CN120917043A - Anti-TDP-43 binding molecules and uses thereof - Google Patents

Abstract

This invention pertains to the field of transactivation response DNA-binding proteins (TARDB, also known as TDP-43) with a molecular weight of 43 kDa. This invention relates to TDP-43-specific binding molecules, particularly anti-TDP-43 antibodies or their antigen-binding fragments or derivatives, and their uses. This invention provides means and methods for diagnosing, preventing, alleviating, 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 limbic-dominant age-related TDP-43 encephalopathy (LATE).

Description

Anti-TDP-43 binding molecules and uses thereof

Technical Field

The present invention is in the field of transactivating response DNA binding proteins (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 a disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteinopathies (proteinopathy) including, but not limited to, frontotemporal dementia (Frontotemporal dementia, FTD), amyotrophic lateral sclerosis (amyotrophic lateral sclerosis, ALS), alzheimer's Disease (AD), parkinson's Disease (PD), chronic traumatic encephalopathy (Chronic Traumatic Encephalopathy, CTE) and edge-dominated age-related TDP-43encephalopathy (limbic-predominant age-RELATED TDP-43enephalopathy, LATE).

Background

Age-related encephalopathy, characterized by pathological aggregation of proteins in the central nervous system (central nervous system, CNS) (proteinopathies) and peripheral organs, is one of the leading causes of disability and mortality worldwide. The best characterized protein for forming aggregates is amyloid beta in alzheimer's disease and related disorders. Other disease-associated, aggregation-prone proteins that lead to neurodegeneration include, but are not limited to, tau, alpha-synuclein (aSyn, a-syn), huntingtin (huntingtin), sarcoma fusion protein (FUS), dipeptide repeat protein (DIPEPTIDE REPEAT protein, DPR) produced by non-conventional translation of C9orf72 repeat amplification, superoxide dismutase 1 (superoxide dismutase 1, sod 1), and TDP-43. Diseases involving TDP-43 aggregates are commonly listed as TDP-43 proteinopathies, including but not limited to ALS and FTD.

I.TDP-43 introduction

The transactivation response (TRANSACTIVE RESPONSE, TAR) DNA binding protein 43kDa (TDP-43) is a protein of 414 amino acids encoded by the TARDBP gene on chromosome 1p36.2 (ALS 10). TARDBP consists of six exons (exon 1 is non-coding; exons 2 to 6 are protein-coded). TDP-43 belongs to the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family (Wang et al.,Trends in Molecular Medicine Vol.14No.11,2008,479-485;Lagier-Tourenne et al.,Human Molecular Genetics,2010,Vol.19,Review Issue 1R46-R64).TDP-43 comprising five functional domains (WARRAICH ET al., the International Journal of Biochemistry & Cell Biology 42 (2010) 1606-1609, FIG. 1) two RNA recognition motifs (RRM 1 and RRM 2) with two highly conserved hexa-ribonucleoprotein 2 (RNP 2) and octa-ribonucleoprotein 1 (RNP 1) regions, nuclear export signals (nuclear export signal, NES) and nuclear localization signals (nuclear localization signal, NLS) enabling them to shuttle between the nucleus and cytoplasm, transporting bound mRNA, and glycine-rich domains at the C-terminus mediating protein interactions. TDP-43 is involved in a number of 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 self-regulating protein whose expression levels shuttle continuously between the nucleus and cytoplasm, but are primarily localized to the nucleus. In 2006, TDP-43 was identified as a protein that accumulated in most cases of frontotemporal lobar degeneration (frontotemporal lobar degeneration, FTLD) with tau-negative, ubiquitin-positive inclusion bodies (hereinafter referred to as FTLD-TDP), and in most cases of Amyotrophic Lateral Sclerosis (ALS) (Arai etal.,Biochemical and Biophysical Research Communications 351(2006)602–611;Neumann et al.,Science 314,(2006),130-133).

38 TDP-43 negative dominant mutations have been identified in sporadic and familial ALS patients as well as in patients with hereditary FTD, which are predominantly located in the glycine-rich domain (Lagier-Tourenne AND CLEVELAND, cell 136,2009,1001-1004, fig. 1). TDP-43 is inherently prone to aggregation, as shown by the sedimentation assay, and this trend is further enhanced by some ALS-related TARDBP mutations (Ticozzi et al., CNS neurol.

TDP-43 in neurodegeneration

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), such as sporadic or familial, with or without motor-neuron disease (MND), with Granulin (GRN) mutations, With C9orf72 mutation, with TARDBP mutation, with valin-containing protein (valosine-containing protein, VCP) mutation, linkage to the 9p chromosome, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin positive TDP-43 inclusion bodies (FTLD-TDP), silvered granulosis (Argyrophilic GRAIN DISEASE), pick's disease, semantic variant primary progressive aphasia (SEMANTIC VARIANT PRIMARY progressive aphasia, svPPA), Behavioral variant FTD (behavioural VARIANT FTD, BVFTD), non-fluent variant primary progressive aphasia (Nonfluent VARIANT PRIMARY Progressive Aphasia, nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenic protein (ANG) mutations), alexander disease (Alexander disease, axD), edge-dominated age-related TDP-43 encephalopathy (LATE), Chronic Traumatic Encephalopathy (CTE), pecry syndrome (Perry syndrome), alzheimer's disease (AD, including sporadic and familial forms of AD), down's syndrome (Down syndrome), familial british dementia (Familial British dementia), polyglutamine disease (huntington's disease and spinocerebellar ataxia type3 (SCA 3 (spinocerebellar ataxia type 3); also known as madado-joseph disease (Machado Joseph Disease))), and the like, sea horse sclerotic dementia and myopathies (sporadic inclusion Body myositis, inclusion Body myopathies with mutations containing valcasein ((VCP); associated with Paget's disease (PAGET DISEASE of bone) and frontotemporal dementia), ocular pharyngeal muscular dystrophy with bordering vacuoles, myocontractile protein (myotilin, MYOT) gene mutations or myofibrillar myopathies with mutations in the gene encoding desmin (desmin, DES)), traumatic brain injury (Traumatic Brain Injury, TBI), lewy Body dementia (DEMENTIA WITH LEWY Body, DLB) or Parkinson's Disease (PD). The term LATE is intended to cover several previously used names associated with TDP-43 proteinopathies, which may be associated with cognitive impairment, including hippocampal sclerosis, senile hippocampal sclerosis, hippocampal sclerotic dementia, brain age-related TDP-43 and Cirrhosis (CARTS), and TDP-43 pathological conditions in the elderly (for reviews see Kuslansky et al.,2004;Lippa and Dickson,2004;Nelson et al, 2013,2016b;Dutra et al, 2015).

Aggregated TDP-43 from the brain of patients shows a number of abnormal modifications including hyperphosphorylation, ubiquitination, acetylation and aggregation mechanism of C-terminal fragment (Arai et al.,Biochemical and Biophysical Research Communications 351(2006)602–611;Neumann etal.,Science 314,(2006),130-133;Neumann et al.,Acta Neuropathol.(2009)117:137–149;Hasegawa et al.,(2008)Annals of Neurology Vol 64No 1,60–70;Cohen et al.,Nat Commun.6:5845,2015).TDP-43 by proteolytic cleavage and the kind of TDP-43 involved in aggregation are not fully understood. However, there is evidence that the C-terminal region of TDP-43 is important in the pathogenesis. The C-terminal domain, known as the Low Complexity Domain (LCD), is disordered in nature and comprises regions rich in glycine, hydrophobic residues, glutamine and asparagine (Afroz et al., 2019). While this region of TDP-43 has intrinsic properties (Gasset-Rosa et al, 2019) that form higher order physiological assemblies (e.g., stress particles), in disease, irreversible intermolecular and intramolecular interactions within this region can lead to pathological aggregates (Gasset-Rosa et al, 2019). In fact, recent structural and biochemical studies have shown that in the brains of ALS and FTD patients, in the C-terminal region, the region consisting of amino acids 272 to 360 of TDP-43, a stable protease resistant amyloid core structure is employed (Arseni et al, 2021;Arseni et al, 2023). Furthermore, disease-specific proteolytic cleavage exposing the amyloid core was shown to further enhance its seeding activity 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 the brain of patients are disease-specific pathological features.

