterograde axonal transport through corticofugal axons (Brettschneider et al., Ann Neurol. 2013 July;74(l):20-38.). Recent experimental evidence supports the hypothesis of protein propagation in neuronal tissue by the prion-like mechanism for amyloid-beta, tau, alpha-synuclein and TDP-43 (Hasegawa et al., 2017), with different starting points and topographical spread patterns for the four proteins (Brettschneider J et al., Nature Rev. Neuroscience, 2015, 109). A common disease unifying mechanism is thought to be the cell-to-cell spread of pathological protein aggregates. This mechanism consists of the release of aggregates from diseased cells, their uptake by naive cells, and the seeding of pathological protein conformations by templating conformational changes of endogenous proteins. Pathological TDP-43 capable of inducing physiological (i.e., non-pathological TDP-43) aggregation is defined as TDP-43 with seeding capacity. In fact, TDP-43 has been found to misfold and aggregate into seeds that are propagating agents with the ability to initiate de novo misfolding. This “prion-like” paradigm is suspected to be one of the key factors in disease development.

[0018] TDP-43 intercellular spreading has been studied at the molecular level in a few in vitro models, where insoluble TDP-43 preparations from patient brains were able to induce intracellular aggregate formation in recipient cells (Nonaka et al., Cell Reports 4 (2013), 124-134; Feiler et al., 2015; Porta et al., Nat. Comm., 2018). Moreover, intracellular TDP-43 aggregates were observed to be released in association with exosomes before spreading to the next cell (Nonaka et al., Cell Reports 4 (2013, 124-134)). Similarly, adenoviral transduced TDP-43 expression leads to cytoplasmic aggregates that are phosphorylated, ubiquitinated and more importantly act as seeds initiating intercellular spreading (Ishii et al., PLoS ONE 12(6): e0179375, 2017). Patient-derived pathological TDP-43 can lead to widespread deposition of endogenous TDP-43 after intracranial inoculation into transgenic mice and wild-type mice (Porta et al., Nat. Comm., 2018). The presence of TDP-43 seeding-competent material in CSF or ALS patients was recently confirmed using a TDP-43 seed amplification assay (Audrain et al., 2023). TDP-43 mAbs targeting the C-terminal domain were able to neutralize these seeding materials (Audrain et al., 2023).

[0019] VII. Prevention and treatment of TDP-43 proteinopathies

[0020] TDP-43 aggregation and pathological spreading are hallmarks of ALS and FTD, currently incurable fatal diseases. Therefore, new approaches are needed to treat and prevent TDP-43 proteinopathies. Mutations in TDP-43 are associated with familial cases of ALS and FTD, providing a causal link between TDP-43 misfolding and disease progression.

[0021] VIII. Diagnosis of TDP-43 proteinopathies

[0022] Diagnosis of FTD based on clinical presentation is insufficient, as clinical presentation can overlap with other diseases, especially in early stages.

[0023] Many approaches aim to develop biomarkers of biochemistry to distinguish between different types of FTD pathology. The development of antibodies against different conformations of TDP-43 can allow the generation of more sensitive and specific diagnostic tools. In parallel to biomarkers of biochemistry, the development of imaging biomarkers can enable early and specific detection of pathology in TDP-43 proteinopathies. The ability to image TDP-43 deposition in the brain can be a major achievement for the diagnosis and drug development of TDP-43 proteinopathies. Such detection can be achieved using cell-permeable antibody fragments.

[0024] The earliest event in neurodegenerative diseases based on misfolding of different proteins is the acquisition of an alternative conformation that renders the protein toxic. In addition, this misfolded conformation can self-propagate by recruiting endogenous normal proteins into the misfolded conformation, as a basis for the observed spread through affected tissues.

[0025] To develop antibodies against different conformational states of a given protein, supramolecular antigenic constructs were designed in which the conformation of the presented antigen is controlled to generate conformation-specific antibodies against a given target in a specific conformational state (WO2012 / 055933 and WO2012 / 020124). Conformation-specific antibodies provide many advantages as they can distinguish between the disease-related conformation of these proteins and the functional endogenous conformation. This approach provides many advantages in therapeutic applications as such antibodies are less likely to be attracted by the normal conformation of the target while targeting the misfolded disease-related isoform of the protein. Similarly, in diagnostic applications, such antibodies only recognize the disease-related structural state of the protein, which is essential for the development of sensitive and specific diagnostics.

[0026] The use of TDP-43-based biomarkers in TDP-43 proteinopathies is still to be established. Such evaluation is hampered, in part, by the lack of high-affinity antibodies that can be used in suitable immunoassays to quantify pathological TDP-43 in biological fluids (Feneberg et al., Molecular Neurobiology, 2018).

[0027] Therefore, there is a clear need for biomarkers that are able to detect, in particular in human samples, misfolded aggregated TDP-43 and non-aggregated physiological TDP-43 for the diagnosis of different types of TDP-43 proteinopathies and / or for monitoring the efficacy of therapeutic drugs for the treatment of diseases, disorders and abnormalities related to TDP-43, in particular to TDP-43 aggregates, or TDP-43 proteinopathies.

[0028] TDP-43 proteinopathies are defined as a group of neurodegenerative diseases characterized by pathological TDP-43.

[0029] IX. Prior Art

[0030] Patent application WO2008 / 151055 discloses methods and materials for using the levels of TDP-43 polypeptides and / or TDP-43 polypeptide cleavage products (e.g., 25 kD and 35 kD TDP-43 polypeptide cleavage products) in a biological fluid to determine whether a mammal has a neurodegenerative disease.

[0031] Patent application WO2013 / 061163 discloses TDP-43 specific binding molecules comprising polypeptides, e.g., human antibodies and fragments, derivatives, and variants thereof.

[0032] Patent application WO2020 / 234473 discloses specific binding molecules comprising polypeptides, e.g., murine antibodies or antigen-binding fragments thereof.

[0033] Patent application WO2022 / 034228 discloses TDP-43 specific binding molecules comprising polypeptides, e.g., humanized antibodies or antigen-binding fragments thereof.

[0034] In view of the foregoing, there is a need for anti-TDP-43 binding molecules that bind both misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43, particularly human TDP-43 (SEQ ID NO: 1). SUMMARY

[0035] Currently, no approved treatment for the treatment and / or prevention of TDP-43 associated diseases is available on the market. Therefore, there is an urgent need to identify new treatments that can treat and / or prevent these diseases. Accordingly, the present invention relates to binding molecules, in particular antibodies or antigen binding fragments thereof, that specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43. In the present invention, misfolded TDP-43 includes misfolded monomeric and / or misfolded oligomeric and / or misfolded aggregated and / or post-translationally modified and / or misfolded truncated TDP-43. Post-translationally modified TDP-43 comprises phosphorylated, ubiquitinated, acetylated, sumoylated and / or methylated TDP-43. Physiological TDP-43 includes soluble nuclear TDP-43. It is shown herein that the binding molecules of the present invention are capable of binding pathological TDP-43, including TDP-43 aggregates and phosphorylated TDP-43. Accordingly, the present invention provides binding molecules, in particular antibodies or antigen binding fragments thereof, that specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43. Such binding molecules are referred to herein as "pan-TDP-43" binding molecules, in particular pan-TDP-43 antibodies. As explained herein, the TDP-43 binding molecules of the present invention can bind misfolded aggregated TDP-43 and non-aggregated physiological TDP-43 equally or preferentially bind one over the other upon specific binding to both types of TDP-43. The present invention also provides binding molecules, in particular antibodies or antigen binding fragments thereof, for use in the prevention, alleviation, treatment and / or diagnosis of diseases, disorders and abnormalities associated with TDP-43, in particular TDP-43 aggregates, or TDP-43 proteinopathies. The present invention also provides binding molecules, in particular antibodies or antigen binding fragments thereof, for use in the detection and / or understanding (i.e. identification) of specific pathological types that cause neurodegeneration. The use as diagnostic biomarker, enabling more effective and precise subject selection for longitudinal monitoring in clinical studies, supporting the development of new treatments for TDP-43 proteinopathies is envisaged.

[0036] The present invention also provides TDP-43 binding molecules, in particular antibodies or antigen binding fragments thereof, as a medicament (therapeutic agent).

[0037] Without wishing to be bound by any particular theory, the present application was developed based on the assumption that modified conformation-specific antigenic peptides and peptide fragments derived from TDP-43 protein or the entire TDP-43 protein and antibodies obtainable by or using said peptides or fragments or the entire TDP-43 protein as antigens block TDP-43 cell-to-cell propagation and / or disaggregate TDP-43 aggregates and / or block TDP-43 seeding and / or neutralize TDP-43 with seeding capacity and / or inhibit aggregation of TDP-43 protein or fragments thereof and / or enhance TDP-43 clearance. The binding molecules of the present application, in particular polypeptides, more particularly antibodies or antigen-binding fragments thereof, bind to misfolded aggregated TDP-43, in particular to cytoplasmic misfolded and extracellular misfolded TDP-43. The humanized binding molecules of the present application, in particular polypeptides, more particularly antibodies or antigen-binding fragments thereof, bind to full-length TDP-43 and / or truncated TDP-43. In one embodiment, the binding molecules of the present application, in particular polypeptides, more particularly antibodies or antigen-binding fragments thereof, specifically bind to cytoplasmic misfolded TDP-43. In one embodiment, the TDP-43 binding molecules of the present application, in particular antibodies or antigen-binding fragments thereof, bind to and neutralize TDP-43 with seeding capacity. In one embodiment, the TDP-43 binding molecules of the present application, in particular antibodies or antigen-binding fragments thereof, bind to extracellular and / or aggregated TDP-43 and enhance its clearance by immune cells such as microglia via antibody-dependent cellular phagocytosis (ADCP).

[0038] Misfolded aggregated or pathologically relevant TDP-43 consists of TDP-43 protein that has lost its normal folding, i.e. is misfolded, and localization. Misfolded aggregated TDP-43 can be found in preinclusion bodies, as well as in neuronal cytoplasmic inclusions and glial cytoplasmic inclusions (NCI and GCI, respectively), neuronal nuclear inclusions (NII) and dystrophic neurites (DN), which are immunoreactive for TDP-43.

[0039] Non-aggregated physiological TDP-43 is the physiologically functional TDP-43 protein that is mainly located in the nucleus and shuttles to the cytoplasm in a state that enables it to display its desired functions in the cellular environment in vivo.

[0040] The TDP-43 binding molecules of the present application, in particular anti-TDP-43 antibodies or antigen-binding fragments thereof, unexpectedly have at least one, preferably two, more preferably three, even more preferably four, still more preferably five, still even more preferably six, still even more preferably seven, most preferably all eight of the following characteristics:

[0041] - blocking TDP-43 intercellular propagation;

[0042] - disaggregating TDP-43 aggregates;

[0043] - inhibiting aggregation of TDP-43 protein or fragments thereof;

[0044] - blocking TDP-43 seeding;

[0045] - neutralizing TDP-43 with seeding capacity;

[0046] - blocking TDP-43 spreading;

[0047] - enhancing TDP-43 clearance; and

[0048] - reducing levels of phosphorylated TDP-43 in vivo.

[0049] Independently of one, two, three, four, five, six, seven or eight of the above-listed features, the anti-TDP-43 binding molecules, preferably anti-TDP-43 antibodies or antigen-binding fragments thereof, of the present application can reduce levels of phosphorylated TDP-43 in the brain and / or ameliorate / inhibit / reduce the formation of TDP-43 pathology in TDP-43 proteinopathy in vivo models and, more importantly, in patients suffering from TDP-43 pathological conditions.

[0050] The anti-TDP-43 binding molecules bind to an epitope within amino acids 304 to 414 of human TDP-43 (SEQ ID NO: 1). The anti-TDP-43 binding molecules bind to an epitope comprising, consisting essentially of, or consisting of amino acid residues 304 to 313, 356 to 361, or 397 to 407 of human TDP-43 (SEQ ID NO: 1). Alternatively, the anti-TDP-43 binding molecules can bind to an epitope within amino acid residues 396 to 414 of human TDP-43 (SEQ ID NO: 1).

[0051] According to the present application, there are provided TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0052] a. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or

[0053] b. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; or

[0054] c. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH-CDR3 comprising the amino acid sequence PC (Pro-Cys); and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0055] d. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or

[0056] e. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0057] The present application especially further relates to (i) immunoconjugates comprising a TDP-43 binding molecule, (ii) labeled antibodies comprising a TDP-43 binding molecule, (iii) pharmaceutical compositions comprising a TDP-43 binding molecule and a pharmaceutically acceptable carrier and / or excipient and / or diluent, (iv) TDP-43 binding molecules for human or veterinary medical use, (v) TDP-43 binding molecules for use in the prevention, alleviation, treatment of a TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy, (vi) TDP-43 binding molecules for diagnostic use, especially for in vivo diagnosis, but also for in vitro testing, (vii) TDP-43 binding molecules for research use, especially as analytical tools or reference molecules, (viii) TDP-43 binding molecules for use as diagnostic tools to monitor a TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy, (ix) methods of preserving or improving cognitive memory capacity or slowing memory loss in an individual suffering from a TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy, by treating said individual with a TDP-43 binding molecule, (x) methods of reducing the level of aggregated TDP-43 and / or phosphorylated TDP-43 in an individual, by treating said individual with a TDP-43 binding molecule, (xi) nucleic acid molecules encoding a TDP-43 binding molecule, (xii) recombinant expression vectors comprising a nucleic acid molecule of the present application, (xiii) host cells comprising a nucleic acid and / or vector of the present application, (xiv) cell-free expression systems comprising a recombinant expression vector of the present application, (xv) methods for producing a TDP-43 binding molecule, (xvi) methods of using a TDP-43 binding molecule for quantifying TDP-43 in a sample obtained from a subject, and (xvii) kits comprising a TDP-43 binding molecule of the present application and / or nucleic acids, expression vectors, host cells and / or cell-free expression systems for producing them.

[0058] The TDP-43 binding molecules, in particular anti-TDP-43 antibodies or antigen binding fragments thereof, of the present application can recruit and / or activate microglia cells. More particularly, the TDP-43 binding molecules of the present application can influence microglia cell morphology in terms of cell size and activation status. This can contribute to a reduction of TDP-43 pathology indicated by the TDP-43 binding molecules of the present application.

[0059] In the present application, the binding molecules, in particular antibodies or antigen binding fragments thereof, specifically recognize TDP-43. The binding molecules of the present application comprise polypeptides and / or antibodies and / or antigen binding fragments thereof that are specific for TDP-43 protein. By "specifically recognize TDP-43" it is meant that the binding molecules of the present application specifically, generally and collectively bind with greater affinity to TDP-43, in particular to some epitopes of TDP-43, in particular to epitopes of the TDP-43 protein that are exposed / accessible in one or more pathological conformations, as compared to other epitopes. The binding molecules of the present application that specifically bind to TDP-43, in particular polypeptides, more particularly antibodies or antigen binding fragments thereof, specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43.

[0060] The TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, bind to both non-aggregated physiological TDP-43 and aggregated TDP-43. Thus, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can bind to soluble TDP-43 and aggregated TDP-43 with about equal ease. The TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can bind to aggregated TDP-43 with about equal ease compared to non-aggregated TDP-43. More specifically, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can bind to aggregated TDP-43 in the cytoplasm with about equal ease compared to non-aggregated TDP-43 in the nucleus. In other embodiments, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can preferentially bind to aggregated TDP-43 compared to non-aggregated TDP-43 when binding to both types of TDP-43. More specifically, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can preferentially bind to aggregated TDP-43 in the cytoplasm compared to non-aggregated TDP-43 in the nucleus when binding to both types of TDP-43. Alternatively, in other embodiments, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can preferentially bind to non-aggregated TDP-43 compared to aggregated TDP-43 when binding to both types of TDP-43. More specifically, the TDP-43 binding molecules of the application, particularly antibodies or antigen-binding fragments thereof, can preferentially bind to non-aggregated TDP-43 in the nucleus compared to aggregated TDP-43 in the cytoplasm when binding to both types of TDP-43. These binding properties can be demonstrated, for example, using immunohistochemistry.

[0061] In some embodiments, the present application encompasses binding molecules of the present application described herein, particularly antibodies and antigen-binding fragments thereof, that specifically bind to TDP-43, and the use of these binding molecules in the diagnosis, prevention, alleviation, and / or treatment of diseases, disorders, and / or abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE). The methods and compositions disclosed herein can be applied in the diagnosis, prevention, alleviation, and / or treatment of diseases, disorders, and / or abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS). Preferably, the use of these binding molecules in the diagnosis, prevention, alleviation, and / or treatment of diseases, disorders, and / or abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies is directed to amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), or frontotemporal dementia (FTD). More preferably, the use is directed to amyotrophic lateral sclerosis (ALS). More preferably, the use is directed to Alzheimer’s disease (AD). More preferably, the use is directed to frontotemporal dementia (FTD).