Another characteristic feature of the pathological condition of TDP-43 is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. The hallmark lesions of FTLD-TDP are neuronal and glial cytoplasmic inclusion bodies (NCI (neuronal cytoplasmic inclusion) and GCI (glial cytoplasmic inclusion), respectively) and dystrophic neurites (dystrophic neurite, DN), which are immunoreactive with TDP-43 and ubiquitin and p62, but negative for other neurodegenerative disease-related proteins. Differences in inclusion body morphology and its tissue distribution are associated with specific mutations and/or clinical manifestations. Four types of TDP-43 pathology have been described by histological classification to date (Mackenzie and Neumann, J.Neurochem. (2016) 138 (journal 1), 54-70). FTLD-TDP type a cases are characterized by a large number of short Dystrophic Neurites (DNs) and compact oval or crescent NCI, mainly in the neocortex II layer (Mackenzie et al, 2016j. Neurochem.138 (journal 1), 54-70, fig. 2 f). This pathological condition usually occurs clinically in the case of frontotemporal dementia with behavioural variants (bvFTD) or non-fluency/grammar variant primary progressive aphasia (nfvPPA) and is associated with granulin precursor (GRN) mutations. Type B cases showed a moderate number of compact or granular NCI in both the superficial and deep cortex, with relatively few DN and NII (neuronal nuclear inclusion bodies (neuronal intranuclear inclusion); mackenzie et al, 2016j. Neurochem.138 (journal 1), 54-70, fig. 2 g). Most cases with simultaneous symptoms of FTD and ALS were found to have FTLD-TDP type B pathology. Type C cases have a large number of long and curved neurites, predominantly in the superficial epidermal plasma layer, with little or no NCI (Mackenzie et al, 2016j. Neurochem.138 (journal 1), 54-70, fig. 2 j). This pathology is found in particular in cases where a semantic variant primary progressive aphasia (svPPA) occurs. FTLD-TDP type D shows rich lenticular neuronal nuclear inclusion bodies (NII) and short DNs in the neocortex layer, and has only rare NCI (Mackenzie et al, 2016j. Neurochem.138 (journal 1), 54-70, fig. 2 k). type E is characterized by particulate filamentous neuronal inclusions (granulofilamentous neuronal inclusion, GFNI) in addition to curvilinear oligodendrocyte inclusions in white matter and very fine punctate neuropil aggregates that affect all neocortex layers (Edward B.Lee et al., acta neuroskill.2017 July;134 (1): 65-78.). This pathological pattern is only found in cases where VCP is associated with inclusion body myositis.

TDP-43 in FTD

Frontotemporal dementia (FTD) is a clinical term that encompasses a broad spectrum of disorders based on the pathological characteristics of degeneration of the frontotemporal lobes-known as frontotemporal lobar degeneration (FTLD). FTD is the second most leading cause of early-stage degenerative dementia in the age group under 65 years (Le beer, revue Neurologique169 (2013) 811-819). FTD is manifested as several syndromes including bvFTD characterized by personality and behavioral changes, semantic dementia (SEMANTIC DEMENTIA, SD) and progressive non-fluency aphasia (progressive nonfluent aphasia, PNFA) characterized by language functional changes, corticobasal syndrome (corticobasal syndrome, CBS) characterized by motor dysfunction, progressive supranuclear palsy syndrome, and motor neuron disease (FTD-MND). The clinical diagnosis of these syndromes is complex and the final conclusions can only be drawn by performing post-mortem histopathological analysis to detect aggregated proteins and to determine the affected brain areas. For pathological protein inclusion bodies, 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.

TDP-43 in ALS

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by premature loss of upper and lower motor neurons. Progression of ALS is characterized by fatal paralysis and respiratory failure, with a course of disease ranging from 1 to 5 years from diagnosis to death. In most sporadic ALS cases, this neuropathology is characterized by abnormal cytoplasmic accumulation of TDP-43 in neurons and glial cells of the primary motor cortex, brain stem motor nucleus, spinal cord and related 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 aid of antibodies that specifically bind to phosphorylated TDP-43 in nuclear and cytoplasmic inclusion bodies, wherein amino acids S379, S403, S404, S409, S410 are the primary sites of TDP-43 phosphorylation (HASEGAWA ET al, ann Neurol 2008;64:60-70; neumann et al, acta Neuropathol (2009) 117:137-149).

TDP-43 in AD and other diseases

TDP-43 pathological conditions occur in the brains of up to 57% of patients with alzheimer's disease, and (Josephs KAet al.,Acta Neuropathol.2014;127(6):811-824;Josephs KA etal.,Acta Neuropathol.2014;127(3):441–450;McAleese et al.,Brain Pathol.2017Jul;27(4):472-479).TDP-43 aggregates are associated with age of the patient and with cognitive decline, memory loss and medial temporal atrophy in AD. Clearly, in AD, TDP-43 represents a second or independent pathology sharing overlapping brain distribution with the amyloid beta and tau pathology in the medial temporal lobe. Pathological TDP-43 follows a conventional progressive deposition pattern, which has been described by a so-called TDP-43 in AD (TAD) staging scheme, where TDP-43 is first deposited in the amygdala (stage I) and then in the hippocampus, limbus, temporo and finally in the frontal striatum (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).

TDP-43 diffusion

Although ALS and FTD episodes and initial symptoms vary significantly from patient to patient, a common feature of disease progression is the spread of pathology from the initial focal region to anatomically connected brain regions. The continued worsening of symptoms can be explained by the progressive spread of TDP-43 pathological conditions. TDP-43 pathological conditions in the brain of ALS patients show spread in a four-stage process and are thought to be transmitted through synaptic triggers using anterograde axonal transport through transmission from cortical axons (Brettschneider et al, ann neurol.2013july;74 (1): 20-38.). Recent experimental evidence supports the hypothesis that amyloid beta, tau, alpha-synuclein and TDP-43 undergo protein transmission in neuronal tissue by prion-like mechanisms (HASEGAWA ET al, 2017), where the origin and topographical diffusion patterns are different for four proteins (Brettschneider J et al, nature rev. Neuroscience,2015,109). The common disease unifying mechanism is thought to be based on intercellular diffusion of pathological protein aggregates. The mechanism consists of the release of aggregates from diseased cells, uptake by naive cells, and seeding (seed) of the pathological protein conformation by a templated conformational change of the endogenous protein. Pathological TDP-43 capable of inducing physiological (i.e., non-pathological) aggregation of TDP-43 is defined as TDP-43 having seeding capability. In fact, it has been found that TDP-43 is misfolded and aggregates into seeds (seed), which are propagation media with the ability to initiate de novo misfolding (propagating agent). This "prion-like" paradigm is suspected to be one of the key factors in disease progression.

TDP-43 intercellular diffusion has been studied at the molecular level in a few in vitro models, in which insoluble TDP-43 preparations from the brain of patients are able to induce intracellular cohesive formation in recipient cells (nomaka et al, cell Reports 4 (2013), 124-134; feiler et al, 2015; pora et al, nat. Comm., 2018). In addition, intracellular TDP-43 aggregates have been observed to be released in combination with exosomes before diffusing to the next Cell (Nonaka et al, cell Reports 4 (2013, 124-134)). Similarly, adenovirus-transduced TDP-43 expression results in cytoplasmic aggregates that phosphorylate, ubiquitinate and more importantly act as seeds that initiate intercellular diffusion (Ishii et al, PLoS ONE 12 (6): e0179375,2017). Patient-derived pathologic TDP-43 can lead to extensive deposition of endogenous TDP-43 after intracranial inoculation into transgenic and wild-type mice (pora et al, nat. The presence of TDP-43 seeding-capable substances in CSF or ALS patients has recently been demonstrated using a TDP-43 seed amplification assay (Audrain et al., 2023). The TDP-43mAb targeting the C-terminal domain is able to neutralize these seeding materials (Audrain et al., 2023).

Prevention and treatment of TDP-43 protein diseases

TDP-43 aggregation and pathological spread are the primary markers of ALS and FTD-currently incurable fatal diseases. Thus, new methods 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.

Diagnosis of TDP-43 protein disease

FTD diagnosis based on clinical manifestations is inadequate because clinical manifestations can overlap with other diseases, especially at early stages.

Many methods aim at developing biochemical biomarkers to distinguish between different types of FTD pathology. Development of antibodies against different conformations of TDP-43 may allow for the creation of more sensitive and specific diagnostic tools. Concurrent with biochemical biomarkers, the development of imaging biomarkers enables early and specific detection of pathology in TDP-43 proteinopathies. The ability to image TDP-43 deposition in the brain can be a significant achievement in diagnosis of TDP-43 proteinopathies and drug development. Such detection can be achieved using cell permeable antibody fragments.

The earliest event in misfolded neurodegenerative diseases based on different proteins was to obtain alternative conformations that make the proteins toxic. Furthermore, this misfolded conformation may self-propagate by recruiting endogenous normal proteins into the misfolded conformation, as a basis for the observed mechanism of diffusion through the affected tissue.

To develop antibodies to different conformational states of a given protein, supramolecular antigenic constructs were designed in which the conformation of the presented antigen was controlled to produce conformation-specific antibodies to a given target in a specific conformational state (WO 2012/055933 and WO 2012/020124). Conformation-specific antibodies offer a number of advantages as they can distinguish disease-associated conformations from functional endogenous conformations of these proteins. This approach provides many advantages in therapeutic applications because such antibodies are less likely to be attracted by their normal conformation while targeting misfolded disease-associated isoforms of the protein. Similarly, in diagnostic applications, such antibodies recognize only disease-related structural states of proteins, which is critical for the development of sensitive and specific diagnostics.

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

Thus, there is a clear need for biomarkers capable of detecting misfolded aggregated TDP-43 and non-aggregated physiological TDP-43, particularly in human samples, for diagnosing different types of TDP-43 proteinopathies and/or for monitoring the efficacy of therapeutic drugs for treating diseases, disorders and abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies.

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

IX. Prior Art

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

Patent application WO2013/061163 discloses TDP-43 specific binding molecules comprising polypeptides, such as human antibodies and fragments, derivatives and variants thereof.

Patent application WO2020/234473 discloses specific binding molecules, including polypeptides, such as murine antibodies or antigen binding fragments thereof.

Patent application WO2022/034228 discloses TDP-43 specific binding molecules, including polypeptides, such as humanized antibodies or antigen binding fragments thereof.

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

Disclosure of Invention

Currently, there is no approved treatment on the market for the treatment and/or prevention of TDP-43 related diseases. Thus, 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 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, ubiquitinated-like (sumoylate) and/or methylated TDP-43. Physiological TDP-43 includes soluble nuclear TDP-43. It is shown herein that the binding molecules of the invention are capable of binding pathological TDP-43, including TDP-43 aggregates and phosphorylated TDP-43. Accordingly, the present invention provides binding molecules, particularly antibodies or antigen binding fragments thereof, that specifically recognize misfolded aggregated 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 illustrated herein, the TDP-43 binding molecules of the present invention can bind both misfolded aggregated and non-aggregated physiological TDP-43 equally or preferentially to one over the other when specifically binding to both types of TDP-43. The invention also provides binding molecules, particularly 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, particularly TDP-43 aggregates, or TDP-43 proteinopathies. The invention also provides binding molecules, particularly antibodies or antigen binding fragments thereof, for detecting and/or knowing (i.e., identifying) the particular pathological type that causes neurodegeneration. The use of the novel therapeutic development of TDP-43 protein disease is contemplated as a diagnostic biomarker, enabling more efficient and accurate selection of subjects for longitudinal monitoring in clinical studies.

The invention also provides TDP-43 binding molecules, particularly antibodies or antigen binding fragments thereof, as medicaments (therapeutic agents).

Without wishing to be bound by any particular theory, the present invention has been developed based on the assumption that modified conformation-specific antigenic peptides and peptide fragments derived from TDP-43 protein or the whole TDP-43 protein and antibodies obtainable or obtained by using said peptides or fragments or the whole TDP-43 protein as antigen block TDP-43 intercellular transmission and/or disaggregate TDP-43 aggregates and/or block TDP-43 seeding and/or neutralize TDP-43 with seeding capability and/or inhibit aggregation of TDP-43 protein or fragments thereof and/or enhance TDP-43 clearance. The binding molecules of the invention, particularly polypeptides, more particularly antibodies or antigen binding fragments thereof, bind to misfolded aggregated TDP-43, particularly to cytoplasmic misfolded and extracellular misfolded TDP-43. The humanized binding molecules of the invention, particularly 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 invention, particularly polypeptides, more particularly antibodies or antigen binding fragments thereof, specifically bind to cytoplasmic misfolded TDP-43. In one embodiment, the TDP-43 binding molecule of the invention, in particular an antibody or antigen binding fragment thereof, binds and neutralizes TDP-43 with seeding capacity. In one embodiment, the TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, bind extracellular and/or aggregated TDP-43 and enhance clearance of immune cells, such as microglia, by Antibody Dependent Cell Phagocytosis (ADCP).

Misfolded aggregated or pathologically related TDP-43 consists of TDP-43 protein that loses its normal folding (i.e., is misfolded) and localization. Misfolded aggregated TDP-43 can be found in pre-inclusion bodies (preinclusion), as well as neuronal and glial cytoplasmic inclusion bodies (NCI and GCI, respectively), neuronal nuclear inclusion bodies (NII), and Dystrophic Neurites (DN), which are immunoreactive with TDP-43.

Non-aggregated physiological TDP-43 is a physiologically functional TDP-43 protein that is located primarily in the nucleus and shuttles to the cytoplasm, in a state that is capable of exhibiting its desired function in the in vivo cellular environment.

The TDP-43 binding molecules of the invention, 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, yet more preferably five, yet even more preferably six, yet even more preferably seven, most preferably all eight of the following features:

-blocking TDP-43 intercellular transmission;

-disaggregating TDP-43 aggregates;

-inhibiting aggregation of TDP-43 protein or a fragment thereof;

-blocking TDP-43 seeding;

-neutralising TDP-43 with seeding capacity;

-blocking TDP-43 diffusion;

Enhanced TDP-43 clearance, and

-Decreasing the level of phosphorylated TDP-43 in vivo.

Independently of the combination of one, two, three, four, five, six, seven or eight of the above listed features, the anti-TDP-43 binding molecules of the invention, preferably anti-TDP-43 antibodies or antigen binding fragments thereof, can reduce the level of phosphorylated TDP-43 in the brain and/or improve/inhibit/reduce the formation of TDP-43 pathological conditions in an in vivo model of TDP-43 protein disease and more importantly in patients suffering from TDP-43 pathological conditions.

The anti-TDP-43 binding molecule binds to an epitope within amino acids 304 to 414 of human TDP-43 (SEQ ID NO: 1). The anti-TDP-43 binding molecule binds 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). Or the anti-TDP-43 binding molecule may bind to an epitope within amino acid residues 396 to 414 of human TDP-43 (SEQ ID NO: 1).

According to the present invention there is provided 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, or

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.

The invention is especially additionally directed to (i) an immunoconjugate comprising a TDP-43 binding molecule, (ii) a labeled antibody comprising a TDP-43 binding molecule, (iii) a pharmaceutical composition comprising a TDP-43 binding molecule and a pharmaceutically acceptable carrier and/or excipient and/or diluent, (iv) a TDP-43 binding molecule for use in a human or veterinary pharmaceutical use, (v) a TDP-43 binding molecule for preventing, alleviating, treating a TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy, (vi) a TDP-43 binding molecule for diagnostic use (in particular for in vivo diagnostics, but also for in vitro testing), (vii) a TDP-43 binding molecule for research use, in particular as an analytical tool or reference molecule, (viii) a TDP-43 binding molecule for use as a diagnostic tool for monitoring a TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy, (ix) a method for reducing the memory loss of a TDP-43-related disease, disorder and/or TDP-43 protein associated with a TDP-43 binding molecule in an individual by using said individual or a method of reducing the memory loss of a memory-43 protein associated with said TDP-43, (xii) A recombinant expression vector comprising a nucleic acid molecule of the invention, (xiii) a host cell comprising a nucleic acid and/or a vector of the invention, (xiv) a cell-free expression system comprising a recombinant expression vector of the invention, (xv) a method for producing a TDP-43 binding molecule, (xvi) a method for quantifying TDP-43 in a sample obtained from a subject using a TDP-43 binding molecule, and (xvii) a kit comprising a TDP-43 binding molecule of the invention and/or a nucleic acid, an expression vector, a host cell and/or a cell-free expression system for producing the same.

The TDP-43 binding molecules of the invention, particularly anti-TDP-43 antibodies or antigen binding fragments thereof, recruit and/or activate microglial cells. More particularly, the TDP-43 binding molecules of the invention can affect microglial morphology in terms of cell size and activation state. This may contribute to a decrease in the pathological condition of TDP-43 as indicated by the TDP-43 binding molecules of the invention.

In the present invention, a binding molecule, in particular an antibody or antigen binding fragment thereof, specifically recognizes TDP-43. Binding molecules of the invention include polypeptides and/or antibodies and/or antigen binding fragments thereof specific for the TDP-43 protein. By "specifically recognizing TDP-43" is meant that the binding molecules of the invention specifically, generally and jointly bind with greater affinity 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, compared to other epitopes. The binding molecules, particularly polypeptides, more particularly antibodies or antigen binding fragments thereof, of the invention that specifically bind to TDP-43 specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43.