[0062] In another embodiment, a TDP-43 binding molecule of the application described herein, particularly an anti-TDP-43 antibody or antigen-binding fragment thereof specific for TDP-43 is contacted with a sample to detect, diagnose and / or monitor a disease, disorder and / or abnormality associated with TDP-43, particularly with TDP-43 aggregates, or a TDP-43 proteinopathy selected from the group consisting of frontotemporal dementia (FTD, e.g. sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g. sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in myotilin (MYOT) or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB) or Parkinson's disease (PD).

[0063] In one embodiment, the present application encompasses a binding molecule of the present application, particularly an antibody or antigen-binding fragment thereof, described herein that specifically binds to TDP-43, and the use of these binding molecules, particularly these antibodies, to detect the presence of TDP-43 in a sample. Thus, a TDP-43 binding molecule of the present application, e.g., an anti-TDP43 antibody described herein, can be particularly useful for screening a clinical sample, particularly human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF), and / or urine, for the presence of TDP-43, e.g., by using an ELISA-based assay or surface plasmon resonance assay. In some cases, a tissue sample, e.g., a brain tissue sample, can be used. The methods and compositions of the present application can also be applied to diagnose pre-symptomatic disease and / or monitor disease progression and / or treatment efficacy. According to some embodiments, an antibody specific for TDP-43 (e.g., a full-length antibody or a TDP-43 binding fragment or derivative of an antibody) is contacted with a sample (e.g., blood, urine, cerebrospinal fluid (CSF), interstitial fluid (ISF), or brain tissue) to detect, diagnose, and / or monitor frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia, and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).The TDP-43 binding molecule of the present invention can be used to quantify TDP-43 in suitable samples, particularly clinical samples such as blood, brain tissue, CSF, ISF, or urine, wherein a relatively high TDP-43 level compared to a suitable control indicates disease and / or a later stage of disease. Many suitable immunoassays are known. Therefore, this method can be performed for diagnostic purposes (e.g., ELISA, MSD (Mesoscale Discovery), HTRF (Homogeneous Time Resolved Fluorescence)). (Single Molecule Array) and High TDP-43 levels indicate disease. Alternatively, this method can be performed for monitoring purposes. Levels that increase over time may indicate disease progression. Levels that decrease over time may indicate disease regression. This method can also be used to monitor treatment, particularly the efficacy of a particular treatment. Successful treatment can be measured by reference to stable or declining TDP-43 levels after treatment. Example 12 of WO2020 / 234473 shows that TDP-43 levels in CSF samples from patients with TDP-43 proteinopathy are higher than in control samples taken from healthy subjects (healthy controls). Control samples may or may not be run in parallel with test samples. In some embodiments, control levels are determined by a series of control samples taken from healthy subjects under similar or identical experimental conditions and used as a comparison level for levels determined in test samples. The method of quantifying TDP-43 in suitable samples using the binding molecules of the present invention can also be used to select treatments (for additional treatment of the subject). Therefore, personalized treatment methods are envisioned. Sampling is performed before and after treatment. If the treatment results in a stable or preferably reduced TDP-43 level after treatment, the treatment may be selected for the subject. If the treatment does not result in a stable or preferably reduced TDP-43 level after treatment, the treatment may not be selected for the subject. The treatment may be any suitable candidate therapeutic agent for treating TDP-43 protein disorders. In some preferred embodiments, the treatment comprises the TDP-43 binding molecule of the present invention, typically in the form of a pharmaceutical composition as described herein.

[0064] The TDP-43 binding molecule of the present invention can also be used to classify diseases into specific types or subtypes. Therefore, methods are provided for classifying diseases, disorders, and / or abnormalities associated with TDP-43, particularly with TDP-43 aggregates, or for classifying TDP-43 protein disorders, comprising:

[0065] a. performing the method of the application, wherein the level of TDP-43 is quantified in comparison to a suitable control;

[0066] b. optionally identifying a mutation in the sample from the subject, including but not limited to a progranulin (GRN) mutation, a C9orf72 mutation, a TARDBP mutation, an angiogenin (ANG) mutation, a valosin-containing protein (VCP) mutation, a myotilin (MYOT) gene mutation, or a mutation in the gene encoding desmin (DES); and

[0067] c. classifying a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or a TDP-43 proteinopathy.

[0068] Similarly, provided are methods for classifying a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or for classifying a TDP-43 proteinopathy, comprising: performing the method of the application, wherein the level of TDP-43 is quantified in a sample obtained from a subject having a disease, disorder and / or abnormality associated with TDP-43, or a TDP-43 proteinopathy, wherein the level is compared to control samples taken from subjects having a different type or subtype of a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or a TDP-43 proteinopathy (i.e. a set of representative control levels is determined for the type or subtype of interest); and classifying the disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or the TDP-43 proteinopathy based on the comparison result. Thus, the classification is based on determining the closest match between the test sample and one or more control samples. These methods can also comprise identifying a mutation in the sample, including but not limited to a progranulin (GRN) mutation, a C9orf72 mutation, a TARDBP mutation, an angiogenin (ANG) mutation, a valosin-containing protein (VCP) mutation, a myotilin (MYOT) gene mutation, or a mutation in the gene encoding desmin (DES), wherein the identified mutation is also used for classifying the disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or the TDP-43 proteinopathy. For the avoidance of doubt, the identification of a mutation in the sample can be performed by any suitable method; for example, based on nucleic acid sequencing of nucleic acid molecules within the sample. The sample can be separate and distinct from the sample in which the level of TDP-43 is determined, but from the same subject.

[0069] In other embodiments, the present application provides methods for preventing, mitigating and / or treating diseases, disorders and / or abnormalities associated with TDP-43, in particular with TDP-43 aggregates, or TDP-43 proteinopathies. According to one embodiment, the methods of the present application comprise administering to a subject an effective concentration of a binding molecule of the present application specific for TDP-43, in particular an antibody (e.g., a full-length antibody or a TDP-43 binding fragment or derivative of an antibody) described herein. In another embodiment, the present application provides methods for preventing, mitigating and / or treating TDP-43 proteinopathies. According to some embodiments, a binding molecule specific for TDP-43 described herein, in particular an antibody or antigen-binding fragment thereof of the present application, is administered to treat, mitigate and / or prevent frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). In another embodiment, a binding molecule specific for TDP-43 described herein, in particular an antibody or antigen-binding fragment thereof of the present application, is administered to prevent, mitigate and / or treat a neurodegenerative disease selected from the group consisting of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD, including sporadic and familial forms of AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0070] In another embodiment, a binding molecule described herein, in particular an antibody of the application or an antigen-binding fragment thereof specific for TDP-43 is administered to prevent, alleviate and / or treat a disease selected from the group consisting of frontotemporal dementia (FTD, e.g. sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g. sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with myotilin (MYOT) gene mutations or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB) or Parkinson's disease (PD). DETAILED DESCRIPTION

[0071] X. DEFINITIONS

[0072] An "antigen binding molecule" as used herein is any molecule that can specifically or selectively bind to an antigen, in particular TDP-43. The binding molecule can comprise or be an antibody or fragment thereof. An anti-TDP-43 binding molecule is a molecule that binds to the TDP-43 protein at a specific recognition site (epitope), such as an anti-TDP-43 antibody or fragment thereof. That is, the antigen binding molecules of the present application bind to an epitope in the amino acid sequence of SEQ ID NO: 1. The antigen binding molecules, in particular antibodies or antigen binding fragments thereof, provided herein recognize full-length TDP-43. Other anti-TDP-43 binding molecules can also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly produced molecules. Illustrative antigen binding molecules useful in the present application include antibody-like molecules. Antibody-like molecules are molecules that can function by binding to a target molecule (see, e.g., Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18:1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15:14-27), and include, for example, DARPins (WO 2002 / 020565), Affibodies (WO 1995 / 001937), Avimers (WO 2004 / 044011; WO 2005 / 040229), Adnectins (WO 2002 / 032925), and fynomers (WO 2013 / 135588).

[0073] The terms "anti-TDP-43 antibody" and "antibody that binds to TDP-43" or simply "antibody" as used herein refer to an antibody that is capable of binding TDP-43 with sufficient affinity that the antibody is useful as a potential diagnostic and / or therapeutic agent for targeting TDP-43. Generally, the term "antibody" is used herein in the broadest sense and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or biparatopic antibodies), fully human antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity. An antibody within the present application can also be a chimeric antibody, a recombinant antibody, an antigen-binding fragment of a recombinant antibody, a humanized antibody, or an antibody displayed on the surface of a phage or on the surface of a chimeric antigen receptor (CAR) T cell.

[0074] An "antigen-binding fragment" of an antibody, or "functional fragment thereof, refers to a molecule that comprises a portion of an intact or full-length antibody and that binds (completely or partially) to the same antigen as the intact or full-length antibody. Some examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. An antigen-binding fragment can also be referred to as a "functional fragment" as they retain the binding function of the original antibody from which they are derived.

[0075] An "antibody that binds to an epitope in a defined region of a protein" is an antibody that requires the presence of one or more amino acids in that region to bind to the protein.

[0076] In certain embodiments, an "antibody that binds to an epitope in a defined region of a protein" is identified by mutational analysis, wherein an amino acid of the protein is mutated and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 20% of the binding to the unaltered protein. In some embodiments, an "antibody that binds to an epitope in a defined region of a protein" is identified by mutational analysis, wherein an amino acid of the protein is mutated and binding of the antibody to the resulting altered protein (e.g., an altered protein comprising the epitope) is determined to be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding to the unaltered protein. In certain embodiments, binding of the antibody is determined by FACS, WB, or by a suitable binding assay, e.g., ELISA.

[0077] The term "binds to" used in the context of the present invention defines the binding (interaction) of at least two "antigen interaction sites" to each other. According to the present invention, the term "antigen interaction site" defines a motif of a polypeptide, i.e. a part of an antibody or antigen binding fragment of the present invention, which shows the ability to specifically interact with a particular antigen or a particular group of antigens in a TDP-43 antigen. Said binding / interaction is also to be understood to define "specific recognition". According to the present invention, the term "specific recognition" means that the antibody is able to specifically interact and / or bind to at least two amino acids of a TDP-43 as defined herein, in particular to at least two amino acids of amino acid residues 304 to 414 of human TDP-43 (SEQ ID NO: 1), even more particularly to at least two amino acids of amino acid residues 304 to 313, 356 to 361, 397 to 407, or 396 to 414 of human TDP-43 (SEQ ID NO: 1).

[0078] The term "pan-TDP-43 antibody" refers to an antibody that binds to misfolded aggregated TDP-43 and to non-aggregated physiological TDP-43, including monomeric TDP-43, oligomeric TDP-43, post-translationally modified TDP-43 (e.g. phosphorylated, ubiquitinated, acetylated, sumoylated and / or methylated), aggregated TDP-43 and truncated TDP-43.

[0079] The term "specific interaction" as used according to the present application means that the antibody of the present application or an antigen binding fragment thereof does not or essentially not cross-react with (poly)peptides having a similar structure. Thus, the antibody of the present application or an antigen binding fragment thereof specifically binds to / interacts with a TDP-43 structure formed by a specific amino acid sequence in amino acid residues 304 to 414 of human TDP-43 (SEQ ID NO: 1), more particularly with a TDP-43 structure formed by a specific amino acid sequence in amino acid residues 304 to 313, 356 to 361, 397 to 407 or 396 to 414 of human TDP-43 (SEQ ID NO: 1).

[0080] The cross-reactivity of a panel of antigen binding molecules, in particular antibodies or antigen binding fragments thereof, under investigation can be tested, for example, by assessing the binding of said panel of antibodies or antigen binding fragments thereof to the (poly)peptide of interest and to a number of more or less (structurally and / or functionally) closely related (poly)peptides under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). Only those constructs (i.e. antibodies, antigen binding fragments thereof, etc.) that bind to certain TDP-43 structures as defined herein, e.g. to a specific epitope of TDP-43 or to a (poly)peptide / protein as defined herein but not or essentially not to any other epitope or (poly)peptide of the same TDP-43 are considered specific for the epitope or (poly)peptide / protein of interest and selected for further investigation according to the methods provided herein. These methods can include, inter alia, binding studies, blocking and competition studies with structurally and / or functionally closely related molecules. These binding studies also include FACS analysis, surface plasmon resonance (SPR, e.g. with a BIACORE® instrument), ELISA, and Western blot analysis. TManalytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy or by radiolabeled ligand binding assays.

[0081] Thus, specificity can be determined experimentally by methods known in the art and as described herein. Such methods include, but are not limited to, Western blotting, ELISA-, RIA-, ECL-, IRMA-tests and peptide scanning.

[0082] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The advantage of monoclonal antibodies is that they can be synthesized by a hybridoma culture, essentially free from other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. As described above, the monoclonal antibodies to be used in accordance with the present application can be made by the hybridoma method first described by Kohler, Nature 256 (1975), 495.

[0083] The term "polyclonal antibody" as used herein refers to an antibody produced in the presence of or among one or more other different antibodies. Generally, polyclonal antibodies are produced by B lymphocytes in the presence of several other B lymphocytes producing different antibodies. Typically, polyclonal antibodies are obtained directly from immunized animals.

[0084] The term "fully human antibody" as used herein refers to an antibody comprising only human immunoglobulin protein sequences. A fully human antibody can comprise murine sugar chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or "murine antibody" refers to an antibody comprising only mouse / murine immunoglobulin protein sequences. Alternatively, a "fully human antibody" can comprise rat sugar chains if produced in a rat, in a rat cell, in a hybridoma derived from a rat cell. Similarly, the term "rat antibody" refers to an antibody comprising only rat immunoglobulin sequences. Fully human antibodies can also be produced, for example, by phage display, which is a widely used screening technique that enables the generation and screening of fully human antibodies. Phage antibodies can also be used in the context of the present application. Phage display methods are described, for example, in US 5,403,484, US 5,969,108 and US 5,885,793. Another technology that enables the development of fully human antibodies involves an improvement on the mouse hybridoma technology. Mice are transgenic to contain human immunoglobulin loci to exchange their own mouse genes (see, for example, US 5,877,397).

[0085] The term "chimeric antibody" refers to an antibody comprising a variable region of the present application fused or chimerized with regions (e.g., constant regions) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

[0086] The term antibody also relates to recombinant human antibodies, heterologous antibodies, and heteromeric antibodies. The term "recombinant (human) antibody" includes all human sequence antibodies made, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes; antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library; or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. However, such antibodies can be mutagenized in vitro (or, when using animals transgenic for human Ig sequences, mutagenized in vivo in somatic cells), and thus the amino acid sequences of the VH and VL regions of recombinant antibodies are such sequences which, although derived from and related to human germline VH and VL sequences, can not naturally exist within the human antibody germline repertoire in vivo.

[0087] A "heterologous antibody" is defined with respect to the transgenic non-human organism that produces such an antibody. The term refers to an antibody having an amino acid sequence or encoding nucleic acid sequence corresponding to that present in an organism that does not consist of the transgenic non-human animal, and the organism is typically from a species other than that of the transgenic non-human animal.

[0088] The term "heteromeric antibody" refers to an antibody having a light chain and a heavy chain of different organismic origin. For example, an antibody having a human heavy chain associated with a murine light chain is a heteromeric antibody. Some examples of heteromeric antibodies include chimeric antibodies and humanized antibodies.

[0089] The term antibody also relates to humanized antibodies. "Humanized" forms of non-human (e.g., murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 321 (1986), 522-525; Reichmann Nature 332 (1988), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596.

[0090] A popular method for antibody humanization involves CDR grafting, in which a functional antigen binding site from a non-human "donor" antibody is grafted onto a human "recipient" antibody. CDR grafting methods are known in the art and described in, e.g., US 5,225,539, US 5,693,761 and US 6,407,213. Another related method is to produce humanized antibodies from transgenic animals that are genetically engineered to contain one or more humanized immunoglobulin loci capable of gene rearrangement and gene conversion (see, e.g., US 7,129,084).

[0091] Accordingly, in the context of the present application, the term "antibody" relates to intact immunoglobulin molecules as well as to portions of such immunoglobulin molecules (i.e., "antigen binding fragments thereof). Furthermore, as mentioned above, the term relates to modified and / or altered antibody molecules. The term also relates to recombinantly or synthetically produced / synthetic antibodies. The term also relates to intact antibodies and antibody fragments thereof, such as isolated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term antibody also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs, such as single chain Fv (scFv) or antibody fusion proteins.

[0092] In the context of the present application, a "single chain Fv" or "scFv" antibody fragment has the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the scFv to form the desired antigen binding structure. Techniques for producing single chain antibodies are described, e.g., in Pluckthun, The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, N.Y. (1994), 269-315.

[0093] A "Fab fragment", as used herein, comprises one light chain, and a portion of one heavy chain, comprising the V H 1 and variable region. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0094] An "Fc" region comprises two heavy chain fragments that contain the C H 2 and C H 3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C H 3 domains.