The TDP-43 binding molecules of the invention, 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 invention, particularly antibodies or antigen binding fragments thereof, bind substantially equally well to soluble TDP-43 and aggregated TDP-43. The TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, bind substantially equally to aggregated TDP-43 as compared to non-aggregated TDP-43. More specifically, the TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, bind substantially equally to aggregated TDP-43 in the cytoplasm as compared to non-aggregated TDP-43 in the nucleus. In other embodiments, when binding to both types of TDP-43, the TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, can preferentially bind to aggregated TDP-43 as compared to non-aggregated TDP-43. More specifically, when binding to both types of TDP-43, the TDP-43 binding molecule of the present invention, particularly an antibody or antigen binding fragment thereof, can preferentially bind to aggregated TDP-43 in the cytoplasm as compared to non-aggregated TDP-43 in the nucleus. Or in other embodiments, when binding to both types of TDP-43, the TDP-43 binding molecule of the invention, particularly an antibody or antigen binding fragment thereof, may preferentially bind to non-aggregated TDP-43 compared to aggregated TDP-43. More specifically, when binding to both types of TDP-43, the TDP-43 binding molecule of the present invention, particularly an antibody or antigen binding fragment thereof, can preferentially bind to non-aggregated TDP-43 in the nucleus of a cell, as compared to aggregated TDP-43 in the cytoplasm. These binding properties can be demonstrated, for example, using immunohistochemistry.

In some embodiments, the invention encompasses the binding molecules of the invention described herein, particularly antibodies and antigen binding fragments thereof, that specifically bind TDP-43, and the use of these binding molecules to diagnose, prevent, alleviate and/or treat 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 edge-dominated age-related TDP-43 encephalopathy (LATE). The methods and compositions disclosed herein are useful for diagnosing, preventing, alleviating and/or treating 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 for diagnosing, preventing, alleviating and/or treating a disease, disorder and/or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or TDP-43 proteinopathies is directed against 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).

In another embodiment, a TDP-43 binding molecule of the invention described herein, in particular 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, in particular with TDP-43 aggregates, or TDP-43 proteinopathies selected from frontotemporal dementia (FTD), e.g. sporadic or familial, with or without Motor Neuron Disease (MND), with particle protein precursor (GRN) mutation, with C9orf72 mutation, with TARDBP mutation, with valin-containing peptide protein (VCP) mutation, with 9p chromosome-linked, corticobasal degeneration, frontotemporal degeneration with ubiquitin-positive TDP-43 inclusion bodies (FTLD) (FTLD-TDP), silver-philic granulosis, pick disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bFTD), non-fluen primary progressive aphasia (nfvPPA), etc.), amyotrophic Lateral Sclerosis (ALS), such as sporadic ALS, TARDBP mutations, angiogenic protein (ANG) mutations, alexander disease (AxD), edge-dominated age-related TDP-43 encephalopathy (LATE), chronic Traumatic Encephalopathy (CTE), paget's syndrome, alzheimer's disease (AD, including sporadic and familial forms of AD), down's syndrome, familial british dementia, dementia of the type, polyglutamine disease (huntington's disease and spinocerebellar ataxia type 3 (SCA 3; also known as maduo-joseph disease)), hippocampal sclerotic dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathies with mutations containing valcasein ((VCP); associated with paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophies with borderline vacuoles, myofibrillar myopathies with mutations in the gene of myocontracture protein (MYOT) or in the gene encoding Desmin (DES)), traumatic Brain Injury (TBI), lewy body Dementia (DLB) or Parkinson's Disease (PD).

In one embodiment, the invention encompasses the binding molecules of the invention described herein, particularly antibodies or antigen binding fragments thereof, that specifically bind to TDP-43, and the use of these binding molecules, particularly these antibodies, to detect the presence of TDP-43 in a sample. Thus, the TDP-43 binding molecules of the invention, e.g., anti-TDP 43 antibodies described herein, can be particularly useful for screening clinical samples, particularly human blood, cerebrospinal fluid (cerebrospinal fluid, CSF), interstitial fluid (INTERSTITIAL FLUID, ISF) and/or urine, for example, for the presence of TDP-43 in the sample, e.g., by using an ELISA-based assay or a surface adaptation assay. in some cases, tissue samples, such as brain tissue samples, may be used. The methods and compositions of the invention may also be applied to diagnose pre-symptomatic disease and/or monitor disease progression and/or therapeutic 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 a granule protein precursor (GRN) mutation, with a C9orf72 mutation, with a TARDBP mutation, with a valcasein-containing protein (VCP) mutation, linked to the 9p chromosome, corticobasal degeneration, a, Frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusion bodies (FTLD-TDP), silver-philic granulosis, pick disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g. sporadic ALS, with TARDBP mutation, with angiogenic protein (ANG) mutation), alexander disease (AxD), edge-dominant age-related TDP-43 encephalopathy (LATE), chronic Traumatic Encephalopathy (CTE), amyotrophic Lateral Sclerosis (ALS), Pair's 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 (SCA 3; also known as Marchaetoposi-Joseph disease)), hippocampal dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathies with mutations containing valcasein peptide protein ((VCP); associated with Paget's disease and frontotemporal dementia), hypopharyngeal muscular dystrophies with borderline vacuoles, myoconstrictor (MYOT) gene mutations or myofibrillar myopathies with mutations of the gene encoding junction protein (DES)), and combinations thereof, Traumatic Brain Injury (TBI), dementia with lewy bodies (DLB) or Parkinson's Disease (PD). The TDP-43 binding molecules of the invention can be used to quantify TDP-43 in suitable samples, particularly clinical samples, such as blood, brain tissue, CSF, ISF or urine, wherein relatively high levels of TDP-43 compared to suitable controls are indicative of a disease and/or a more advanced disease. Many suitable immunoassay formats are known. Thus, the method (e.g., ELISA, MSD (mesoscale discovery (Meso Scale Discovery)), HTRF (homogeneous time-resolved fluorescence (Homogeneous Time Resolved Fluorescence)), and the like) can be performed for diagnostic purposes,(Single molecule array (Single Molecule Array))) Wherein a high level of TDP-43 indicates a disease. Or the method may be performed for monitoring purposes. An elevated level over time may be indicative of disease progression. A level that decreases over time may indicate disease regression. The method can also be used to monitor treatment, particularly to monitor the efficacy of a particular treatment. Successful treatment may be measured with reference to stabilized or decreased levels of TDP-43 following treatment. In example 12 of WO2020/234473 it is shown that the level of TDP-43 in a CSF sample from a patient suffering from TDP-43 protein is higher than in a control sample taken from a healthy subject (healthy control). The control sample may or may not run in parallel with the test sample. In some embodiments, the control level is determined from a series of control samples taken from healthy subjects under similar or identical experimental conditions and used as a comparison level to the level determined in the test sample. Methods of quantifying TDP-43 in a suitable sample using the binding molecules of the invention may also be used to select for treatment (for additional treatment of a subject). Thus, personalized treatment methods are contemplated. Samples were taken before and after treatment. If treatment with the treatment results in a stable or preferably reduced level of TDP-43 after treatment, the treatment may be selected for the subject. If the treatment does not result in a stable or preferably reduced level of TDP-43 after treatment, the treatment is not selected for the subject. The treatment may be any suitable candidate therapeutic agent for the treatment of TDP-43 proteinopathies. In some preferred embodiments, the treatment comprises a TDP-43 binding molecule of the invention, typically in the form of a pharmaceutical composition as described herein.

The TDP-43 binding molecules of the invention can also be used to classify diseases as a specific type or subtype. Accordingly, there is provided a method 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:

a. the method of the invention is performed wherein the level of TDP-43 is quantified as compared to a suitable control;

b. Optionally identifying mutations in a sample from the subject, including but not limited to, granulin precursor (GRN) mutations, C9orf72 mutations, TARDBP mutations, angiogenic protein (ANG) mutations, valcasein-containing (VCP) mutations, myocontractile protein (MYOT) gene mutations, or mutations of genes encoding Desmin (DES), and

C. diseases, disorders and/or abnormalities associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteopathies are classified.

Similarly, a method for classifying a disease, disorder and/or abnormality associated with TDP-43, particularly an aggregate of TDP-43, or for classifying a TDP-43 proteinopathy is provided, comprising performing a method of the invention, wherein the level of TDP-43 in a sample obtained from a subject suffering from a disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy is quantified, wherein the level is compared to a control sample taken from a subject suffering from a different type or subtype of disease, disorder and/or abnormality associated with TDP-43, particularly an aggregate of TDP-43, or 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 aggregate, or TDP-43 proteinopathy based on the comparison. Thus, classification is based on determining the closest match between the test sample and one or more control samples. These methods may also include identifying mutations in the sample, including but not limited to, granulin precursor (GRN) mutations, C9orf72 mutations, TADBP mutations, angiogenic protein (ANG) mutations, valcasein (VCP) -containing mutations, myocontractile protein (MYOT) gene mutations, or mutations of genes encoding Desmin (DES), wherein the identified mutations are also used to classify a disease, disorder, and/or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteopathy. For the avoidance of doubt, the identification of mutations in a sample may be carried out by any suitable method, for example, nucleic acid sequencing based on nucleic acid molecules within the sample. The sample may be separate and distinct from the sample in which the level of TDP-43 is determined, but from the same subject.

In other embodiments, the invention provides methods for preventing, alleviating and/or treating a disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteinopathies. According to one embodiment, the method of the invention comprises administering to a subject an effective concentration of a binding molecule of the invention, particularly an antibody (e.g., a full length antibody or a TDP-43 binding fragment or derivative of an antibody) specific for TDP-43 as described herein. In another embodiment, the present invention provides a method for preventing, alleviating and/or treating TDP-43 proteinopathies. According to some embodiments, a binding molecule described herein, in particular an antibody or antigen binding fragment thereof of the invention, specific for TDP-43 is administered to treat, reduce and/or prevent frontotemporal degeneration (FTD) or Amyotrophic Lateral Sclerosis (ALS). In another embodiment, a binding molecule described herein, particularly an antibody of the invention or antigen binding fragment thereof, specific for TDP-43 is administered to prevent, reduce and/or treat a neurodegenerative disease selected from frontotemporal dementia (FTD), amyotrophic Lateral Sclerosis (ALS), alzheimer's disease (AD, including sporadic and familial forms of AD), parkinson's Disease (PD), chronic traumatic brain disease (CTE), edge-dominated age-related TDP-43 brain disease (LATE).

In another embodiment, the binding molecules described herein, particularly antibodies or antigen binding fragments thereof, specific for TDP-43 are administered to prevent, reduce and/or treat a disease selected from frontotemporal dementia (FTD), e.g., sporadic or familial, with or without Motor Neuron Disease (MND), with granule protein precursor (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valcasein-containing protein (VCP) mutations, with 9p chromosome linkage, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-43 inclusion bodies (silver-philic granulomatosis, pick's disease, progressive semantic variant aphasia (svPPA), behavioural variant FTD (bvFTD), non-fluentic primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenic protein (ANG), with Valcasein (VCP) mutations, with ataxia, ataxia (Grandin), brain-related forms including the forms of the brain-ataxia, the brain-related diseases (Grandin), the brain-related forms (szebra-3, the brain-related forms (szechuan-type), the brain-related forms (szein the brain-disorder (szechuan-3, the brain-associated with the brain-type) and the like; also known as Marchado-Joseph disease), sea horse sclerotic dementia and myopathy (sporadic inclusion body myositis, there are inclusion body myopathies containing mutations in valcasein ((VCP); associated with Paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophy with bordering vacuoles, myocontractile protein (MYOT) gene mutations or myofibrillar myopathies with mutations in the gene encoding Desmin (DES), traumatic Brain Injury (TBI), dementia with lewy bodies (DLB) or Parkinson's Disease (PD).

Detailed Description

Definition of X

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 may comprise or be an antibody or fragment thereof. An anti-TDP-43 binding molecule is a molecule that binds to a 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 invention bind to an epitope in the amino acid sequence of SEQ ID NO. 1. The antigen binding molecules provided herein, particularly antibodies or antigen binding fragments thereof, recognize full-length TDP-43. Other anti-TDP-43 binding molecules may also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptamers, affibodies (avimers), or other naturally occurring or recombinantly produced molecules. Exemplary antigen binding molecules useful in the present invention include antibody-like molecules. Antibody-like molecules are molecules that can function by binding to a target molecule (see, e.g., ,Current Opinion in Biotechnology2006,17:653-658;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, e.g., DARPin (WO 2002/020565), affibody (Affibody) (WO 1995/001937), affibody (Avimer) (WO 2004/044011; WO 2005/040229), adnectin (WO 2002/032925), and fynomer (WO 2013/135588).

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 such that further evaluation of the antibody as a potential diagnostic and/or therapeutic agent that targets TDP-43 is contemplated. In general, the term "antibody" is used herein in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or biconjugate antibodies), fully human antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity. Antibodies within the invention may also be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies, or antibodies displayed on the surface of phage or on the surface of Chimeric Antigen Receptor (CAR) T cells.

An "antigen binding fragment" or "functional fragment" of an antibody refers to a molecule that comprises a portion of a full or full length antibody that is different from a full or full length antibody and that binds (in whole or in part) to an antigen to which the full or full length antibody binds. 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. Antigen binding fragments may also be referred to as "functional fragments" because they retain the binding function of the original antibody from which they were derived.

An "antibody that binds to a 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.

In certain embodiments, an "antibody that binds to an epitope in a defined region of a protein" is identified by a mutation analysis in which the amino acid of the protein is mutated and the binding of the antibody to the resulting altered protein (e.g., 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 a mutation analysis in which the amino acid of the protein is mutated and the binding of the antibody to the resulting altered protein (e.g., 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, such as ELISA.

The term "bind to" as 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 exhibits the ability to specifically interact with a specific antigen or a specific group of TDP-43 antigens. The binding/interaction should also be understood as defining a "specific recognition". According to the present invention, the term "specifically recognizes" means that the antibody is capable of specifically interacting with and/or binding to at least two amino acids of 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 in particular 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).

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

The term "specific interaction" as used according to the invention means that the antibodies of the invention or antigen binding fragments thereof do not cross-react or substantially do not cross-react with (poly) peptides having a similar structure. Thus, the antibodies or antigen binding fragments thereof of the present invention specifically bind/interact with the TDP-43 structure formed by the specific amino acid sequence of amino acid residues 304 to 414 of human TDP-43 (SEQ ID NO: 1), more specifically, with the TDP-43 structure formed by the specific amino acid sequence 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).

The cross-reactivity of the antigen binding molecules under investigation, in particular of a group of Antibodies or antigen binding fragments thereof, can be tested, for example, by assessing the binding of said group 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, for example, 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. specific epitopes or (poly) peptides/proteins of TDP-43 as defined herein, but do not or substantially not bind 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 are selected for further investigation according to the methods provided herein. These methods may include, inter alia, binding studies, blocking and competition studies of molecules closely related to structure and/or function. These binding studies also include FACS analysis, surface plasmon resonance (surface plasmon resonance, SPR, e.g., with BIACORE ^TM), analytical ultracentrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy, or by radiolabeled ligand binding assays.

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-testing, and peptide scanning.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures and are substantially free of contamination by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being in a substantially homogeneous population of antibodies, and is not to be construed as requiring that the antibody be produced by any particular method. As described above, the monoclonal antibodies used according to the present invention can be prepared by the hybridoma method described by Kohler, nature 256 (1975), 495.

The term "polyclonal antibody" as used herein refers to an antibody that is produced among or in the presence of one or more other different antibodies. In general, polyclonal antibodies are produced by B lymphocytes in the presence of several other B lymphocytes that produce different antibodies. Typically, polyclonal antibodies are obtained directly from the immunized animal.

The term "fully human antibody" as used herein refers to an antibody comprising only human immunoglobulin protein sequences. Fully human antibodies may comprise murine sugar chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, "mouse antibody" or "murine antibody" refers to an antibody that comprises only mouse/murine immunoglobulin protein sequences. Or "fully human antibodies" may comprise a rat sugar chain 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 may also be produced, for example, by phage display, a widely used screening technique that is capable of producing and screening fully human antibodies. Phage antibodies can also be used in the context of the present invention. Phage display methods are described, for example, in US 5,403,484, US 5,969,108 and US 5,885,793. Another technique that enables the development of fully human antibodies involves improvements to the mouse hybridoma technique. Mice are transgenic to contain human immunoglobulin loci in exchange for their own mouse genes (see, e.g., US 5,877,397).

The term "chimeric antibody" refers to an antibody comprising a variable region of the invention fused or chimeric to an antibody region (e.g., constant region) from another, human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

The term antibody also relates to recombinant human antibodies, heterologous antibodies and heterohybrid antibodies. The term "recombinant (human) antibody" includes all human sequence antibodies prepared, expressed, produced or isolated by recombinant means, e.g., antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes, antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant, combinatorial human antibody libraries, or antibodies prepared, expressed, produced or isolated by any other means that involves splicing 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 may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in the human antibody germline repertoire in vivo.