[0095] A "Fab fragment" comprises one light chain, and a portion of one heavy chain, comprising the V H domain and the C H 1 domain and also having a region between the C H 1 and C H 2 domains, such that an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0096] An "F(ab')2 fragment" comprises two light chains and two heavy chains comprising a portion of the constant region between the C H 1 and C H 2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, an F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0097] An "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0098] The antibodies, antibody constructs, antibody fragments, antibody derivatives (all of Ig origin), or their corresponding immunoglobulin chains used according to the present application can be further modified using conventional techniques known in the art, for example by using amino acid deletions, insertions, substitutions, additions and / or recombination alone or in combination and / or any other modification known in the art. Methods for introducing such modifications in DNA sequences based on the amino acid sequences of the immunoglobulin chains are well known to the person skilled in the art; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2ndedition (1989) and 3rdedition (2001). The term "Ig-derived domain" relates in particular to (poly)peptide constructs comprising at least one CDR. The fragments or derivatives of the Ig-derived domains listed define (poly)peptides which are part of the above antibody molecules and / or are modified by chemical / biochemical or molecular biological methods. The corresponding methods are known in the art and are described, inter alia, in laboratory manuals (see, Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2ndedition (1989) and 3rdedition (2001); Gerhardt et al., Methods for General and Molecular Bacteriology ASM Press (1994); Lefkovits, Immunology Methods Manual: The Comprehensive Sourcebook of Techniques; Academic Press (1997); Golemis, Protein-Protein Interactions: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

[0099] The term "CDR" as used herein relates to "complementarity determining region" which is well known in the art. CDRs are part of immunoglobulins which determine the specificity of the molecule and contact a particular ligand. CDRs are the most variable part of the molecule and contribute to the diversity of these molecules. There are three CDR regions in each V domain: CDR1, CDR2 and CDR3. CDR-H shows the CDR regions of the variable heavy chain, while CDR-L relates to the CDR regions of the variable light chain. VH means variable heavy chain and VL means variable light chain. The CDR regions of Ig-derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest", 5th Ed. NIH Publication no. 91-3242 U.S. Department of Health and Human Services (1991). The CDR sequences provided herein are defined according to Kabat. However, the skilled person will appreciate that the present application is intended to encompass binding molecules in which the CDR sequences are defined according to any useful identification / numbering scheme.For example, the following numbering schemes can be employed to define CDRs: Chothia (Canonical structures for the hypervariable regions of immunoglobulins. Chothia C, Lesk AM. J Mol Biol. 1987 Aug 20;196(4):901-17); IMGT (IMGT, the international ImMunoGeneTics database. Giudicelli V, Chaume D, Bodmer J, Muller W, Busin C, Marsh S, Bontrop R, Marc L, Malik A, Lefranc MP. Nucleic Acids Res. 1997 Jan 1;25(1):206-11 and Unique database numbering system for immunogenetic analysis. Lefranc MP. Immunol Today. 1997 Nov;18(11):509); MacCallum (MacCallum RM, Martin AC, Thornton JM, J Mol Biol. 1996 Oct 11;262(5):732-45) and Martin (Abhinandan KR, Martin ACR. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. (2008) 45:3832-9. 10.1016 / j.molimm.2008.05.022).

[0100] Thus, in the context of the present application, the antibody molecules described above herein are selected from the group consisting of intact antibodies (immunoglobulins, e.g. IgGl, IgG2, IgAl, IgGA2, IgG3, IgG4, IgA, IgM, IgD or IgE), F(ab)-, Fab'-SH-, Fv-, Fab'-, F(ab')2- fragments, chimeric antibodies, CDR-grafted antibodies, fully human antibodies, diabody constructs, antibody fusion proteins, synthetic antibodies, di-valent single chain antibodies, tri-valent single chain antibodies and multi-valent single chain antibodies.

[0101] "Humanization methods" are well known in the art and are described in particular for antibody molecules, e.g. Ig-derived molecules. The term "humanized" refers to a humanized form of a non-human (e.g., murine) antibody or fragment thereof (e.g. Fv, Fab, Fab', F(ab'), scFv or other antigen binding portion sequence of an antibody) that contains some portions of the sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues from a CDR of the human immunoglobulin are replaced by residues from a CDR of a non-human species (e.g. mouse, rat or rabbit) that have the desired binding specificity, affinity, and capacity. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, the humanized antibody also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin; see, in particular, Jones et al., Nature 321 (1986), 522-525, Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596. Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acids introduced into it from a non-human source that retain the original binding activity of the antibody. Methods for humanizing antibodies / antibody molecules are also described in detail in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536. Some specific examples of humanized antibodies, e.g. against EpCAM, are known in the art (see e.g. LoBuglio, Proceedings of the American Society of Clinical Oncology Abstract (1997), 1562 and Khor, Proceedings of the American Society of Clinical Oncology Abstract (1997), 847).

[0102] Thus, in the context of the present application, there is provided an antibody molecule or antigen binding fragment thereof, which can be humanized and can be successfully used in a pharmaceutical composition.

[0103] It will be understood by the skilled person that the epitope can be comprised in the TDP-43 protein, but also in its degradation products or can be a chemically synthesized peptide. The indication of the amino acid position is only to show the position of the respective amino acid sequence in the TDP-43 protein sequence. The present invention encompasses all peptides comprising the epitope. The peptide can be part of a polypeptide of more than 100 amino acids in length or can be a small peptide of less than 100, preferably less than 50, more preferably less than 25 amino acids, even more preferably less than 16 amino acids. The amino acids of such a peptide can be natural amino acids or unnatural amino acids (e.g. beta amino acids, gamma amino acids, D-amino acids) or combinations thereof. Furthermore, the present invention can encompass the respective retro-inverso peptide of the epitope. The peptide can be unbound or bound. It can be bound to e.g. small molecules (e.g. drugs or fluorophores), high molecular weight polymers (e.g. polyethylene glycol (PEG), polyethylene imine (PEI), hydroxypropylmethacrylate (HPMA) etc.) or proteins, fatty acids, sugar moieties or can be inserted into a membrane.

[0104] To test whether the antibody in question and the antibody of the present invention recognize the same epitope, the following competition study can be performed: Vero cells infected at 3 MOI (multiplicity of infection) are incubated after 20 hours for 1 hour with different concentrations of the antibody in question as competitor. In a second incubation step, the antibody of the present invention is applied at a constant concentration of 100 nM and its binding is detected by flow cytometry using a fluorescently labeled antibody against the constant domain of the antibody of the present invention. Inverse proportionality (inversely proportional) of the binding to the concentration of the antibody in question indicates that both antibodies recognize the same epitope. However, many other assays known in the art can be used.

[0105] The present application also relates to the production of specific antibodies against the native polypeptides and the recombinant polypeptides of TDP-43. This production is for example based on the immunization of animals such as mice. However, other animals for the production of antibodies / antisera are also envisaged in the present application. For example, monoclonal and polyclonal antibodies can be produced from rabbits, mice, goats, donkeys, etc. A polynucleotide encoding a respective selected polypeptide of TDP-43 can be subcloned into a suitable vector, wherein the recombinant polypeptide is expressed in an organism capable of expression, for example in bacteria. The expressed recombinant protein can thus be injected intraperitoneally into mice, and the resulting specific antibodies can be obtained for example from the mouse serum provided by intracardial blood puncture. The present application also envisages the production of specific antibodies against the native polypeptides and the recombinant polypeptides by using DNA / RNA vaccine strategies as exemplified in the appended examples. DNA vaccine strategies are well known in the art and encompass liposome-mediated delivery, injection by gene gun or jet injection and intramuscular or intradermal injection. Thus, antibodies against a polypeptide or protein or epitope of TDP-43, in particular an antibody epitope provided herein, can be obtained by direct immunization of an animal with a vector expressing the desired polypeptide or protein or epitope of TDP-43 by intramuscular direct injection, in particular an antibody epitope of the present application which is located in the amino acid residues 304 to 414 of SEQ ID NO: 1; more particularly an antibody epitope of the present application which is located in the amino acid residues 304 to 313, 356 to 361, 397 to 407 or 396 to 414 of SEQ ID NO: 1. The amount of specific antibodies obtained can be quantified using ELISA, which is also described hereinafter. Further methods for the production of antibodies are well known in the art, see, e.g., Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

[0106] Thus, under designated assay conditions, a particular antibody binds to its corresponding epitope of TDP-43 and not to other components present in a sample in significant amounts. Specific binding to the target analyte under such conditions can require a binding moiety selected for its specificity for a particular target analyte. A variety of immunoassay formats can be used to select antibodies that specifically react with a particular antigen. For example, a solid-phase ELISA immunoassay is routinely used to select monoclonal antibodies that immunospecifically react with an analyte. See Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press and / or Howard and Bethell for a description of immunoassay formats and conditions that can be used to determine immunospecific reactivity. In general, a specific or selective reaction will be at least two-fold greater than the background signal and more typically more than 10 to 100 times greater than the background. Persons skilled in the art will be able to provide and generate specific binding molecules to new polypeptides. For specific binding assays, they can readily be used to avoid undesired cross-reactivity, e.g., polyclonal antibodies can readily be purified and selected by known methods (see Shepherd and Dean, loc. cit.).

[0107] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0108] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it can be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion(s), insertion(s), and / or substitution(s) can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding.

[0109] In some embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 1. Further substitutional variations are provided under the heading “Exemplary Substitutions” in Table 1 and are further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody to target desired activities, such as, preserving / improving antigen binding, reducing immunogenicity, or improving antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0110] Table 1

[0111]

[0112] Amino acids can be grouped according to their common side chain characteristics:

[0113] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0114] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0115] (3) Acidity: Asp, Glu;

[0116] (4) Alkaline: His, Lys, Arg;

[0117] (5) Residues that affect chain orientation: Gly, Pro;

[0118] (6) Aromatics: Trp, Tyr, Phe.

[0119] Non-conservative replacement would require replacing members of one of these categories with members of another category.

[0120] One type of substitution variant involves replacing one or more hypervariable residues in a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further research will have improvements (e.g., enhancements) in certain biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques, such as those described herein. In short, it involves mutating one or more CDR residues and displaying the variant antibody on a phage and screening for specific biological activities (e.g., binding affinity).

[0121] Alterations (e.g., substitutions) can be made in the CDR, for example, to improve antibody affinity. Such alterations can be made in CDR “hotspots,” which are residues encoded by codons that mutate at high frequencies during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or the SDR (a-CDR), and the binding affinity of the resulting variant VH or VL is tested. Affinity maturation by constructing a secondary library and reselecting from it has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand mixing, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves a CDR-guided method, where several CDR residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0122] In some embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as provided herein) may be made in the CDRs. Such changes may occur outside of CDR "hot spots" or SDRs. In some embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0123] A useful method for identification of residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions can be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for substitution. Variants can be screened for desired properties.

[0124] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Another example of an insertion variant is an antibody in which the N or C terminus has been fused with an enzyme (e.g., for ADEPT) or a polypeptide which enhances serum half-life.

[0125] In certain embodiments, an antibody provided herein is altered to improve or diminish the extent to which it is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0126] When the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is typically linked to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can include various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the application can be made in order to create antibody variants with certain improved properties.

[0127] In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297, relative to the sum of all structures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues; see Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)); however, Asn297 can also be located about ± 3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to minor sequence variations in antibodies. Such fucosylation variants can have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Some examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Some examples of cell lines capable of producing afucosylated antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 Al, Presta, L; and WO 2004 / 056312 Al, Adams et al., especially in Example 11), and knockout cell lines, such as a-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotech. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0128] Also provided are antibody variants with bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can have reduced fucosylation and / or improved ADCC function. Some examples of such antibody variants are described in, e.g., WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Such antibody variants are described in, e.g., WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0129] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. Fc region variants can comprise human Fc region sequences (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0130] In certain embodiments, the antibodies provided herein bind to pathological TDP-43 and form immune complexes that can be cleared by antibody-dependent cellular phagocytosis (ADCP) with the result that TDP-43 clearance is enhanced. ADCP is mediated by the interaction of the antibody Fc fragment with Fc receptors (such as Fcy receptors) expressed on the surface of innate immune cells (such as microglia or dendritic cells). By modifying the Fc portion of the antibody, Fc-mediated functions can be modulated to achieve the desired effect.

[0131] In certain embodiments, the application contemplates the following antibody variants, which have some, but not all, effector functions, making them desirable candidates for applications in which the half-life of the antibody in vivo is important, while certain effector functions (e.g., complement activation and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to determine the loss / exhaustion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cell for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes and granulocytes express FcyRI, FcyRII, and FcyRIII. A summary of FcR expression on hematopoietic cells can be found in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)).

[0132] Alternatively, non-radioactive assays methods can be used (see, e.g., ACTI TM CellTiter 96® Reagent (Promega, Madison, WI) following the manufacturer's Non-radioactive cell toxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.

[0133] Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0134] C1q binding assays also can be conducted to determine if an antibody is unable to bind C1q, and hence lacks CDC activity. See, e.g., WIPO 2006 / 029879 and WO 2005 / 100402 for C1q and C3c binding ELISAs. To assess complement activation, a CDC assay can be conducted (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0135] Antibodies with reduced effector function include antibodies in which one or more of residues 234, 235, 238, 265, 269, 270, 297, 327, and 329 of the Fc region are substituted (U.S. Patent No. 6,737,056). Certain antibody variants described that have increased or decreased binding to FcRs. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). Such Fc mutants include Fc mutants with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581), or the so-called “DANG” FC mutant in which residue 265 is substituted with alanine and residue 297 is substituted with glycine. Alternatively, antibodies with reduced effector function include antibodies in which one or more of residues 234, 235, and 329 of the Fc region are substituted, i.e., the so-called “PG-LALA” Fc mutant in which residues 234 and 235 are substituted with alanine and 329 is substituted with glycine (Lo, M. et al., Journal of Biochemistry, 292, 3900-3908). Other known mutations at positions 234, 235, and 321 can be used, i.e., the so-called TM mutant comprising mutations L234F / L235E / P331S in the CH2 domain (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies from the human IgG4 isotype comprise the mutations S228P / L235E to stabilize the hinge and reduce FgR binding (Schlothauer et al, PEDS, 29(10):457-466). Constant domains are numbered according to the EU numbering system.

[0136] Other Fc variants include Fc variants having one or more substitutions in the following Fc region residues: position 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., Fc variants in which the residue at position 434 of an Fc region is substituted (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821.

[0137] In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0 and thus to prolong the half-life of the antibody. Antibodies with enhanced affinity for FcRn include those in which one or more of the following Fc region residues are substituted: 252, 253, 254, 256, 428, 434, including so-called YTE mutations M252Y / S254T / T256E (Dall’Acqua et al, J Immunol. 169:5171-5180 (2002)) or LS mutations M428L / N434S (Zalevsky et al, Nat Biotechnol. 28(2): 157-159 (2010)).

[0138] In certain embodiments, it can be desirable to generate a cysteine- engineered antibody, e.g., a “thioMAb,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated as described in, e.g., U.S. Patent No. 7,521,541.

[0139] In some embodiments, the antibodies provided herein may also be modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Some non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, and poly-1,3,6-trimethyloxolane. Alkane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers linked to the antibody can vary, and if more than one polymer is linked, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in treatment under the following defined conditions, etc.

[0140] In another embodiment, an antibody is provided as a conjugate with a non-protein portion that can be selectively heated by exposure to radiation. In one embodiment, the non-protein portion is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to wavelengths that do not damage normal cells but heat the non-protein portion to a temperature that kills cells adjacent to the antibody-non-protein portion.

[0141] Antibodies can be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-misfolded TDP-43 antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or the heavy chain of the antibody). In another embodiment, one or more vectors (e.g., recombinant expression vectors) comprising such a nucleic acid are provided. In another embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody; or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment, a method of making an anti-misfolded TDP-43 antibody is provided, wherein the method comprises: culturing a host cell comprising a nucleic acid encoding the antibody as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0142] For recombinant production of an anti-misfolded TDP-43 antibody, a nucleic acid encoding the antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell or cell-free expression system. Such a nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to genes encoding the heavy and light chains of the antibody).

[0143] In one embodiment, a method of making a TDP-43 binding molecule, particularly an antibody or antigen binding fragment thereof, is provided, wherein the method comprises culturing a host cell or cell-free expression system comprising a nucleic acid encoding the TDP-43 binding molecule as provided above, under conditions suitable for expression of the TDP-43 binding molecule, and isolating the TDP-43 binding molecule.

[0144] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245- 254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0145] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expression of antibody-encoding vectors, including a fungal or yeast strain in which the glycosylation pathway has been "humanized," resulting in production of an antibody having a partially or fully human glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0146] Suitable host cells for expression of glycosylated antibodies also are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0147] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES TM technology for producing antibodies in transgenic plants).

[0148] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CVl line (COS-7) transformed by SV40 (ATCC CRL 1651); human embryonic kidney line 293 or 293 cells (Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells derived from the bowels of adult male mice); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HeLa); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (Wl38); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells [see, e.g., Mather et al., Annals N.Y Acad. Sci. 383:44-68 (1982)]; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as YO, NSO and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0149] For delivery of molecules across the blood brain barrier (BBB), several methods known in the art exist, such as alteration of the route of administration, disruption of the BBB and alteration of its permeability, nanoparticle delivery, Trojan horse approach, receptor-mediated transport, and cell and gene therapy.

[0150] The route of administration can be altered by: direct injection into the brain (see, for example, Papanastassiou et al., Gene Therapy 9:398-406 (2002)), or implantation of a delivery device into the brain (see, for example, Gillet et al., Nature Med. 9:589-595 (2003); and Gliadel Wafers). TM Guildford Pharmaceutical), and intranasal administration bypassing the BBB (Mittal et al, Drug Deliv. 21(2): 75-86. (2014)).

[0151] Methods of barrier disruption include, but are not limited to: ultrasound (see, for example, U.S. Patent Publication No. 2002 / 0038086); osmotic pressure (e.g., by application of hypertonic mannitol (Neuwelt, EA, Implication of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press, NY (1989))); and permeation, by, for example, bradykinin or permeation agent A-7 (see, for example, U.S. Patent Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416).

[0152] Methods for altering BBB permeability include, but are not limited to: using glucocorticoid blockers to increase blood-brain barrier permeability (see, for example, U.S. Patent Application Publication Nos. 2002 / 0065259, 2003 / 0162695, and 2005 / 0124533); activating potassium channels (see, for example, U.S. Patent Application Publication No. 2005 / 0089473); and inhibiting ABC drug transporters (see, for example, U.S. Patent Application Publication No. 2003 / 0073713).

[0153] Trojan horse delivery methods for delivering antibodies or antibody fragments across the blood-brain barrier include, but are not limited to: cationizing the antibody (see, for example, U.S. Patent No. 5,004,697), and using cell-penetrating peptides such as Tat peptides to allow entry into the CNS (see, for example, Dietz et al., J. Neurochem. 104:757–765 (2008)).

[0154] Nanoparticle delivery methods to deliver antibodies or antigen-binding fragments thereof across the blood-brain barrier include, but are not limited to, encapsulating the antibody or antigen-binding fragment thereof in a liposome or extracellular vesicle, such as an exosome, coupled (without limitation) to an antibody or antigen-binding fragment or, as an alternative, a peptide that binds to a receptor on the vascular endothelium of the blood-brain barrier (see, e.g., U.S. Patent Application Publication No. 20020025313); and coating the antibody or antigen-binding fragment thereof in low-density lipoprotein particles (see, e.g., U.S. Patent Application Publication No. 20040204354) or in apolipoprotein E (see, e.g., U.S. Patent Application Publication No. 20040131692).

[0155] The antibodies of the present application can be additionally modified to enhance blood-brain barrier penetration.

[0156] The antibodies of the present application or antigen-binding fragments thereof can be fused to a polypeptide that binds to a receptor of the blood-brain barrier. BBB receptors include, but are not limited to, transferrin receptor, insulin receptor, or low-density lipoprotein receptor. The polypeptide can be a peptide, a receptor ligand, a single domain antibody (VHH), a scFv, or a Fab fragment.

[0157] The antibodies of the present application can also be delivered as a corresponding nucleic acid encoding the antibody. Such nucleic acid molecules can be part of a viral vector for targeted delivery to the blood-brain barrier or any other cell type in the CNS. One non-limiting example is a viral vector comprising a nucleic acid molecule encoding an antibody of the present application for targeted delivery to endothelial cells of the BBB, pericytes of the BBB, or astrocytes. In some embodiments, the endothelial cells of the BBB, pericytes of the BBB, or astrocytes express and secrete the antibody into the brain parenchyma. One preferred example is a viral vector comprising a nucleic acid molecule encoding an antibody of the present application for targeted delivery in endothelial cells of the BBB, wherein the endothelial cells of the BBB express and secrete the antibody into the brain parenchyma. The viral vector can be a recombinant adeno-associated viral vector (rAAV) selected from any of the AAV serotypes known in the art (including, but not limited to, AAV1 through AAV12) to enable expression of the antibody or antibody fragment or antibody derivative either intracellularly or in the brain parenchyma.

[0158] Cellular therapy methods of delivering the antibodies or antibody fragments or antibody derivatives of the present application across the blood brain barrier include, but are not limited to, the use of the homing ability of endothelial progenitor cells (EPCs) transfected ex vivo with vectors, and the secretion of antibodies or antibody fragments by these cells and delivery of the antibodies or antibody fragments to the brain to overcome the powerful filtering activities of the BBB (see, e.g., Heller and al., J Cell Mol Med. 00: 1-7 (2020)); or the use of polymeric cell-implant devices loaded with genetically engineered cells to secrete antibodies or antibody fragments (see, e.g., Marroquin Belaunzaran et al. PLoS ONE 6(4): e18268 (2011)).

[0159] Pharmaceutical Sciences, 15th or 18th Edition. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th Edition. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd Edition. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th Edition. (Walter Lund ed.; Pharmaceutical Press, London, 1994); The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention); Fiedler's "Lexikon der Hilfstoffe" 5th Edition, Edition Cantor Verlag Aulendorf 2002; "The Handbook of Pharmaceutical Excipients", 4th Edition, American Pharmaceuticals Association, 2003; and Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992), the disclosures of which are incorporated herein by reference.

[0160] The carriers, diluents, adjuvants and pharmaceutical excipients can be chosen according to the intended route of administration and standard pharmaceutical practice. These compounds must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient thereof. See Remington's Pharmaceutical Sciences, 15th or 18th edition. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th edition. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd edition. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th edition. (Walter Lund ed.; Pharmaceutical Press, London, 1994); The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention); Fiedler's "Lexikon der Hilfstoffe" 5th edition, Edition Cantor Verlag Aulendorf 2002; "The Handbook of Pharmaceutical Excipients", 4th edition, American Pharmaceuticals Association, 2003; and Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992), the disclosures of which are incorporated herein by reference.

[0161] An "effective amount" of a compound to be administered to a subject is a dose appropriate for treating, preventing, or reducing a condition, disease, or disorder according to sound medical judgment. Specific dosage levels and dosage frequency can depend on, for example, a variety of factors, including: the activity of the particular compound used, the metabolic stability and length of action of that compound, the mode and time of administration, the rate of excretion, and the combination with other drugs. Patient-specific factors, such as age, body weight, general health, sex, diet, and the severity of the particular condition can also affect the amount to be administered.

[0162] The term "clearance" (also referred to as "clearance value" or "CL" or "systemic clearance") relates to the efficiency of elimination of a substance from the body. Clearance of a substance (in this case a binding molecule of the present application) is the sum of urinary and extrarenal clearance; for substances cleared by both renal and extrarenal routes, plasma clearance exceeds urinary clearance. The PK characteristics of mAbs are a function of their large size (150 kDa), relative polarity, Fc-receptor binding, and specific binding to target antigens. The primary elimination pathway for mAbs is cellular uptake followed by proteolytic degradation. The low clearance of mAbs from the systemic circulation makes their dosing frequency lower than for peptides or small molecules, which is generally more convenient for patients (Betts et al., MABs. 2018).

[0163] XI. Some inventive embodiments of TDP-43 specific binding molecules

[0164] In some embodiments, provided are TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0165] a. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; or

[0166] b. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; or

[0167] c. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH-CDR3 comprising the amino acid sequence of PC (Pro-Cys); or

[0168] d. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; or

[0169] e. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13.

[0170] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0171] a. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or

[0172] b. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; or

[0173] c. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0174] d. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or

[0175] e. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0176] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0177] a) a heavy chain variable region (VH) comprising:

[0178] i. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:51, a VH- CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:53; or

[0179] ii. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:41, a VH- CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:43; or

[0180] iii. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:31, a VH- CDR2 comprising the amino acid sequence of SEQ ID NO:32, and a VH-CDR3 comprising the amino acid sequence PC (Pro-Cys); or

[0181] iv. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:21, a VH- CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:23; or

[0182] v. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:11, a VH- CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:13; and

[0183] b) a light chain variable region (VL) comprising:

[0184] i. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:55, a VL- CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:57; or

[0185] ii. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:45, a VL- CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:47; or

[0186] iii. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; or

[0187] iv. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or

[0188] v. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0189] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0190] a) a heavy chain variable region (VH) comprising:

[0191] i. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 51 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 51; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 52; and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 53; or

[0192] ii. a VH-CDR1 that comprises an amino acid sequence of SEQ ID NO: 41 or a VH- CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 41; a VH-CDR2 that comprises an amino acid sequence of SEQ ID NO: 42 or a VH-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 42; and a VH-CDR3 that comprises an amino acid sequence of SEQ ID NO: 43 or a VH-CDR3 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 43; or

[0193] iii. a VH-CDR1 that comprises an amino acid sequence of SEQ ID NO: 31 or a VH- CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 31; a VH-CDR2 that comprises an amino acid sequence of SEQ ID NO: 32 or a VH-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 32; and a VH-CDR3 that comprises an amino acid sequence PC (Pro-Cys); or

[0194] iv. a VH-CDR1 that comprises an amino acid sequence of SEQ ID NO: 21 or a VH- CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 that comprises an amino acid sequence of SEQ ID NO: 22 or a VH-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 22; and a VH-CDR3 that comprises an amino acid sequence of SEQ ID NO: 23 or a VH-CDR3 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 23; or

[0195] v. a VH-CDR1 comprising an amino acid sequence of SEQ ID NO: 11 or a VH-CDR1 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 11; a VH-CDR2 comprising an amino acid sequence of SEQ ID NO: 12 or a VH-CDR2 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 12; and a VH-CDR3 comprising an amino acid sequence of SEQ ID NO: 13 or a VH-CDR3 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 13; and

[0196] b) a light chain variable region (VL) comprising:

[0197] i. a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 55 or a VL-CDR1 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 55; a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 56 or a VL-CDR2 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 56; and a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 57 or a VL-CDR3 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 57; or

[0198] ii. a VL-CDR1 comprising an amino acid sequence of SEQ ID NO: 45 or a VL-CDR1 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 45; a VL-CDR2 comprising an amino acid sequence of SEQ ID NO: 46 or a VL-CDR2 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 46; and a VL-CDR3 comprising an amino acid sequence of SEQ ID NO: 47 or a VL-CDR3 comprising an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 47; or

[0199] iii. a VL-CDR1 that comprises the amino acid sequence of SEQ ID NO: 35 or a VL-CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 35; a VL-CDR2 that comprises the amino acid sequence of SEQ ID NO: 36 or a VL-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 36; and a VL-CDR3 that comprises the amino acid sequence of SEQ ID NO: 37 or a VL-CDR3 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 37; or

[0200] iv. a VL-CDR1 that comprises the amino acid sequence of SEQ ID NO: 15 or a VL-CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 15; a VL-CDR2 that comprises the amino acid sequence of SEQ ID NO: 26 or a VL-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 26; and a VL-CDR3 that comprises the amino acid sequence of SEQ ID NO: 17 or a VL-CDR3 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 17; or

[0201] v. a VL-CDR1 that comprises the amino acid sequence of SEQ ID NO: 15 or a VL-CDR1 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 15; a VL-CDR2 that comprises the amino acid sequence of SEQ ID NO: 16 or a VL-CDR2 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 16; and a VL-CDR3 that comprises the amino acid sequence of SEQ ID NO: 17 or a VL-CDR3 that comprises an amino acid sequence that has at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 17.

[0202] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0203] a. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:51, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:53; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:55, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:56, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:57; or

[0204] b. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:43; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:45, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:47; or

[0205] c. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:31, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:32, and a VH-CDR3 comprising the amino acid sequence PC (Pro-Cys); and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:35, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:36, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:37; or

[0206] d. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:21, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:23; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:26, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:17; or

[0207] e. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0208] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0209] a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50 or a heavy chain variable region (VH) having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; or

[0210] b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 or a heavy chain variable region (VH) having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40; or

[0211] c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 30 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; or

[0212] d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 20 or a heavy chain variable region (VH) having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20; or

[0213] e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 10 or a heavy chain variable region (VH) having at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0214] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0215] a. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or

[0216] b. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 or a light chain variable region (VL) having at least 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or

[0217] c. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 34 or a light chain variable region (VL) having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or

[0218] d. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 24 or a light chain variable region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 24; or

[0219] e. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 14.

[0220] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0221] a) a heavy chain variable region (VH) selected from:

[0222] i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50 or a heavy chain variable region (VH) having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; or

[0223] ii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a heavy chain variable region (VH) having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:40; or

[0224] iii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:30 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:30; or

[0225] iv. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:20 or a heavy chain variable region (VH) having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:20; or

[0226] v. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 10 or a heavy chain variable region (VH) having at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and

[0227] b) a light chain variable region (VL) selected from:

[0228] i. a light chain variable region (VL) comprising the sequence of SEQ ID NO:54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:54; or

[0229] ii. a light chain variable region (VL) comprising the sequence of SEQ ID NO:44 or a light chain variable region (VL) having at least 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:44; or

[0230] iii. a light chain variable region (VL) comprising the sequence of SEQ ID NO:34 or a light chain variable region (VL) having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:34; or

[0231] iv. a light chain variable region (VL) comprising the sequence of SEQ ID NO:24 or a light chain variable region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:24; or

[0232] v. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 14.

[0233] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0234] a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:50 or a heavy chain variable region (VH) having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:50; and a light chain variable region (VL) comprising the sequence of SEQ ID NO:54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:54; or

[0235] b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:40 or a heavy chain variable region (VH) having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:40; and a light chain variable region (VL) comprising the sequence of SEQ ID NO:44 or a light chain variable region (VL) having at least 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:44; or

[0236] c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:30 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:30; and a light chain variable region (VL) comprising the sequence of SEQ ID NO:34 or a light chain variable region (VL) having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:34; or

[0237] d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO:20 or a heavy chain variable region (VH) having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:20; and a light chain variable region (VL) comprising the sequence of SEQ ID NO:24 or a light chain variable region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:24; or

[0238] e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 10 or a heavy chain variable region (VH) having at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 14.

[0239] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, comprising:

[0240] a. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:50, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:54; or

[0241] b. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:40, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:44; or

[0242] c. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:30, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:34; or

[0243] d. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO:20, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:24; or

[0244] e. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 14.

[0245] In some embodiments, TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, are provided which bind to misfolded aggregated TDP-43 and to non-aggregated physiological TDP-43.

[0246] In some embodiments, TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, are provided which bind to monomeric and / or oligomeric and / or aggregated and / or post-translationally modified and / or truncated TDP-43, preferably human TDP-43.

[0247] In some embodiments, TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, are provided which bind to misfolded aggregated human TDP-43 and to non-aggregated physiological human TDP-43.

[0248] In some embodiments, TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, are provided which exhibit one or more, up to all, of the following characteristics:

[0249] a. inhibit aggregation of TDP-43 protein or fragments thereof,

[0250] b. block TDP-43 intercellular propagation;

[0251] c. disaggregate TDP-43 aggregates;

[0252] d. block TDP-43 seeding;

[0253] e. neutralize TDP-43 with seeding capacity;

[0254] f. block TDP-43 spreading;

[0255] g. enhance TDP-43 clearance; and

[0256] h. reduce levels of phosphorylated TDP-43 in vivo.

[0257] In some embodiments, TDP-43 binding molecules, in particular TDP-43 antibodies or antigen-binding fragments thereof, are provided which exhibit one or more, up to all, of the following characteristics:

[0258] a. inhibit aggregation of TDP-43 protein or fragments thereof,

[0259] b. block TDP-43 intercellular propagation;

[0260] c. block TDP-43 seeding;

[0261] d. block TDP-43 spreading;

[0262] e. enhance TDP-43 clearance; and

[0263] f. reduce levels of phosphorylated TDP-43 in vivo.

[0264] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that enhance TDP-43 clearance.

[0265] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that reduce TDP-43 pathology in vivo.

[0266] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that reduce levels of misfolded, aggregated TDP-43 and / or phosphorylated TDP-43 in vivo.

[0267] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that reduce levels of phosphorylated TDP-43 in the hippocampus.

[0268] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to an epitope within amino acid residues 304-414 of human TDP-43 (SEQ ID NO: 1).

[0269] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to an epitope within amino acid residues 304-313 of human TDP-43 (SEQ ID NO: 1).

[0270] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to an epitope consisting of amino acid residues 304-313 of human TDP-43 (SEQ ID NO: 1).

[0271] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to an epitope within amino acid residues 356-361 of human TDP-43 (SEQ ID NO: 1).

[0272] In some embodiments, provided are TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to an epitope consisting of amino acid residues 356-361 of human TDP-43 (SEQ ID NO: 1).

[0273] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which binds to an epitope within amino acid residues 397 to 407 of human TDP-43 (SEQ ID NO: 1).

[0274] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which binds to an epitope consisting of amino acid residues 397 to 407 of human TDP-43 (SEQ ID NO: 1).

[0275] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which binds to an epitope within amino acid residues 396 to 414 of human TDP-43 (SEQ ID NO: 1).

[0276] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which binds to an epitope consisting of amino acid residues 396 to 414 of human TDP-43 (SEQ ID NO: 1).