"Heterologous antibodies" are defined with respect to transgenic non-human organisms that produce such antibodies. The term refers to antibodies having an amino acid sequence or coding nucleic acid sequence corresponding to that present in an organism that does not consist of a transgenic non-human animal, and the organism is typically from a species other than the species of the transgenic non-human animal.

The term "heterohybrid antibody" refers to antibodies having light and heavy chains of different biological origin. For example, an antibody having a human heavy chain associated with a murine light chain is a heterohybrid antibody. Some examples of heterohybrid antibodies include chimeric antibodies and humanized antibodies.

The term antibody also relates to humanized antibodies. A "humanized" form of a non-human (e.g., murine or rabbit) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., fv, fab, fab ', F (ab') 2 or other antigen-binding subsequence of the antibody) that contains minimal sequence derived from a non-human immunoglobulin. Typically, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (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 cases, fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or in the introduced CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a 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 may further 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 (1998), 323-327 and Presta Curr Op Struct Biol 2 (1992), 593-596.

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

Thus, in the context of the present invention, the term "antibody" relates to an intact immunoglobulin molecule as well as to a portion of such an immunoglobulin molecule (i.e. "antigen binding fragment thereof"). Furthermore, as described above, the term relates to modified and/or altered antibody molecules. The term also relates to recombinantly or synthetically produced/synthesized antibodies. The term also relates to intact antibodies and antibody fragments thereof, e.g., 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.

In the context of the present invention, 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, scFv polypeptides also comprise a polypeptide linker between the V _H and V _L domains, which enables the scFv to form the desired antigen binding structure. Techniques described for producing single chain antibodies are described, for example, in Plückthun,The Pharmacology of Monoclonal Antibodies,Rosenburg and Moore eds.Springer-Verlag,N.Y.(1994),269-315.

As used herein, a "Fab fragment" comprises a light chain, and the C _H and variable regions of a heavy chain. The heavy chain of a Fab molecule is unable to form disulfide bonds with another heavy chain molecule.

The "Fc" region comprises two heavy chain fragments containing the C _H and C _H 3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the C _H domains.

"Fab fragments" comprise 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 and C _H 2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab 'fragments to form a F (ab') ₂ molecule.

The "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, the F (ab ') ₂ fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

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

The antibodies, antibody constructs, antibody fragments, antibody derivatives (all of Ig origin), or their corresponding immunoglobulin chains used according to the invention may be further modified using conventional techniques known in the art, for example by using amino acid deletions, insertions, substitutions, additions and/or recombinations, 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 sequence of 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, 2 nd edition (1989) and 3 rd edition (2001). The term "Ig-derived domain" relates in particular to a (poly) peptide construct comprising at least one CDR. Fragments or derivatives of the listed Ig-derived domains define the following (polypeptide) peptides, which are part of the above antibody molecules and/or are modified by chemical/biochemical or molecular biological methods. Corresponding methods are known in the art and are described in particular in the laboratory Manual (see, sambrook et al, molecular Cloning: A Laboratory Manual; cold Spring Harbor Laboratory Press, 2 nd edition (1989) and 3 rd edition (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:AMolecular Cloning Manual Cold Spring Harbor Laboratory Press(2002))).

The term "CDR" as used herein relates to "complementarity determining regions", which are well known in the art. CDRs are part of immunoglobulins that determine the specificity of the molecule and are in contact with specific ligands. CDRs are the most variable parts of the molecules and contribute to the diversity of these molecules. Three CDR regions are present in each V domain, CDR1, CDR2 and CDR3.CDR-H shows the CDR regions of the variable heavy chain, while CDR-L refers to the CDR regions of the variable light chain. VH means variable heavy chain and VL means variable light chain. CDR regions of Ig derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest", 5 th edition NIH publication No.91-3242U.S.Department of Health and Human Services (1991). CDR sequences provided herein are defined according to Kabat. However, the skilled artisan will appreciate that the invention is intended to encompass binding molecules in which CDR sequences are defined according to any useful identification/numbering scheme. For example, the following numbering scheme may be employed to define CDR：Chothia(Canonical structures for the hypervariable regions ofimmunoglobulins.Chothia C,Lesk AM.J Mol Biol.1987Aug 20;196(4):901-17);IMGT(IMGT,the international ImMunoGeneTics database.Giudicelli V,Chaume D,Bodmer J,Müller W,Busin C,Marsh S,Bontrop R,Marc L,Malik A,Lefranc MP.Nucleic Acids Res.1997Jan 1;25(1):206-11 and Unique database numbering system for immunogenetic analysis.Lefranc MP.Immunol Today.1997Nov;18(11):509);MacCallum(MacCallum RM,Martin AC,Thornton JM,J Mol Biol.1996Oct 11;262(5):732-45) Martin(Abhinandan KR,Martin ACR.Analysis and improvements to Kabat and structurally correct numbering of antibody variabledomains.Mol Immunol.(2008)45:3832–9.10.1016/j.molimm.2008.05.022).

Thus, in the context of the present invention, the antibody molecules described herein above are selected from the group consisting of whole antibodies (immunoglobulins, e.g.IgG 1, igG2, igA1, 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, bivalent antibody constructs, antibody fusion proteins, synthetic antibodies, bivalent single chain antibodies, trivalent single chain antibodies and multivalent single chain antibodies.

"Humanization methods" are well known in the art and are described specifically with respect to antibody molecules, e.g., ig derived molecules. The term "humanized" refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (e.g., fv, fab, fab ', F (ab'), scFv, or other antigen-binding portion sequences of antibodies) that comprise portions of sequences derived from non-human antibodies. Humanized antibodies include human immunoglobulins in which residues from a Complementarity Determining Region (CDR) of the human immunoglobulin are replaced by residues from a CDR of a non-human species (e.g., mouse, rat or rabbit) having the desired binding specificity, affinity and capacity. In general, a 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. Most preferably, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin, see, inter alia, jones et al, nature321 (1986), 522-525, presta, curr.Op.Structure.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, yet retains the original binding activity of the antibody. Methods for humanizing antibodies/antibody molecules are also described in detail in Jones et al, nature321 (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, such as antibodies to 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).

Thus, in the context of the present invention, an antibody molecule or antigen binding fragment thereof is provided, which may be humanized and may be successfully used in pharmaceutical compositions.

It will be appreciated by those skilled in the art that the epitope may be comprised in the TDP-43 protein, but may also be comprised in its degradation products or may be a chemically synthesized peptide. The amino acid positions are indicated only in order to show the positions of the corresponding amino acid sequences in the TDP-43 protein sequence. The present invention encompasses all peptides comprising an epitope. The peptide may be part of a polypeptide of more than 100 amino acids in length, or may be a small peptide of less than 100, preferably less than 50, more preferably less than 25, even more preferably less than 16 amino acids in length. The amino acids of such peptides may be natural amino acids or unnatural amino acids (e.g., β amino acids, γ amino acids, D-amino acids), or combinations thereof. Furthermore, the invention may cover the corresponding retro-peptide (retro-inverso peptide) of the epitope. The peptide may be unbound or bound. It may be conjugated with, for example, small molecules (e.g., drugs or fluorophores), high molecular weight polymers (e.g., polyethylene glycol (polyethylene glycol, PEG), polyethylenimine (polyethylene imine, PEI), hydroxypropyl methacrylate (hydroxypropylmethacrylate, HPMA), etc.), or proteins, fatty acids, sugar moieties, or may be intercalated into a membrane.

To test whether the antibody in question and the antibody of the invention recognize the same epitope, competition studies can be performed in which Vero cells infected with 3 MOI (multiplicity of infection) were incubated with different concentrations of the antibody in question as competitors for 1 hour after 20 hours. In the second incubation step, the antibody of the invention is applied at a constant concentration of 100nM and its binding is detected by flow cytometry using a fluorescently labeled antibody directed against the constant domain of the antibody of the invention. Binding in inverse proportion (inversely proportional) to the concentration of the antibody in question indicates that both antibodies recognize the same epitope. However, many other assays known in the art may be used.