[0277] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which binds to a protease-resistant amyloid core of TDP-43. The protease-resistant amyloid core of TDP-43 consists of amino acids 272 to 360 of TDP-43.

[0278] In some embodiments, the TDP-43 binding molecule is an antibody or antigen-binding fragment thereof.

[0279] In some embodiments, there is provided a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, which has a dissociation constant (KD) of 1 nM or less, preferably 750 pM or less, 500 pM or less, 380 pM or less, 230 pM or less, 200 pM or less or 110 pM or less for binding to soluble TDP-43 (SEQ ID NO: 1). Further details on suitable assays to determine KD can be found in Example 3.

[0280] In some embodiments, the TDP-43 binding molecule is an IgA, IgD, IgE, IgM, IgGl, IgG2, IgG3 or IgG4 antibody or antigen-binding fragment thereof.

[0281] In a preferred embodiment, the TDP-43 binding molecule is an IgGl or IgG4 antibody or antigen binding fragment thereof.

[0282] In some embodiments, a TDP-43 binding molecule, in particular a TDP-43 antibody or antigen binding fragment thereof, is provided which comprises a Fc mutation, preferably a S228P mutation.

[0283] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid encodes a TDP-43 binding molecule, in particular a TDP-43 antibody and fragments thereof, as described herein.

[0284] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 18 encoding a heavy chain variable region (VH) of an anti- TPD-43 antibody as described herein.

[0285] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 19 encoding a light chain variable region (VL) of an anti- TPD-43 antibody as described herein.

[0286] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 28 encoding a heavy chain variable region (VH) of an anti- TPD-43 antibody as described herein.

[0287] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 29 encoding a light chain variable region (VL) of an anti- TPD-43 antibody as described herein.

[0288] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 38 encoding a heavy chain variable region (VH) of an anti- TPD-43 antibody as described herein.

[0289] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 39 encoding a light chain variable region (VL) of an anti- TPD-43 antibody as described herein.

[0290] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 48 encoding a heavy chain variable region (VH) of an anti- TPD-43 antibody as described herein.

[0291] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 49 encoding a light chain variable region (VL) of an anti- TPD-43 antibody described herein.

[0292] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 58 encoding a heavy chain variable region (VH) of an anti- TPD-43 antibody described herein.

[0293] In some embodiments, a (isolated) nucleic acid is provided, wherein the (isolated) nucleic acid comprises SEQ ID NO: 59 encoding a light chain variable region (VL) of an anti- TPD-43 antibody described herein.

[0294] XI. Kits

[0295] The present application also relates to a pharmaceutical composition comprising a TDP-43 binding molecule of the present application, in particular an antibody or antigen binding fragment thereof, as described herein, and a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0296] In some embodiments, a pharmaceutical composition is provided, comprising an (isolated) antibody described herein and a pharmaceutically acceptable carrier.

[0297] In some embodiments, binding molecules comprising the following conjugates are provided, particularly antibodies or antigen-binding fragments thereof: binding molecules described herein, particularly antibodies or antigen-binding fragments thereof, and conjugate molecules. The conjugates of the present invention may be referred to as immunoconjugates. Any suitable conjugate molecule may be used according to the present invention. Some suitable examples include, but are not limited to: enzymes (e.g., alkaline phosphatase or horseradish peroxidase), avidin, streptavidin, biotin, protein A / G, magnetic beads, fluorophores, radioisotopes (i.e., radioactive conjugates), nucleic acid molecules, detectable labels, therapeutic agents, toxins, and blood-brain barrier penetration portions. Conjugation methods are well known in the art, and several techniques for conjugating antibodies to labels or other molecules are commercially available. Conjugation is generally carried out by amino acid residues (e.g., lysine, histidine, or cysteine) contained within the binding molecules of the present invention. These may rely on methods such as the NHS (succinimide) ester method, isothiocyanate method, carbodiimide method, and periodate method. Conjugation can be achieved, for example, by generating fusion proteins. This is suitable in cases where the binding molecule is conjugated to another protein molecule. Therefore, suitable genetic constructs can be formed that allow expression of fusions of the binding molecule of the present invention with a marker or other molecules. Conjugation can be performed via suitable adapter portions to ensure proper spatial separation of the antibody and the conjugated molecule (e.g., a detectable marker). However, adapters are not required in all cases. In some embodiments, the TDP-43-specific binding molecule of the present invention is linked to a detectable marker.

[0298] This invention also relates to immunoconjugates comprising the TDP-43 binding molecule described herein, conjugated to one or more therapeutic agents, such as: chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant or animal-derived protein toxins, enzyme-active toxins, or fragments thereof), radioactive isotopes (i.e., radioactive conjugates), blood-brain barrier penetrating portions, or detectable markers. Various techniques exist for improving drug delivery across the blood-brain barrier (BBB) ​​as discussed herein, which are applied with necessary modifications. Non-invasive techniques include the so-called “Trojan horse approach,” in which the conjugate molecule delivers the binding molecule of the invention by binding to and mediating transport to a BBB receptor. Suitable molecules may comprise endogenous ligands or antibodies, particularly monoclonal antibodies, that bind to specific epitopes on the BBB receptor.

[0299] In some embodiments, an immunoconjugate is provided, wherein the immunoconjugate comprises the (isolated) antibody and therapeutic agent described herein. In some embodiments, a labeled antibody is provided, comprising the antibody described herein and a detectable label.

[0300] In some embodiments, the TDP-43 specific binding molecule is part of an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent.

[0301] In some embodiments, the TDP-43 specific binding molecule or the immunoconjugate comprising the same is present as a composition comprising the TDP-43 specific binding molecule.

[0302] In some embodiments, the TDP-43 specific binding molecule is part of a pharmaceutical composition comprising the TDP-43 specific binding molecule, or an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the TDP-43 specific binding molecule, in combination with a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0303] In some embodiments, the immunoconjugate comprising the TDP-43 binding molecule of the application crosses the blood brain barrier using a delivery vehicle or a blood brain barrier moiety. In some embodiments, the delivery vehicle comprises a liposome or an extracellular vesicle. In some embodiments, the TDP-43 binding molecule is linked to a blood brain barrier moiety. In some embodiments, the blood brain barrier moiety is a polypeptide or a small molecule, preferably a peptide, a receptor ligand, a single domain antibody (VHH), a scFv or a Fab fragment. In some embodiments, the blood brain barrier moiety binds to a blood brain barrier receptor, which can comprise a transferrin receptor, an insulin receptor or a low density lipoprotein receptor.

[0304] In some embodiments, the TDP-43 specific binding molecule is part of a detection and / or diagnostic kit comprising the TDP-43 specific binding molecule, or an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the TDP-43 specific binding molecule.

[0305] In some embodiments, the TDP-43 binding molecule described herein is used in a paired assay comprising the following steps:

[0306] a. incubating the sample with a capture antibody and a detection antibody;

[0307] b. incubating the mixture obtained in step a with a reagent suitable for detection by the detection antibody;

[0308] c. measuring the signal emitted by the detection antibody;

[0309] wherein the capture antibody is a TDP-43 binding molecule of the application.

[0310] In some embodiments, the TDP-43 binding molecules described herein are used in a paired assay comprising the following steps:

[0311] a. incubating the sample with a capture antibody and a detection antibody;

[0312] b. incubating the mixture obtained in step a with reagents suitable for detection by the detection antibody;

[0313] c. measuring the signal emitted by the detection antibody;

[0314] wherein the detection antibody is a TDP-43 binding molecule of the present application.

[0315] In some embodiments, the TDP-43 binding molecules described herein are used in a paired assay comprising the following steps:

[0316] a. incubating the sample with a capture antibody and a detection antibody;

[0317] b. incubating the mixture obtained in step a with reagents suitable for detection by the detection antibody;

[0318] c. measuring the signal emitted by the detection antibody;

[0319] wherein the capture antibody and the detection antibody are TDP-43 binding molecules of the present application.

[0320] In some embodiments, the present application provides a method of detecting TDP-43 in a sample comprising the following steps:

[0321] a. incubating the sample with a capture antibody and a detection antibody to produce a mixture;

[0322] b. incubating the mixture obtained in step a. with reagents suitable for detection of TDP-43 by the detection antibody; and

[0323] c. measuring the signal emitted by the detection antibody.

[0324] In one embodiment of the method of detecting TDP-43 in a sample, the capture antibody is a TDP-43 binding molecule of the present application. In another embodiment of the method of detecting TDP-43 in a sample, the detection antibody is a TDP-43 binding molecule of the present application. In another embodiment of the method of detecting TDP-43 in a sample, the capture antibody and the detection antibody are TDP-43 binding molecules of the present application. The capture antibody and the detection antibody can be the same antibody or different antibodies of the present application.

[0325] In some embodiments, a paired assay kit for detecting TDP-43 in a sample is provided. The paired assay kit comprises one or more TDP-43 binding molecules of the application. The kit can be an Enzyme-Linked Immunosorbent Assay (ELISA) kit. The kit can be a Single Molecule Array (SIMA) ) kit. The kit comprises a capture agent and / or a detection agent. The kit optionally further comprises a detection reagent. The TDP-43 binding molecules of the application can be provided in the kit as a capture agent (e.g. a capture antibody) and / or as a detection agent (e.g. a detection antibody).

[0326] In some embodiments, one or more TDP-43 binding molecules described herein are used in a paired assay comprising the step of incubating a sample with a capture antibody and a detection antibody, wherein the sample is human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF) and / or urine, preferably CSF.

[0327] Kits comprising the binding molecules of the application are also provided. In particular, such kits can be used to perform the diagnostic methods of the application, including the classification, monitoring and treatment selection methods. Thus, a kit for diagnosing a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or a TDP-43 proteinopathy, or for use in a method of the application, comprising a TDP-43 specific binding molecule of the application is provided. Such a kit can comprise all the necessary components for performing the methods provided herein. Typically, each component is stored separately in a single unit package. Suitable additional components to be included in the kit are, for example, buffers, detectable dyes, laboratory equipment, reaction vessels, instructions, etc. The instructions for use can be tailored to the specific method for which the kit is to be used. A TDP-43 binding molecule of the application, suitably labeled, is also provided, which can be included in such a kit.

[0328] In some embodiments, the TDP-43 specific binding molecules are used in immuno diagnostic methods for the prevention, diagnosis or treatment of a TDP-43 proteinopathy.

[0329] In some embodiments, a TDP-43 specific binding molecule, or an immunoconjugate in which a TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising a TDP-43 specific binding molecule is administered to a subject in need thereof, or used in a method of diagnosing or monitoring a TDP-43 related, in particular TDP-43 aggregate related, disease, disorder and / or abnormality, or TDP-43 proteinopathy selected from the following: frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia, and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in myotilin (MYOT) or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0330] In some embodiments, a TDP-43 specific binding molecule, or an immunoconjugate in which a TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising a TDP-43 specific binding molecule is administered to a subject in need thereof, or used in a method of diagnosing or monitoring a TDP-43 related, in particular TDP-43 aggregate related, disease, disorder and / or abnormality, or TDP-43 proteinopathy selected from the following: frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia, and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in myotilin (MYOT) or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0331] In other embodiments, the present application relates to any method for detecting, diagnosing or monitoring a TDP-43-related, in particular TDP-43-aggregate-related, disease, disorder and / or abnormality, or TDP-43 proteinopathy, selected from the group consisting of frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE) and limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0332] Preferably, the TDP-43-related, in particular TDP-43-aggregate-related, disease, disorder and / or abnormality, or TDP-43 proteinopathy is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and frontotemporal dementia (FTD). More preferably, the TDP-43-related, in particular TDP-43-aggregate-related, disease, disorder and / or abnormality, or TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS). More preferably, the TDP-43-related, in particular TDP-43-aggregate-related, disease, disorder and / or abnormality, or TDP-43 proteinopathy is Alzheimer's disease (AD). More preferably, the TDP-43-related, in particular TDP-43-aggregate-related, disease, disorder and / or abnormality, or TDP-43 proteinopathy is frontotemporal dementia (FTD).

[0333] In some embodiments, the TDP-43-specific binding molecule is used in a method for diagnosing a pre-symptomatic disease or for monitoring disease progression and treatment efficacy, or for predicting responsiveness, or for selecting subjects likely to respond to a treatment with a TDP-43-specific binding molecule. The method is preferably performed using a sample of human blood or urine. Most preferably, the method involves an ELISA-based assay or a surface adaptation assay.

[0334] In some embodiments, the TDP-43-specific binding molecule is used in a method wherein the TDP-43-specific binding molecule of the present application is contacted with a sample (e.g., blood, urine, cerebrospinal fluid, interstitial fluid (ISF) or brain tissue) for detecting, diagnosing or monitoring frontotemporal degeneration (FTD) or amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), Perry syndrome, limbic-predominant age-related TDP-43 encephalopathy (LATE) and / or Parkinson's disease (PD).

[0335] In some embodiments, the TDP-43 specific binding molecule is used in a method wherein the TDP-43 specific binding molecule of the application is contacted with a sample (e.g., blood, urine, cerebrospinal fluid, interstitial fluid (ISF), or brain tissue) to detect, diagnose, or monitor a disease selected from frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia, and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia, oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0336] In some embodiments, the TDP-43 specific binding molecule, or an immunoconjugate wherein the TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the TDP-43 specific binding molecule, is administered to a subject in need thereof, or is used to prevent, alleviate, or treat a disease, disorder, and / or abnormality associated with TDP-43, particularly with TDP-43 aggregates, or a TDP-43 proteinopathy, or frontotemporal degeneration (FTD) or amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD, including sporadic and familial forms of AD), chronic traumatic encephalopathy (CTE), Perry syndrome, and limbic-predominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0337] In some embodiments, a TDP-43 specific binding molecule, or an immunoconjugate in which a TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising a TDP-43 specific binding molecule is administered to a subject in need thereof, or is used in the treatment of a disease selected from frontotemporal dementia (FTD, e.g., sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine disease (Huntington's disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia, and myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD). Preferably, the disease treatment helps to maintain or improve psychological cognition, and / or to reduce the level of TDP-43 aggregates in the brain.

[0338] In some embodiments, a TDP-43 specific binding molecule, or an immunoconjugate in which a TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising a TDP-43 specific binding molecule is administered to a subject in need thereof, or is used in the manufacture of a medicament for the treatment of a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or a TDP-43 proteinopathy, or frontotemporal degeneration (FTD) or amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD, including sporadic and familial forms of AD), chronic traumatic encephalopathy (CTE), Perry syndrome, and limbic-predominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0339] Pharmaceutical formulations of an anti-TDP-43 antibody (a preferred type of TDP-43- specific binding molecule) or immunoconjugate as described herein are prepared by mixing such antibody or immunoconjugate having the desired degree of purity with one or more optional pharmaceutically acceptable carriers and / or excipients and / or diluents, (Remington's Pharmaceutical Sciences 16thEdition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Generally, the antibodies or fragments thereof are prepared as injectables, as either liquid solutions or lyophilized formulations. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include an interstitial drug dispersion agent such as a soluble neutral-active hyaluronidase glycoprotein (sHASEGP), e.g., a human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Halozyme). Baxter International, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase. Pharmaceutically acceptable excipients that can be used to formulate 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, such as sodium carboxymethylcellulose, polyethylene glycol, polyacangles, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. The diluent can be a buffer. It can comprise a salt selected from the group consisting of phosphate, acetate, citrate, succinate and tartrate, and / or wherein the buffer comprises histidine, glycine, TRIS glycine, Tris or mixtures thereof. It is further envisaged in the context of the present application that the diluent is a buffer selected from the group consisting of potassium phosphate, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, and histidine / histidine HC1 or mixtures thereof.

[0340] Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0341] The formulations herein can also contain more than one active ingredient, preferably those with complementary activities.

[0342] Active ingredients can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0343] Sustained-release preparations can be prepared. Some suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate in which the matrix of the preparation is in the form of shaped articles, e.g., films, or microcapsules. The preparations for intravenous administration are usually sterile. Sterility is readily achieved, for example, by filtration through a bacterial-retaining filter, e.g., a 0.2 μιη filter.

[0344] Any of the antigen binding molecules, anti-TDP-43 antibodies or immunoconjugates provided herein can be used in a method, e.g., a therapeutic method.

[0345] In another aspect, an anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is provided for use as a medicament. In a further aspect, an anti-misfolded TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is provided for use in a method of treatment. In certain embodiments, an anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is provided for use in the prevention, diagnosis and / or treatment of a TDP-43 proteinopathy. In a preferred embodiment of the application, an anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is provided for use in the prevention, diagnosis and / or treatment of a disease, disorder and / or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or a TDP-43 proteinopathy, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE) and / or limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0346] In another aspect, the present application provides the use of an anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate in the manufacture or preparation of a medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, e.g., as described below.

[0347] According to any of some embodiments, the “subject” or “individual” can be an animal, a mammal, preferably a human.

[0348] In another aspect, the application provides a pharmaceutical formulation comprising any of the anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates provided herein, e.g., for use in any of the methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates provided herein, and a pharmaceutically acceptable carrier and / or excipient and / or diluent (as discussed elsewhere herein). In another embodiment, the pharmaceutical formulation comprises any of the anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates provided herein, and at least one additional therapeutic agent, e.g., as described below.

[0349] The antibodies or immunoconjugates of the application can be used alone in therapy or in combination with other agents. For example, the antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application can be co-administered with at least one additional therapeutic agent that targets alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or a neuroinflammatory protein.

[0350] For example, the antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application can be co-administered with at least one additional therapeutic agent selected from, but not limited to, a neuropharmaceutical, an anti-beta amyloid antibody, an anti-tau antibody, a tau aggregation inhibitor (including small molecules), a beta-amyloid aggregation inhibitor (including small molecules), an anti-BACE1 antibody, a BACE1 inhibitor, an anti-alpha-synuclein inhibitor, an anti-alpha-synuclein antibody, and a neuroinflammatory inhibitor.

[0351] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, where administration of the antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application can occur prior to, simultaneously with, and / or following, administration of the additional therapeutic and / or adjunct agents. The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application can also be used in combination with radiation therapy.

[0352] The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application (and any additional therapeutic agents) can be administered by any suitable means, including parenterally, intrapulmonary, and intranasally, and, if desired, for local treatment, intralesionally, intrauterine, or intravesically. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be performed by any suitable route, for example, by injection, for example, intravenous or subcutaneous injection, depending in part on whether short- or long-term administration is involved. A variety of dosing schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at different time points, bolus administration, and pulse infusion.

[0353] The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the application can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors for consideration in this context include the particular disease, disorder, and / or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy that is being treated, the particular mammal that is being treated, the clinical condition of the individual subject, the cause of the disease, disorder, and / or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibodies or immunoconjugates need not, but optionally can be, formulated with one or more agents currently used in the prevention or treatment of the disease, disorder, and / or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy in question. The effective amount of such other agents depends on the amount of antibodies or immunoconjugates present in the formulation; the type of disease, disorder, and / or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy; or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes and schedules as in the methods described herein or about from 1 to 99% of the dosages described herein, or any dosage and any route of administration as is empirically / clinically determined to achieve the intended effect.

[0354] For the prevention or treatment of disease, the appropriate dosage of an antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the application (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and cause of the disease, whether the antibody or immunoconjugate is administered for preventative or therapeutic purposes, previous therapies, the clinical history and response to antibody or immunoconjugate of the subject, and the judgment of the attending physician. The antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is suitably administered to the subject at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate can be an initial candidate dosage, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or higher, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms is achieved. An exemplary dosage of the antibody or immunoconjugate is about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses (or any combination thereof) of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg can be administered to the subject. Such doses can be administered intermittently, e.g. every week or every three weeks (e.g. the subject receives about two to about twenty, or e.g. about six doses of the antibody). Higher initial dosages followed by one or more lower dosages can be employed. Other dosage regimens can be useful. The progress of the therapy is easily monitored by conventional techniques and assays.

[0355] It is understood that any of the above formulations or methods of treatment can be carried out using both the immunoconjugates and the anti-TDP-43 antibodies (preferred type of TDP-43 specific binding molecule) of the application.

[0356] In another aspect of the application, an article of manufacture containing materials useful for the treatment, prevention and / or diagnosis of a disease, disorder or abnormality associated with TDP-43, particularly TDP-43 aggregates, or a TDP-43 proteinopathy is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers can be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or in combination with another composition effective for treating, preventing and / or diagnosing a disease, disorder and / or abnormality associated with TDP-43, particularly TDP-43 aggregates, or a TDP-43 proteinopathy, and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate of the application. The label or package insert indicates that the composition is used for treating the condition of choice.

[0357] Moreover, the article of manufacture can comprise: (a) a first container comprising a composition, wherein the composition comprises an antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the application; and (b) a second container comprising a composition, wherein the composition comprises an additional therapeutic agent. The article of manufacture in this embodiment of the application can further comprise a package insert indicating that the compositions can be used to treat the particular condition. The article of manufacture can also, or instead, comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0358] In another embodiment, the application relates to a method of maintaining or improving cognitive memory capacity, motor and language function, or preventing and / or slowing the decline of cognitive memory capacity, motor and language function in a subject, comprising administering a binding molecule of the application, an immunoconjugate of the application, a composition of the application, or a pharmaceutical composition of the application.

[0359] In another embodiment, the application relates to a method of reducing TDP-43 levels, comprising administering a binding molecule of the application, an immunoconjugate of the application, a composition of the application, or a pharmaceutical composition of the application.

[0360] The methods of the application can comprise administering at least one additional treatment, preferably wherein the additional treatment is selected from, but not limited to: antibodies or small molecules targeting alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43 or neuroinflammatory proteins, in particular neuropharmaceuticals, anti-beta-amyloid antibodies, anti-tau antibodies, tau aggregation inhibitors, beta-amyloid aggregation inhibitors, anti-BACE1 antibodies, BACE1 inhibitors, anti-alpha-synuclein antibodies and neuroinflammatory inhibitors.

[0361] The present application also relates to a method of detecting TDP-43 comprising contacting a sample with a binding molecule of the present application, preferably an antibody of the present application, wherein the sample is a brain sample, a cerebrospinal fluid sample, an interstitial fluid (ISF) sample, a urine sample or a blood sample.

[0362] In other embodiments, the present application relates to a method of detecting and / or measuring the level of TDP-43 comprising contacting a sample with a binding molecule of the present application, preferably an antibody of the present application, using a single molecule array (SIMA) ) technology, wherein the sample is a human blood sample, a cerebrospinal fluid sample (CSF), an interstitial fluid (ISF) sample or a urine sample, preferably a CSF sample.

[0363] As described herein, the binding molecules of the present application, preferably the antibodies, target (i.e. bind to) specific domains or fragments of TDP-43. For example, the ACI-7071-810 H12-Ab1 antibody binds to the protease-resistant amyloid core of TDP-43. Accordingly, the methods can be based on detecting and / or measuring the level of specific domains or fragments of TDP-43. For example, the methods can be based on detecting and / or measuring the level of the protease-resistant amyloid core of TDP-43 in the C-terminal fragment. This can provide an indication of the disease or disease state, given that disease-specific proteolytic cleavage exposing this amyloid core was observed to further enhance its seeding activity, which is important for templated aggregation.

[0364] In certain embodiments, the dissociation constant (KD) of the TDP-43 binding molecules, in particular the TDP-43 antibodies and fragments thereof, as provided herein is ≤ 1 μΜ, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM or ≤ 0.001 nM (e.g. 10 -8 M or less, e.g. 10 -8 M to 10 -13 M, e.g. 10 -9 M to 10 -13In particular embodiments, the TDP-43 binding molecules, particularly TDP-43 antibodies and fragments thereof, can bind to TDP-43, particularly soluble TDP-43. For example, the TDP-43 binding molecules of the application can have a KD of 2 nM or less, in some embodiments 1 nM or less, in some more particular embodiments a KD of 750 pM or less, 500 pM or less, 380 pM or less, 230 pM or less, 200 pM or less, or 110 pM or less for binding to soluble full length TDP-43. This is demonstrated for the TDP-43 binding molecules of the application in Example 3 with reference to Table 4. In one embodiment, the binding affinity for full length (FL) TDP-43 can be evaluated by determining the dissociation constant (KD) using surface plasmon resonance (SPR; Biacore 8K, GE Healthcare Life Sciences). For a detailed description of a suitable SPR method that can be employed, reference is made to Example 3.

[0365] In one embodiment, the TDP-43 binding molecules, particularly TDP-43 antibodies and fragments thereof, can reduce the levels of pathological TDP-43 in the brain and improve / inhibit / reduce the formation of TDP-43 pathological conditions in vivo.

[0366] In another embodiment, the TDP-43 binding molecules, particularly TDP-43 antibodies and fragments thereof, can reduce the levels of phosphorylated TDP-43 in the brain and improve / inhibit / reduce the formation of TDP-43 pathological conditions in vivo.

[0367] In yet another embodiment, the TDP-43 binding molecules, particularly TDP-43 antibodies and fragments thereof, can reduce the levels of phosphorylated TDP-43 in the hippocampus and improve / inhibit / reduce the formation of TDP-43 pathological conditions in vivo.

[0368] The TDP-43 binding molecules, particularly antibodies or antigen binding fragments thereof, of the application typically bind to TDP-43 with high affinity. For example, they can show an EC50 value of 200 pM or less, 40 pM or less, 20 pM or less, or 10 pM or less, as determined by a Luminex assay. For further details of a suitable assay, reference is made to Example 2.

[0369] The TDP-43 binding molecules, particularly antibodies or antigen binding fragments thereof, of the application can have a half-life in mice of at least 10 days or at least 16 days. For further details of a suitable method for measuring the half-life in mice, reference is made to Example 9. BRIEF DESCRIPTION OF DRAWINGS

[0370] Figure 1. Graphical representation of phospho-TDP-43 (pTDP-43) quantification from ipsilateral hippocampus (A) or contralateral hippocampus (B) of TDP-43 proteinopathy mouse model treated with ACI-7071-806H5-Ab1 (B), ACI-7071-810H12-Ab1 (C) or mAb negative control antibody (D). (E) Uninoculated mice. (F) Monogenic CamK2a mice lacking the human TDP-43 transgene (WT-tTA). Mean ± SD. Figure 1a Figure 1b

[0371] Figure 2 Figure 4. Graphical representation of antibody plasma exposure at study endpoint following 13 weekly i.p. administrations of ACI-7071-810H12-Ab1 at 60 mg / kg each in CamKIIa-hTDP-43NLSm mice. Mean ± SD.

[0372] Figure 3 Figure 5. Graphical representation of linear relationship between pTDP-43 levels and ACI- 7071-810H12-Ab1 plasma concentration at study endpoint in ipsilateral (black line and circle) and contralateral (light gray line and triangle).

[0373] Figure 4 Figure 6. Immunoblotting performed with (A) ACI-7071-810H12-Ab1, (B) anti-total TDP-43 antibody or (C) anti-pTDP-43 (S409 / S410) antibody to detect TDP-43 in sarkosyl- insoluble brain extracts prepared from frontal cortex of FTLD-TDP case type A before (-) or after (+) limited proteolysis. Top arrow indicates expected molecular weight of full-length TDP-43 and pTDP-43. Bottom arrow indicates expected molecular weight of protease-resistant fragments. Bracket indicates expected molecular weight of TDP-43 C-terminal fragment (CTF). Bottom part of the blot provides an enhanced contrast image of the immunoblot at the expected molecular weight of protease-resistant fragments (about 8 to 15 kDa).

[0374] Example

[0375] Example 1. Preparation of TDP-43 vaccine composition

[0376] Liposome-based vaccines were prepared according to the protocol published in WO2012 / 055933. Vaccines comprising full-length TDP-43 (FL TDP-43) protein as antigen (Table 2, SEQ ID NO: 1) were used for antibody production.

[0377] ​​Table 2: TDP-43 protein and peptide antigen description

[0378]

[0379] Example 2. Generation of anti-TDP-43 antibodies

[0380] A. Immunization of mice

[0381] Female C57BL / 6JOlaHsd (C57BL / 6) and BALB / c OlaHsd (BALB / c) wild type mice (Harlan, USA) were received at the age of 9 weeks. Vaccination was started at the age of 10 weeks. Mice were vaccinated with full-length TDP-43 protein displayed on the surface of liposomes in the presence of monophosphoryl hexaacyl lipid A, 3-deacylated (synthetic) (3D-MPL®) as adjuvant. ) as adjuvant.

[0382] Mice were vaccinated by 200 μl subcutaneous injection (s.c.) at days 0, 4, 8, 21, 35 and 70. Mice were bled and heparinized plasma was prepared 7 days prior to immunization (preimmune plasma) and at days 15, 28, 42, 77 and 136 after the first immunization. Mice for myeloma fusion were additionally vaccinated with three daily boost injections of TDP-43 protein i.p. without adjuvant. Vaccine responses were measured in mouse plasma. Binding of plasma-derived antibodies from immunized mice to immobilized recombinant full-length (FL) TDP-43 indicated high titers of antibodies against TDP-43.

[0383] B. Generation of hybridomas and selection of subclones

[0384] Mice were euthanized and spleen cells from four individual mice were used for fusion with myeloma cells. Antibodies were screened from successfully fused hybridoma cell lines as follows. Diluted (1 :32) cell culture supernatants were analyzed using a Luminex bead-based multiplex assay (Luminex, The Netherlands). Luminex beads were conjugated with FL TDP-43 and IgG was captured with anti-mouse IgG-Fc antibodies (Jackson Immunoresearch, USA) specific for IgGl, IgG2a, IgG2b, IgG2c and IgG3 subclasses. Binding to beads conjugated with FL TDP-43 identified 386 hits derived from mice immunized with FL TDP-43 liposome vaccine.

[0385] Live hybridomas were cultured using serum-containing selection medium. Clones that preferentially bound to TDP-43 inclusions in human FTD brains and clones that bound to the C-terminus of TDP-43 were selected for further subcloning. After limiting dilution, clonal hybridomas were cultured in low immunoglobulin-containing medium and stable colonies were selected for antibody screening and selection. Antibodies shown in Table 3 were identified from this screening.

[0386] Table 3: EC50 values determined by Luminex assay

[0387]

[0388] Example 3. Characterization of antibodies by surface plasmon resonance (SPR)

[0389] Measurements were performed on a Biacore 8K instrument (GE Healthcare Life Sciences) by immobilizing soluble TDP-43 on a CM5 series S sensor chip (GE Healthcare, BR-1005-30).

[0390] KD determination by SPR against soluble TDP-43

[0391] The instrument was prepared with running buffer PBS-P+ and flow cells (Fc) 1 and 2 of channels 1 to 8 were activated with a fresh solution of EDC / NHS (amine coupling kit, ratio of both reagents 1 :1, GE Healthcare Life Sciences, BR-1006-33) at 10 pL / min for 420 seconds. Soluble TDP-43 (Selvita) was diluted in sodium acetate pH 4.5 to a final concentration of 5 pg / mL and injected at a flow rate of 10 pL / min onto Fc 2 for 80 seconds. All flow cells were quenched with 1 M ethanolamine (GE Healthcare Life Sciences, BR-1006-33) at 10 pL / min for 420 seconds. The immobilization level after ethanolamine quenching was approximately 370 RU on all eight channels. Three start-up cycles were run prior to analysis. Increasing mAb concentrations of 1.2 to 100 nM were injected in single cycle kinetics prepared from 3-fold serial dilutions in running buffer with a contact time of 300 seconds and a dissociation time of 3600 seconds at a flow rate of 30 pL / min. Each cycle was followed by a regeneration using 10 mM glycine-HCI pH 1.7 at 10 pL / min for a contact time of 30 seconds followed by a stabilization period of 300 seconds. Results obtained from single cycle kinetics were double referenced using blank Fc 1 and buffer cycles and evaluated using a 1 :1 kinetic fitting model with RI and global Rmax using Biacore 8K evaluation software. The following kinetic parameters were obtained (Table 4): association rate constant (ka), dissociation rate constant (kd), affinity constant (KD).

[0392] Table 4: ka, kd, KD values of antibodies to soluble TDP-43

[0393]

[0394] Example 4: Determination of the binding region

[0395] A. Epitope mapping of ACI-7071-704H9-Ab1, ACI-7071-707A6-Ab1, ACI-7071-801H1-Ab1 and ACI-7071-810H12-Ab1 using peptide arrays

[0396] Epitope mapping was determined using a custom-made peptide array library (Pepscan, Netherlands). Briefly, an overlapping linear peptide array covering the entire TDP-43 was used to define epitopes.

[0397] Synthesis of peptides

[0398] To reconstitute the epitope of the target molecule, a library of peptide-based mimics was synthesized using Fmoc-based solid phase peptide synthesis. Amino-functionalized polypropylene supports were obtained by grafting using a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt), and subsequent cleavage of the Boc-group using trifluoroacetic acid (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on the amino-functionalized solid supports by means of a custom-modified JANUS liquid handling station (Perkin Elmer).

[0399] ELISA screening

[0400] Antibodies were tested for binding to each synthetic peptide in a pepscan-based ELISA. The peptide array was incubated with primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated with a 1 / 1000 dilution of the appropriate antibody peroxidase conjugate for one hour at 25°C. After washing, peroxidase substrate 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl / ml of 3% H2O2 were added. After one hour, the color development was measured. Color development was quantified with a charge coupled device (CCD) camera and image processing system. The determined epitopes are provided in Table 5.

[0401] Table 5: Epitopes for the tested antibodies

[0402]

[0403] B. Determination of the binding region for ACI-7071-806H5-Ab1 using an ELISA assay

[0404] ACI-7071-806H5-Ab1 was screened by ELISA assay to determine the binding region using a library of biotinylated peptides on the N-terminus. The peptide sequences are provided in Table 6.