The invention also relates to the production of antibodies specific for the native and recombinant polypeptides of TDP-43. The generation is for example based on immunization of animals such as mice. However, other animals for producing antibodies/antisera are also contemplated in the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, and the like. The polynucleotide encoding the corresponding selected polypeptide of TDP-43 may be subcloned into a suitable vector, wherein the recombinant polypeptide is expressed in an organism capable of expression, e.g. in bacteria. Thus, the expressed recombinant protein may be injected intraperitoneally into mice, and the resulting specific antibodies may be obtained, for example, from mouse serum provided by intracardiac blood puncture. The present invention also contemplates the generation of specific antibodies to native and 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, and intramuscular or intradermal injection. Thus, antibodies directed against a polypeptide or protein or epitope of TDP-43, particularly an antibody epitope provided herein, can be obtained by direct immunization of animals by intramuscular direct injection of a vector expressing the desired polypeptide or protein or epitope of TDP-43, particularly an antibody epitope of the invention located in amino acid residues 304 to 414, more particularly an antibody epitope of the invention located in 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 antibody obtained can be quantified using ELISA, which is also described below. Additional methods for producing Antibodies are well known in the art, see, e.g., harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, cold Spring Harbor,1988.

Thus, under the indicated assay conditions, a particular antibody binds to the corresponding epitope of TDP-43 to each other, but not to other components present in the sample in significant amounts. Specific binding to a target analyte under such conditions may require a binding moiety that is 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, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with an analyte. See Shepherd and Dean(2000),Monoclonal Antibodies:APractical Approach,Oxford University Press and/or Howard and Bethell, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, the specific or selective response will be at least twice the background signal to noise ratio, and more typically more than 10 to 100 times greater than the background. Those skilled in the art are able to provide and generate specific binding molecules for the novel polypeptides. For specific binding assays, they can be readily used to avoid undesired cross-reactivity, e.g., polyclonal antibodies can be readily purified and selected by known methods (see SHEPHERD AND DEAN, loc.cit.).

"Class" of antibodies refers to the type of constant domain or constant region that the heavy chain has. Antibodies exist in five main classes, igA, igD, igE, igG and IgM, and several of these can be further divided into subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively.

In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions of residues in the amino acid sequence of the antibody and/or insertions therein and/or substitutions thereof. Any combination of deletions, insertions, and substitutions may be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include CDRs and FR. Conservative substitutions are shown under the heading of "preferred substitutions" in table 1. Further substitution-type variations are provided under the heading of "exemplary substitutions" in table 1, and as further described below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the Antibody of interest and directed against a desired activity, e.g., retained/improved antigen binding, reduced immunogenicity or improved Antibody-dependent cellular cytotoxicity (anti-body-DEPENDENT CELLULAR CYTOTOXICITY, ADCC) or complement-dependent cytotoxicity (Complement-DEPENDENT CYTOTOXICITY, CDC).

TABLE 1

Amino acids can be grouped according to common side chain characteristics:

(1) Hydrophobicity, norleucine Met, ala, val, leu, ile;

(2) Neutral hydrophilicity Cys, ser, thr, asn, gln;

(3) Acid, asp, glu;

(4) Basicity His, lys, arg;

(5) Residues affecting chain orientation, gly, pro;

(6) Aromatic Trp, tyr, phe.

Non-conservative substitutions will require a member of one of these categories to be replaced with another category.

One type of substitution variant involves substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variants selected for further investigation will have improvements (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and/or will substantially retain certain biological properties of the parent antibody relative to the parent antibody. One exemplary alternative variant is an affinity matured antibody, which may be conveniently produced, for example, using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Alterations (e.g., substitutions) may be made in the CDRs, for example, to increase antibody affinity. Such changes may be made in CDR "hot spots", i.e. residues encoded by codons that are mutated at high frequencies during the somatic maturation process (see, e.g., chowdhury, methods mol. Biol.207:179-196 (2008)) and/or SDR (a-CDRs), and the resulting variant VH or VL tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from secondary libraries has been described in Hoogenboom et al.,Methods in Molecular Biology178:1-37(O'Brien et al.,ed.,Human Press,Totowa,NJ,(2001)), for example. 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 for introducing diversity involves CDR-directed approaches in which 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 scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are typically targeted.

In certain embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, provided that such alterations do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in the CDRs that do not substantially reduce binding affinity. Such changes may be outside of CDR "hot spots" or SDR. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or comprises no more than one, two, or three amino acid substitutions.

A useful method for identifying targetable mutagenic residues or regions of an antibody is known as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues, such as Arg, asp, his, lys and Glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Additional substitutions may be introduced at amino acid positions, showing functional sensitivity to the initial substitutions. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the point of contact between the antibody and the antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for replacement. Variants may be screened to determine whether they contain the desired trait.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions of one residue in length to polypeptides comprising 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Some examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the N-or C-terminus of an antibody with an enzyme (e.g., for ADEPT) or polypeptide that increases the serum half-life of the antibody.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree to which the antibodies are glycosylated. The 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 are created or removed.

When an antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically comprise branched double-antennary oligosaccharides that are typically linked by an N-bond to Asn297 of the CH2 domain of the Fc region. See, for example, wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include a variety of carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, as well as fucose linked to GlcNAc in the "stem" of a double-antennary oligosaccharide structure. In some embodiments, oligosaccharides in the antibodies of the invention may be modified to produce antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure lacking fucose linked (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the sugar chains of Asn297, relative to the sum of all sugar structures (e.g. complex, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (Eu numbering of residues in the Fc region; see Edelman, g.m.et al., proc.Natl. Acad. Usa,63,78-85 (1969)), however Asn297 may also be located about + -3 amino acids upstream or downstream of position 297, i.e., at positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylated variants may have improved ADCC function. See, for example, U.S. patent publication No. US2003/0157108 (Presta, l.), US2004/0093621 (Kyowa Hakko Kogyo co., ltd). Some examples of publications relating to "defragmentation" or "fucose deficient" antibody variants include ：US2003/0157108;WO2000/61739;WO2001/29246;US2003/0115614;US2002/0164328;US2004/0093621;US2004/0132140;US2004/0110704;US2004/0110282;US2004/0109865;WO2003/085119;WO2003/084570;WO2005/035586;WO2005/035778;WO2005/053742;WO2002/031140;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 defragmentation antibodies include protein fucosylation deficient Lec13 CHO cells (Ripka et al., arch. Biochem. Biophys.249:533-545 (1986); U.S. patent application No. US2003/0157108a1, presta, l; and WO2004/056312A1,Adams et al, especially in example 11), and knockout cell lines, e.g., alpha-1, 6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., ,Yamane-Ohnuki et al.,Bioteeh.Bioeng.87:614(2004);Kanda,Y.et al.,Bioteehnol.Bioeng.,94(4):680-688(2006); and WO2003/085l 07).

Antibody variants having bisected oligosaccharides are also provided, e.g., wherein a double antennary oligosaccharide linked to the Fc region of an antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Some examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-mair et al.), U.S. Pat. No.6,602,684 (Umana et al.), and US 2005/0123946 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al), WO 1998/58964 (Raju, S.), and WO 1999/22764 (Raju, S.).

In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, igG2, igG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

In certain embodiments, the antibodies provided herein bind to pathological TDP-43 and form an immune complex that is cleared by Antibody Dependent Cellular Phagocytosis (ADCP), which results in enhanced TDP-43 clearance. ADCP is mediated by the interaction of an antibody Fc fragment with an Fc receptor (e.g., fcγ receptor) that is expressed on the surface of a innate immune cell (e.g., microglial or dendritic cell). By modifying the Fc portion of the antibody, fc-mediated functions can be modulated to achieve the desired effect.

In certain embodiments, the present invention contemplates antibody variants that have some, but not all, effector functions, making them desirable candidates for applications in which the in vivo half-life of the antibody is important and certain effector functions (e.g., complement activation and ADCC) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays may be performed to determine a reduction/depletion of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγr binding (and thus may lack ADCC activity), but retains FcRn binding capability. The primary cells mediating ADCC, NK cells, express fcyriii only, whereas monocytes and microglia express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in Table 3 at page 464 of RAVETCH AND KINET, ANNU.REV.IMMUNOL.9:457-492 (1991). Some non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. 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)).

Alternatively, non-radioactive assays (see, e.g., ACTI ^TM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, inc.Mountain View, calif.), and Cytotox may be usedNonradioactive cytotoxicity assay (Promega, madison, wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs) and Natural Killer (NK) cells.

Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al, proc.Nat' l Acad.sci.USA 95:652-656 (1998).

A C1q binding assay may also be performed to determine that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3C binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (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, blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determination 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)).