[0405] A 96-well streptavidin-coated ELISA plate was incubated with 5 pg / mL of biotinylated peptides. The plate was washed 4 times with 0.05% Tween-20 / PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20 / PBS for 1 hour at 37°C. Antibodies purified from hybridoma supernatant were then added at 1 pg / ml and incubated for 2 hours at 37°C, after which the plate was washed. An AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) was added at a dilution of 1 / 1000 in 0.05% Tween-20 / PBS for 1 hour at 37°C. After a final wash, the plate was incubated with pNPP (Sigma-Aldrich, Switzerland) AP substrate solution and read at 405 nm using an ELISA plate reader (Tecan). The determined binding regions are provided in Table 7. The test antibodies were found to bind to the following peptides: TP-52, TP-95, TP-96, TP-102 corresponding to regions 402-414, 396-414, 396-414 (pS403 / 404), and 402-414 (pS409 / 410), respectively.

[0406] Table 6: Peptides used to determine binding regions by ELISA

[0407] Peptide number a.a. position in SEQ ID NO: 1 TP-52 402 to 414 TP-95 396 to 414 TP-96 396 to 414 (with phosphorylated S403 / 404) TP-102 402 to 414 (with phosphorylated S409 / 410)

[0408] Table 7: Binding regions for test antibodies

[0409]

[0410] Example 5: Detection of TDP-43 in brain tissue from FTD / ALS subjects by immunohistochemistry

[0411] Target engagement was evaluated in immunohistochemistry experiments on tissue from FTD subject brains. Human FTD brain tissue was obtained from the UCSF Neurodegenerative Disease Brain Bank. All materials were collected from donors from whom the brain bank had obtained written informed consent for brain autopsy and use of materials and clinical information for research purposes. Immunohistochemistry was performed on 10 pm thick frozen sections using fluorescently labeled secondary antibodies for detection. The following antibodies were used as controls: rat monoclonal anti-phospho-TDP-43 p409 / 410 antibody (Biolegend, 829901) for detection of phosphorylated TDP-43; and secondary antibody without primary antibody (No 1° Ab) for detection of non-specific background.

[0412] All antibodies of the present invention bind to both non-aggregated physiological TDP-43 as well as aggregated TDP-43. A detailed evaluation of the binding characteristics is outlined in Table 8.

[0413] Table 8: Detection of TDP-43 in brain tissue from FTLD-TDP subjects

[0414] NA: data not available; - : not present; + / - : unclear; + : weak; ++ : moderate; +++ : abundant

[0415] Example 6: In vitro function in a recombinant TDP-43 aggregation assay

[0416] To evaluate the in vitro function of the antibodies, the ability of the antibodies to inhibit TDP-43 aggregation was tested. FL TDP-43 was produced recombinantly with a maltose binding protein (MBP) fusion separated at the C-terminus by a Tobacco Etch Virus (TEV) protease cleavage site. Aggregation of 2.5 mM TDP-43-TEV-MBP fusion protein in 30 mM Tris, 150 mM NaCl, pH 7.4 was induced by the addition of TEV protease (AcTEV, Invitrogen) in the presence of 2.5 mM of each anti-TDP-43 antibody or a negative control mAb that does not bind to TDP-43 and the absorbance was monitored at 600 nm in a pclear 96-well plate (Greiner) over 1.5 hours. For evaluation, the end point was normalized to the negative control mAb and the percentage of aggregated TDP-43 was calculated for each antibody. All antibodies significantly inhibited TDP-43 aggregation compared to the negative control mAb with more than 97% inhibition (Table 9). The p-value obtained for each mAb when compared to the control mAb was < 0.0001 using statistical analysis with one-way ANOVA followed by Dunnett’s multiple comparison test.

[0417] Table 9. Inhibition of TDP-43 aggregation

[0418] Antibody name % inhibition of TDP-43 aggregation ACI-7071-704 H9-Ab1 99.3 ACI-7071-707 A6-Ab1 98.1 ACI-7071-801 H1-Ab1 100 ACI-7071-806 H5-Ab1 100 ACI-7071-810 H12-Ab1 97.4

[0419] Example 7: In vitro function of FTLD-TDP brain-derived TDP-43 seeds in immunodepletion

[0420] Sarkosyl insoluble brain fractions (Sarko-spin) were prepared according to published protocols (Laferriere et al., 2019). Using immunodepleted with Protein G magnetic beads. For a single reaction, 20 pL of beads were used with 3 pg of antibody for 10 pg of sarkosyl insoluble brain extract (total protein). Prior to the addition of the antibody, the beads were first washed twice with 500 pL of PBS-0.05% Tween®-20 -20 and then resuspended with 100 pL of PBS-0.05% Tween®-20 -20. The beads were resuspended with 100 pL of PBS-0.05% Tween®-20 -20 with 30 pg / mL of antibody. The beads / antibody reaction was incubated for 30 minutes at room temperature under constant rotation and shaking (Hula Mixer 15920D, Thermofisher). The beads / antibody complex was washed twice with PBS-0.05% Tween®-20 TM -20 and once with PBS before the addition of the sarkosyl insoluble brain extract. The FTLD-TDP type A brain extract pool was diluted to 100 pg / mL. The beads / antibody complex was resuspended with 100 pL of extract and incubated for 30 minutes at room temperature under constant rotation and shaking. The supernatant was collected using the magnetic support as the immunodepleted fraction and characterized by western blot. ACI-7071-806H5-Ab1 was able to efficiently immunodeplete TDP-43 seeds from FTLD-TDP brain extract (Table 10).

[0421] Table 10. Immunodepletion of TDP-43 seeds in patient brain extracts

[0422] Antibody name Immune depletion ACI-7071-806 H5-Ab1 +++

[0423] NA: data not available; - : not present; + / - : unclear; + : weak; ++ : moderate; +++ : abundant

[0424] Example 8: In vitro function of TDP-43 aggregates uptake by microglia cells

[0425] For the preparation of mouse primary microglial cells, the cortex was enzymatically dissociated and mechanically dissociated from CD1 mice (Charles River, France) at postnatal day 5 (P5) as described in the Neural Tissue Dissociation Kit (P) (Miltenyi, 130-092-628). From the obtained cell suspension, microglial cells were purified using CD11b / c microbeads according to the manufacturer’s instructions (Miltenyi, 130-093-634). Microglial cells were plated at 3 x 105cells per well in 6-well plates in 2 mL of DMEM medium supplemented with 10% FBS, 2 mM glutamax, 1% penicillin / streptomycin, 50 ng / mL mouse recombinant M-CSF (R&D Systems, 416-MC-010) and 10 ng / mL mouse recombinant IL-34 (R&D Systems, 1110-MC-010) for 7 days. 5 ​Cells were seeded at a density of 60,000 cells per well into 60 inner wells of a 96-well tissue culture plate (Falcon, 353219) and maintained in complete growth medium adapted from (5). Growth medium consisted of DMEM / F12 (Gibco, 31331-093) supplemented with: 2.5% heat-inactivated FBS, 1% PS, 200 ng / mL tumor growth factor beta 2 (TGF-b2; Peprotech, 100-35B), 100 ng / mL interleukin 34 (IL-34; R&D Systems), 5 pg / ml N-acetyl cysteine (Sigma, A9165), 5 pg / ml insulin (Sigma, I6634), 100 pg / mL apotransferrin (Sigma, T1147), 100 ng / mL sodium selenite (Sigma, S-5261), and ovine wool cholesterol (1.5 pg / mL, Avanti Polar Lipids). During the experiment, basal medium was used, i.e., DMEM / F12 (Gibco, 31331-093) supplemented with: 1% penicillin / streptomycin, 5 pg / ml N-acetyl cysteine (Sigma, A9165), 5 pg / ml insulin (Sigma, I6634), 100 pg / mL apotransferrin (Sigma, T1147), and 100 ng / mL sodium selenite (Sigma, S-5261).

[0426] Microglia cells were plated at 30,000 cells per well in growth medium and incubated for 48 hours. The immunocomplex was prepared at 2x final concentration in basal medium by mixing pHrodo TM labeled TDP-43 aggregates and ACI-7071-806 H5-Ab1, ACI-7071-810 H12-Ab1 or negative control mAb and incubated overnight at 4°C. The dilution plate was equilibrated at room temperature while the cells were washed three times with basal medium. After the last wash, 100 pL of basal medium was left on the cells to which 100 pL of pHrodo TMLabeled TDP-43 aggregates. Cells were immediately placed inside the Incucyte for 24 hours of real-time imaging of phase contrast (to delineate microglia) and green fluorescence (to quantify labeled TDP-43 within microglia). Uptake of TDP-43 aggregates by microglia was significantly increased in the presence of both mAbs ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1 (Table 11). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison when compared to the negative control mAb, with p-value < 0.001 obtained for each mAb.

[0427] Table 11. Percentage increase in uptake of immune complexes by microglia

[0428]

[0429] Example 9: Pharmacokinetics in mice

[0430] Nine female mice (C57BL / 6 strain, 7 to 11 weeks old) were used for each antibody tested in this study. Animals were purchased from Lingchang / Vital River Laboratory Animal Co., Ltd. Free plasma concentrations of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1 were determined after a single intraperitoneal (i.p.) administration of 60 mg / kg of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1, respectively. Plasma samples were collected from the tail vein of 3 mice per time point at the following time points after dose administration: 0.25 hours, 1 hour, 8 hours, 24 hours, 72 hours and at days 7, 10, 14, 21 and 28, except at the time point 72 hours, where 6 mice were sampled. Table 12 shows the half-life obtained for both antibodies.

[0431] Table 12. Half-life of antibodies in mice

[0432] Antibody name Half-life (days) (mouse, i.p.) ACI-7071-806 H5-Ab1 10.3 ACI-7071-810 H12-Ab1 16.8

[0433] Both antibodies exhibit good and similar PK parameters, making them desirable candidates for applications where antibody half-life in vivo is important, such as therapeutic use in humans.

[0434] Example 10: Functional potency in vivo

[0435] The objective of this study was to evaluate the therapeutic effect of antibodies delivered intraperitoneally (i.p.) in a mouse model of TDP-43 proteinopathy.

[0436] Methods

[0437] In-life phase

[0438] Biallelic CamKIIa-hTDP43NLSm animals were generated by crossing hemizygous females (JAX Stock #14650: B6; C3-Tg(tetO-TARDBP*)4Vle / J) with hemizygous males (JAX Stock #007004: B6.Cg-Tg(CamKIIa-tTA)1Mmay / DboJ). Breeders and mice were kept on a 200 mg / kg doxycycline (DOX) diet until 12.5 > 2 weeks of age. At 13.5 > 2 weeks of age, CamKIIa-hTDP43NLSm mice were deeply anesthetized with 1 mg / kg buprenorphine and fixed in a stereotaxic frame. Sarkosyl-insoluble extracts from FTLD-TDP cases brains were sonicated prior to injection in the dorsal hippocampus. Each injection site (needle introduced in the left hemisphere according to bregma coordinates: -2.0 mm anterior to the midline and 1.3 mm left; three dorsal hippocampus locations with an initial depth of -1.95 mm subdural and subsequent partial withdrawal of the needle to -1.55 mm for the second injection and again to -1.15 mm for the last injection) received 1 μΐ of Sarkosyl-insoluble extract at a rate of 0.3 μΐ / min with a 4 min rest period after injection. One day later, intraperitoneal injections of mAb (60 mg / kg) were initiated and continued weekly for 13 consecutive weeks. Three different antibodies were tested: ACI-7071-806H5-Ab1, ACI-7071-810H12-Ab 1 and a negative control mAb. Terminal tissue collection was performed 3 months after injection.

[0439] Tissue fixation, sectioning, immunofluorescence staining and quantification

[0440] Frozen tissue blocks were sectioned at a thickness of 20 pm per slice. Staining was performed on a Leica BOND-RX. Coronal sections from 4 levels covering the hippocampus were processed for immunolabeling. For triple pTDP43 / NeuN / Iba1 immunofluorescence (IF) staining, slides were first subjected to a fixation / permeabilization step in methanol / acetone (1 :1) for 10 min and washed in PBS. Epitopes were then repaired in Leica ER1 buffer pH 6 (AR 9640) at 100°C for 10 min, followed by incubation with a protein blocker (PowerVision IHC / ISH Super Blocking, Leica, Ref. PV6122). Slides were then incubated with primary antibodies in two steps, first with phospho-TDP43 Ab (Biolegend, Ref. 829901, rat Ab, 1 / 500), followed by a mixture of NeuN (Millipore, Ref. MAB377(CH), mouse Ab, 1 / 500) and Iba1 (Wako, Ref. 019-19741, rabbit Ab, 1 / 1000). Next, secondary antibodies were incubated in two steps, first with a mixture of three antibodies anti-mouse-Cy3 (Jackson, goat Ab, 1 / 200), anti-rabbit-Alexa488 (Jackson, goat Ab, 1 / 200) and anti-rat-biotin (Jackson, goat Ab, 1 / 250), and second with streptavidin-Cy5 (Jackson, 1 / 300). Finally, slides were incubated with DAPI (1 / 300). All antibodies were diluted in BOND antibody diluent (Cat. No. AR9352) and slides were mounted in anti-fade and coverslipped.

[0441] IF slides were digitized using an Axio Scan.Z1 digital whole slide scanner (Carl Zeiss, Canada). Images were reviewed for quality control (QC) and final images were transferred to a Biopensive server for image processing and analysis. ROIs were defined using a U-Net convolutional neural network trained on manually drawn tissue section datasets. ROIs were then reviewed for visual QC and manually adjusted when needed. Using Biospective PERMITS software, the number of pTDP43-positive cells was quantified in the CA1 and CA3 subfields of the hippocampus. TMSoftware performed IHC staining quantification for each digitized IHC slide (shown as mean staining density in Figure 1). In addition, double co-localization of pTDP-43 and NeuN and triple co-localization of pTDP-43, NeuN and Ibal were calculated from the segmented images. IHC analysis and quantification were performed in a blinded fashion with respect to the cohort. Any potential outliers due to technical reasons were removed prior to data unblinding. Data are expressed as mean ± standard deviation.

[0442] Statistical analysis

[0443] Statistical analysis was performed in MATLAB. First, normality of data was assessed using normal probability plots, followed by assessment of variance homogeneity if applicable. Treatment groups were compared using one-way ANOVA (anova1) with post-hoc comparisons using Tukey honest significant difference (multcompare), or for non-normally distributed clinical data, Kruskal-Wallis (kruskalwallis). For body weight measurements, two-way mixed ANOVA was also used to investigate any interaction between group and time point. As an additional exploratory measure, direct t-tests or Mann-Whitney comparisons were performed between the two groups. (P values < 0.05 are indicated by an asterisk *).

[0444] Results

[0445] Inoculation of brain extract in double transgenic mice (CamKIIa-hTDP43NLSm) resulted in pTDP-43 pathology in both ipsilateral (same brain hemisphere where brain extract was injected) and contralateral (brain hemisphere opposite to the brain extract injection site) brain hippocampus when compared to non-inoculated mice (E in Figure 1) or single gene CamK2a mice lacking the human TDP-43 transgene (WT-tTA) (F in Figure 1).

[0446] Thirteen weekly intraperitoneal administrations of ACI-7071-810H12-Ab1 resulted in steady state antibody plasma levels of 1787 μg / mL (R-range 1430- 2080 μg / mL) measured at the end of the study. Figure 2). Assuming a blood to brain permeability of 0.1%, the theoretical concentration in the brain was calculated to be 1787 ng / mL. Comparison of the ACI-7071-810H12-Ab1 KD (0.38 pM or 57 ng / ml) to the theoretical brain concentration (1787 ng / mL ~ 30 times higher than the KD) highlights the favorable ratio of ACI-7071-810H12-Ab1 for therapeutic use. ACI 7071-810H12-Ab1 treated mice showed statistically significant reductions in pTDP-43 in both ipsilateral and contralateral hippocampus compared to negative control mAb (Fig. 1C) compared to negative control mAb (Fig. 1D). In addition, linear regression analysis showed a negative trend between ACI-7071-810H12-Ab1 antibody exposure and pTDP-43 pathology (in both ipsilateral and contralateral) confirming an exposure-response relationship (Ipsilateral: r 2 = 0.37 and p = 0.06; Contralateral: r 2 = 0.23 and p = 0.17; Figure 3 ). ACI-7071-806H5-Ab1 showed a trend of reduction in pTDP-43 pathology in both ipsilateral and contralateral (Fig. 1B). These data suggest that the monoclonal antibodies tested can effectively capture extracellular TDP-43 that leads to spread of TDP-43 pathology in this mouse model.

[0447] Example 11 Antibody Sequencing

[0448] Clonal hybridoma cell lysates were used for gene sequencing of the variable regions. Mouse hybridomas were harvested and lysed using a lysis buffer containing guanidium salts to inactivate RNases. cDNA was obtained by reverse transcription of total mRNA. DNA fragments encoding the antibody variable regions were amplified by RACE-PCR (Takara Bio, Cat# 634839) using specific primers annealing in the antibody constant regions. PCR products were gel purified and cloned into a shuttle vector for Sanger sequencing. Sequencing was performed in both directions to provide overlap at both ends. Sequences were analyzed using a multiple sequence alignment (Clustal tool) and annotated using the Kabat algorithm as described in Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242 (1991). The nucleotide sequences of the heavy chain (VH) and light chain (VL) variable regions are shown in Table 13. The translated protein sequences of the selected heavy chain (VH) and light chain (VL) variable regions, and their complementarity determining regions (CDRs) are shown in Table 14.

[0449] Example 12. Binding of ACI-7071-810H12-Ab1 to the protease-resistant amyloid core of TDP-43

[0450] The binding of the ACI-7071-810H12-Ab1 antibody to the protease-resistant amyloid core of TDP-43 was evaluated.

[0451] Immunoblotting was performed on sarkosyl-insoluble brain extracts from FTLD-TDP type A patients (prepared as described above; Laferriere et al., 2019) with and without streptoprotein treatment. Briefly, the sarkosyl-insoluble sample was treated with 0.4 mg / mL streptoprotein (Sigma, 10165921001) at 21°C for 1 hour, followed by centrifugation at 20,000 g for 30 minutes at 4°C. The supernatant was discarded, and the precipitate was resuspended in PBS by sonication 30 times at an amplitude of 30 using a Q-Sonica probe.

[0452] Sarkosyl insoluble extract was mixed with 4× sample loading buffer and 0.1 mM dithiothreitol (DTT) and boiled at 95 °C for 10 min. The sample was loaded onto a 4% to 12% Bis-Tris gel and migrated at 100 volts (V) for 90 min. Proteins were transferred to a nitrocellulose membrane using the iBlot2 system (20 mA, 7 min). Block the membrane in blocking buffer at room temperature for 1 hour with stirring. Add primary antibody (ACI-7071-810H12-Ab1, TDP-43 (Proteintech, 60019-2-IG) or pTDP-43 (Biolegend, 829901)) to PBS-0.1% Tween-20 / Dilute to 1:1000 in blocking buffer (1:1) and incubate overnight on the membrane at 4°C with stirring. Wash the membrane three times with stirring in PBS-0.1% Tween-20. The secondary antibodies, donkey anti-mouse IRDye680CW and donkey anti-rat IRDye800CW, were then added. Dilute 1:10,000 in the same buffer as the primary antibody and incubate the membrane at room temperature for 1 hour with constant stirring. Wash the membrane three times in PBS containing 0.1% Tween-20 before using... The Odyssey imager was used for scanning.

[0453] Immunoblotting of the FTLD-TDP type A sarkosyl insoluble brain extract showed that, in addition to the 43 kDa band corresponding to full-length TDP-43, (A)ACI-7071-810H12-Ab1 also bound to the C-terminal fragment. Figure 4). Similar signals were obtained with the antibody binding the TDP-43 phosphorylation epitope pS409 / 410 using (C), indicating that the C-terminal fragment bound by ACI-7071-810H12-Ab1 retains the disease-specific phosphorylation site Figure 4 ). However, after limited proteolysis using pronase treatment of sarkosyl-insoluble FTLD-TDP type A brain extracts, only ACI-7071-810H12-Ab1 showed binding to the protected core of TDP-43 Figure 4 ) in contrast, antibodies binding to the N-terminal ((B) TDP-43 antibody binding in the RRM2 region) or C-terminal region (pS409 / 410 antibody, (C)) of the amyloid core did not show signals on immunoblots of samples after limited proteolysis.

[0454] These data establish that ACI-7071-810H12-Ab1 binds to the protease-resistant amyloid core of full-length TDP-43 and TDP-43 fragments (expected to be about 8 to 9 kDa based on the reported structure; Arseni et al., 2021; Arseni et al., 2023). Such binding properties are valuable for use in therapy, as the exposure of the amyloid core after disease-specific proteolytic cleavage has been shown to further enhance TDP-43 seeding activity (Kumar et al., 2023). In addition, these binding properties are valuable for use in diagnostics, as the proteolytic processing of TDP-43 and its enrichment in patient brains has proven to be a disease-specific pathological feature. In summary, these data further support the potential of ACI-7071-810H12-Ab1 for use as a therapeutic or diagnostic antibody.

[0455] Table 13: Nucleotide sequences of heavy chain (VH) and light chain (VL) variable regions

[0456]

[0457]

[0458]

[0459] Table 14: Amino acid sequences of heavy chain (VH) and light chain (VL) variable regions and their CDRs

[0460]

[0461]

[0462]

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[0544] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes and purposes.

[0545] The scope of the application is not intended to be limited to the particular embodiments described in this document. Indeed, various modifications of the application, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Furthermore, all aspects and embodiments of the application described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken in isolation from other aspects of the application.

Claims

1. A TDP-43 binding molecule, in particular a TDP-43 antibody or antigen-binding fragment thereof, comprising: a. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 51, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 52, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 56, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 57; or b. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; or c. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32, and a VH-CDR3 comprising the amino acid sequence PC (Pro-Cys); and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; or d. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:

17. e. a heavy chain variable region (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable region (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

2. The TDP-43 binding molecule of claim 1, comprising: a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50 or a heavy chain variable region (VH) having at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 or a heavy chain variable region (VH) having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 or a light chain variable region (VL) having at least 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 30 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 34 or a light chain variable region (VL) having at least 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 20 or a heavy chain variable region (VH) having at least 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 24 or a light chain variable region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 24; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 10 or a heavy chain variable region (VH) having at least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

14.

3. The TDP-43 binding molecule of any one of the preceding claims, comprising: a. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 40, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 30, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 34; or d. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 20, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 24; or e. a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 10, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO:

14.

4. The TDP-43 binding molecule of any one of the preceding claims, which binds to misfolded, aggregated TDP-43 and to non-aggregated physiological TDP-43.

5. The TDP-43 binding molecule of any one of the preceding claims, which binds to monomeric and / or oligomeric and / or aggregated and / or post-translationally modified and / or truncated TDP-43, preferably wherein the TDP-43 is human TDP-43.

6. The TDP-43 binding molecule of any one of the preceding claims, which binds to misfolded, aggregated human TDP-43 and to non-aggregated physiological human TDP-43.

7. The TDP-43 binding molecule of any one of the preceding claims, which exhibits one or more, up to all, of the following characteristics: a. inhibits aggregation of TDP-43 protein or fragments thereof, b. blocks TDP-43 intercellular propagation; c. disaggregating TDP-43 aggregates; d. blocking TDP-43 seeding; e. neutralizing TDP-43 with seeding capacity; f. blocking TDP-43 spreading; g. enhancing TDP-43 clearance; and h. reducing levels of phosphorylated TDP-43 in vivo.

8. The TDP-43 binding molecule of any one of the preceding claims, which exhibits one or more, up to all, of the following characteristics: a. inhibits aggregation of TDP-43 protein or fragments thereof, b. blocks TDP-43 intercellular transmission; c. blocks TDP-43 seeding; d. blocks TDP-43 spreading; e. enhances TDP-43 clearance; and f. reduces levels of phosphorylated TDP-43 in vivo.

9. The TDP-43 binding molecule of any one of the preceding claims, which enhances TDP-43 clearance.

10. The TDP-43 binding molecule of any one of the preceding claims, which alleviates a TDP-43 pathological condition in vivo.

11. The TDP-43 binding molecule of any one of the preceding claims, which reduces levels of misfolded aggregated TDP-43 and / or phosphorylated TDP-43 in vivo.

12. The TDP-43 binding molecule of any one of the preceding claims, which reduces levels of phosphorylated TDP-43 in the hippocampus.

13. The TDP-43 binding molecule of any one of the preceding claims, which binds to an epitope within amino acid residues 304 to 414 of human TDP-43 (SEQ ID NO: 1).

14. The TDP-43 binding molecule of any one of the preceding claims, which binds to an epitope within amino acid residues 304 to 313 of human TDP-43 (SEQ ID NO: 1).

15. The TDP-43 binding molecule of any one of claims 1 to 13, which binds to an epitope within amino acid residues 396 to 414 of human TDP-43 (SEQ ID NO: 1).

16. The TDP-43 binding molecule of any one of claims 1 to 14, which binds to a protease-resistant amyloid core of TDP-43.

17. The TDP-43 binding molecule of any one of the preceding claims, which is an antibody or antigen-binding fragment thereof.

18. The TDP-43 binding molecule of any one of the preceding claims, which has a dissociation constant (KD) of 1 nM or less, preferably 750 pM or less, 500 pM or less, 380 pM or less, 230 pM or less, 200 pM or less, or 110 pM or less for binding to soluble TDP-43 (SEQ ID NO: 1).

19. The TDP-43 binding molecule of any one of the preceding claims, which is an IgA, IgD, IgE, IgM, IgGl, IgG2, IgG3, or IgG4 antibody or antigen-binding fragment thereof.

20. The TDP-43 binding molecule of any one of the preceding claims, which is an IgGl or IgG4 antibody or antigen-binding fragment thereof.

21. The TDP-43 binding molecule of any one of the preceding claims, comprising an Fc mutation, preferably a S228P mutation.

22. An immunoconjugate comprising the TDP-43 binding molecule according to any one of the preceding claims.

23. The immunoconjugate of claim 22, wherein the immunoconjugate crosses the blood brain barrier using a delivery vehicle or a blood brain barrier moiety.

24. The immunoconjugate of claim 23, wherein the delivery vehicle comprises a liposome or an extracellular vesicle.

25. The immunoconjugate of claim 23, wherein the TDP-43 binding molecule is linked to the blood brain barrier moiety.

26. The immunoconjugate of claim 23 or 25, wherein the blood brain barrier moiety is a polypeptide or a small molecule, preferably a peptide, a receptor ligand, a single domain antibody (VHH), a scFv or a Fab fragment.

27. The immunoconjugate of claim 23, 25 or 26, wherein the blood brain barrier moiety binds to a blood brain barrier receptor.

28. The immunoconjugate of claim 27, wherein the blood brain barrier receptor comprises a transferrin receptor, an insulin receptor or a low density lipoprotein receptor.

29. A labeled binding molecule, in particular a labeled antibody, comprising the TDP-43 binding molecule according to any one of claims 1 to 21.

30. A pharmaceutical composition comprising the TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28; and a pharmaceutically acceptable carrier and / or excipient and / or diluent.

31. The TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the pharmaceutical composition of claim 30 for human or veterinary medical use.

32. The TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the pharmaceutical composition of claim 30 for use in the prevention, alleviation, treatment of a TDP-43 associated disease, disorder and / or abnormality, or a TDP-43 proteinopathy.

33. The TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the labeled binding molecule of claim 29 or the pharmaceutical composition of claim 30 for diagnostic use.

34. The TDP-43 binding molecule or immunoconjugate, the labeled binding molecule or the pharmaceutical composition for use according to claim 33 for the diagnosis of a TDP-43 associated disease, disorder and / or abnormality, or a TDP-43 proteinopathy.

35. The TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the labeled binding molecule of claim 29 or the pharmaceutical composition of claim 30 for research use, in particular as an analytical tool or reference molecule.

36. The TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28, the labeled binding molecule of claim 29, or the pharmaceutical composition of claim 30, for use as a diagnostic tool for monitoring a TDP-43 associated disease, disorder and / or abnormality, or a TDP-43 proteinopathy.

37. The TDP-43 binding molecule or immunoconjugate or labeled binding molecule or pharmaceutical composition for use according to any one of claims 32, 34 or 36, wherein the TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy is: frontotemporal dementia (FTD, e.g. sporadic or familial, with or without motor neuron disease (MND), with granulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick’s disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), nonfluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g. sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer’s disease (AD, including sporadic and familial forms of AD), Down’s syndrome, familial British dementia, polyglutamine disease (Huntington’s disease and spinocerebellar ataxia type 3 (SCA3; also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with valosin-containing protein (VCP); associated with Paget disease of the bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with rimmed vacuoles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB) or Parkinson’s disease (PD).

38. The TDP-43 binding molecule or immunoconjugate or labeled binding molecule or pharmaceutical composition for use according to claim 37, wherein the TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy is: frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), chronic traumatic encephalopathy (CTE) or limbic-predominant age-related TDP-43 encephalopathy (LATE).

39. The TDP-43 binding molecule or immunoconjugate or labeled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS).

40. The TDP-43 binding molecule or immunoconjugate or labeled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy is Alzheimer’s disease (AD).

41. The TDP-43 binding molecule or immunoconjugate or labeled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy is frontotemporal dementia (FTD).

42. A method of preserving or improving cognitive memory capacity or slowing memory loss in an individual having a TDP-43 associated disease, disorder and / or abnormality, or TDP-43 proteinopathy, comprising administering to the individual the TDP-43 binding molecule of any one of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the pharmaceutical composition of claim 30.

43. A method of reducing the level of aggregated TDP-43 and / or phosphorylated TDP-43 in an individual, comprising administering to the individual the TDP-43 binding molecule of claims 1 to 21 or the immunoconjugate of any one of claims 22 to 28 or the pharmaceutical composition of claim 30.

44. The method of claim 42 or 43, wherein the method comprises administering at least one additional therapeutic agent.

45. The method of claim 44, wherein the additional therapeutic agent targets alpha-synuclein, BACE1, Tau, beta-amyloid, TDP-43, or a neuroinflammatory protein.

46. A nucleic acid molecule encoding the TDP-43 binding molecule of any one of claims 1 to 21.

47. The nucleic acid molecule of claim 46, comprising the nucleotide sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 58, or SEQ ID NO:

59.

48. A nucleic acid molecule encoding a TDP-43 binding molecule, comprising the nucleotide sequence set forth in: a. a heavy chain variable region (VH) encoded by SEQ ID NO: 58 and a light chain variable region (VL) encoded by SEQ ID NO: 59; or b. a heavy chain variable region (VH) encoded by SEQ ID NO: 48 and a light chain variable region (VL) encoded by SEQ ID NO: 49; or c. a heavy chain variable region (VH) encoded by SEQ ID NO: 38 and a light chain variable region (VL) encoded by SEQ ID NO:

39. ​ ​ c. a heavy chain variable region (VH) encoded by SEQ ID NO: 38 and a light chain variable region (VL) encoded by SEQ ID NO: 39; or d. a heavy chain variable region (VH) encoded by SEQ ID NO: 28 and a light chain variable region (VL) encoded by SEQ ID NO: 29; or e. a heavy chain variable region (VH) encoded by SEQ ID NO: 18 and a light chain variable region (VL) encoded by SEQ ID NO:

19.

49. The nucleic acid molecule of claim 46, 47 or 48, wherein the nucleic acid is part of a viral vector for targeted delivery into the blood brain barrier or any other cell type in the CNS.

50. The nucleic acid molecule of claim 49, wherein the targeted delivery is to endothelial cells of the blood brain barrier, pericytes of the blood brain barrier or astrocytes, preferably to endothelial cells of the blood brain barrier.

51. The nucleic acid of claim 50, wherein the viral vector is a recombinant adeno-associated viral vector (rAAV), preferably a recombinant adeno-associated viral vector selected from the group consisting of AAV1 to AAV12.

52. A recombinant expression vector comprising the nucleic acid of any one of claims 46 to 51.

53. A host cell comprising the nucleic acid of any one of claims 46 to 51 and / or the vector of claim 52.

54. A cell-free expression system comprising the recombinant expression vector of claim 52.

55. A method for producing a TDP-43 binding molecule, in particular an antibody or an antigen binding fragment thereof, comprising the steps of: a. culturing the host cell of claim 53 or the cell-free expression system of claim 54 under conditions suitable for producing said TDP-43 binding molecule, in particular said antibody or antigen binding fragment thereof; and b. isolating said TDP-43 binding molecule, in particular said antibody or antigen binding fragment thereof.

56. A method of detecting and / or quantifying TDP-43 in a sample obtained from a subject, the method comprising contacting the sample with the TDP-43 binding molecule of any one of claims 1 to 21 and comparing the level of TDP-43 in the sample to the level of TDP-43 in a control sample.

57. Use of the TDP-43 binding molecule of any one of claims 1 to 21 in a paired assay comprising the steps of: a. incubating a sample with a capture antibody and a detection antibody; b. incubating the mixture obtained in step a. with reagents suitable for detection by the detection antibody; c. measuring the signal emitted by the detection antibody; wherein the capture antibody is selected from the group consisting of the antibodies defined in any one of claims 1 to 21.

58. Use of the TDP-43 binding molecule of claim 57, wherein the detection antibody is selected from the group consisting of the antibodies defined in any one of claims 1 to 21.

59. The method of claim 56 or the use of the TDP-43 binding molecule according to claim 57 or 58, wherein the sample is human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF) and / or urine, preferably CSF.

60. A kit for diagnosing a TDP-43 associated disease, disorder and / or abnormality, or a TDP-43 proteinopathy, or for use according to any one of claims 31 to 41, or for the method of any one of claims 42 to 45, the kit comprising a TDP-43 binding molecule according to any one of claims 1 to 21.

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