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 replaced (U.S. Pat. No.6,737,056). Certain antibody variants are described that have increased or decreased binding to FcR. (see, e.g., U.S. Pat. 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 so-called "DANA" Fc mutants with residues 265 and 297 replaced with alanine (U.S. Pat. No.7,332,581), or so-called "DANG" Fc mutants with residue 265 replaced with alanine and residue 297 replaced with glycine. Or antibodies with reduced effector function include antibodies in which one or more of residues 234, 235 and 329 of the Fc region are replaced, i.e. so-called "PG-LALA" Fc mutants in which residues 234 and 235 are replaced with alanine and 329 are replaced with glycine (Lo, m.et al., journal of Biochemistry,292, 3900-3908). Other known mutations at positions 234, 235 and 321, so-called TM mutants comprising the mutation L234F/L235E/P331S in the CH2 domain (Oganesyan et al. Antibodies from the human IgG4 isotype contain the mutation S228P/L235E to stabilize the hinge and reduce FgR binding (Schlothauer et al, PEDS,29 (10): 457-466). The constant domains are numbered according to the EU numbering system.

Other Fc variants include those in which a substitution is made at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region, e.g., an Fc variant in which residue 434 of the Fc region is substituted (U.S. patent No.7,371,826). See also Duncan & Winter, nature322:738-40 (1988), U.S. Pat. No.5,648,260, U.S. Pat. No.5,624,821.

In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0 and thus extend antibody half-life. Antibodies with enhanced affinity for FcRn include those in which one or more of the following Fc region residues are replaced, position 252, 253, 254, 256, 428, 434, including the so-called YTE mutation with substitution M252Y/S254T/T256E (Dall' Acqua et al, J immunol.169:5171-5180 (2002)) or the LS mutation M428L/N434S (Zalevsky et al, nat biotechnol.28 (2): 157-159 (2010)).

In certain embodiments, it may be desirable to produce cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the substituted residue occurs at an accessible site of the antibody. By replacing these residues with cysteines, reactive sulfhydryl groups are thus located at accessible sites of antibodies and can be used to conjugate antibodies with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as described further herein. In certain embodiments, any one or more of the following residues may be replaced with a cysteine, V205 of the light chain (Kabat numbering), A118 of the heavy chain (EU numbering), and S400 of the Fc region of the heavy chain (EU numbering). Cysteine engineered antibodies may be produced as described, for example, in U.S. patent No.7,521,541.

In certain embodiments, the antibodies provided herein can also be modified to include additional non-proteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of antibodies 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), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxaneAlkanes, ethylene/maleic anhydride copolymers, polyamino acids (homo-or random copolymers) and dextran or poly (n-vinylpyrrolidone) polyethylene glycols, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohols, and mixtures thereof. Polyethylene glycol propionaldehyde can be advantageous in manufacturing because it is stable in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular characteristics or function of the antibody to be improved, whether the antibody derivative will be used in a treatment under the conditions defined below, and the like.

In another embodiment, conjugates of antibodies with non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation may be of any wavelength including, but not limited to, a wavelength that does not damage normal cells but heats the non-proteinaceous portion to a temperature that kills cells adjacent to the antibody-non-proteinaceous portion.

Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No.4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-misfolded TDP-43 antibody described herein is provided. Such nucleic acids may encode amino acid sequences comprising the VL of an antibody and/or amino acid sequences comprising the VH of an antibody (e.g., the light chain and/or heavy chain of an antibody). In another embodiment, one or more vectors (e.g., recombinant expression vectors) comprising such nucleic acids 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) a vector comprising (1) a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is eukaryotic, e.g., chinese hamster Ovary (CHINESE HAMSTER Ovary, CHO) cells or lymphoid cells (e.g., YO, NSO, sp). In one embodiment, a method of producing an anti-misfolded TDP-43 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an 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).

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

In one embodiment, a method of preparing a TDP-43 binding molecule, in particular 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 a 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.

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199 and 5,840,523. (see also Charlton, methods in Molecular Biology, val.248 (B.K.C.Lo, ed., humana Press, totowa, NJ, 2003), pages 245 to 254, describing the expression of antibody fragments in E.coli (E.coli)). After expression, the antibodies in the soluble fraction may be isolated from the bacterial cell paste and may be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with 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).

Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Some examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells.

Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^TM techniques for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for suspension culture may be used. Further examples of mammalian host cell lines that can be used are the Kidney CVl line transformed by SV40 (COS-7), the human embryonic kidney cell line (293 or 293 cells, as described, for example, in Graham et al, J.Gen. Visual.36:59 (1977)), baby hamster kidney cells (baby HAMSTER KIDNEY CELL, BHK), mouse Sertoli cells (TM 4 cells, as described, for example, in Mather, biol. Reprod.23:243-251 (1980)), macaque kidney cells (CV l), african green macaque kidney cells (VERO-76), human cervical carcinoma cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (WI 38), human liver cells (Hep G2), mouse mammary tumors (MMT 060562), TRI cells, as described, for example, in Mather et al, annals N.Y Ad. Sei.383:44-68 (1982), and FS4 cells. Other mammalian host cell lines that may be used include Chinese Hamster Ovary (CHO) cells, including DHFR CHO cells (Urlaub et al, proc.Natl.Acad.cii.USA 77:4216 (1980)), and myeloma cell lines such as YO, NSO and Sp2/0. For reviews of certain mammalian host cell lines suitable for antibody production, see, e.g. ,Yazaki and Wu,Methods in Molecular Biology,Val.248(B.K.C.Lo,ed.,Humana Press,Totowa,NJ),pp.255-268(2003).

For delivery of molecules across the blood brain barrier (blood brain barrier, BBB), there are several methods known in the art, such as changes in the route of administration, disruption of the BBB and its permeability, nanoparticle delivery, trojan horse method (Trojan horse approach), receptor-mediated transport, and cell and gene therapy.

The modification of the route of administration can be achieved by direct injection into the brain (see, e.g., papanastassiou et al, GENE THERAPY, 9:398-406 (2002)), implantation of a delivery device in the brain (see, e.g., gillet 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)).

Methods of barrier disruption include, but are not limited to, ultrasound (see, e.g., U.S. patent publication No. 2002/0038086), osmotic pressure (e.g., permeabilization by administration of hypertonic mannitol (Neuwelt,E.A.,Implication of the Blood-Brain Barrier and its Manipulation,Vols 1&2,Plenum Press,N.Y.(1989)));, permeabilization by, e.g., bradykinin or permeabilizing agent A-7 (see, e.g., U.S. patent Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416).

Methods of altering BBB permeability include, but are not limited to, the use of glucocorticoid blockers to increase blood brain barrier permeability (see, e.g., U.S. patent application publication nos. 2002/0065259, 2003/0162695, and 2005/0123533), activation of potassium channels (see, e.g., U.S. patent application publication No. 2005/0089473), and inhibition of ABC drug transporters (see, e.g., U.S. patent application publication No. 2003/0073013).

Trojan horse delivery methods of delivering antibodies or antibody fragments thereof across the blood brain barrier include, but are not limited to, cationizing the antibody (see, e.g., U.S. Pat. No.5,004,697), and using cell penetrating peptides such as Tat peptides to gain access to the CNS. (see, e.g., dietz et al, J. Neurochem.104:757-765 (2008)).

Nanoparticle delivery methods for delivering antibodies or antigen-binding fragments thereof across the blood brain barrier include, but are not limited to, encapsulation of antibodies or antigen-binding fragments thereof in liposomes or extracellular vesicles, such as exosomes, that are conjugated to (without limitation to) antibodies or antigen-binding fragments that bind to receptors on the vascular endothelium of the blood brain barrier or alternatively peptides (see, e.g., U.S. patent application publication No. 20020025313), and encapsulation of antibodies or antigen-binding fragments 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).

The antibodies of the invention may be additionally modified to enhance blood brain barrier penetration.

The antibodies or antigen binding fragments thereof of the invention may be fused to polypeptides that bind to blood brain barrier receptors. BBB receptors include, but are not limited to, transferrin receptor, insulin receptor, or low density lipoprotein receptor. The polypeptide may be a peptide, receptor ligand, single domain antibody (VHH), scFv or Fab fragment.

The antibodies of the invention may also be delivered as the corresponding nucleic acids encoding the antibodies. Such nucleic acid molecules may 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 invention for targeting endothelial cells, pericytes or astrocytes of the BBB for delivery to the BBB. In some embodiments, the endothelial cells of the BBB, the pericytes or astrocytes of the BBB express the antibody and secrete it into the brain parenchyma. A preferred example is a viral vector comprising a nucleic acid molecule encoding an antibody of the invention for targeted delivery in endothelial cells of the BBB, wherein the endothelial cells of the BBB express the antibody and secrete it into the brain parenchyma. The viral vector may be a recombinant adeno-associated viral vector (recombinant adeno-associated viral vector, rAAV) selected from any AAV serotype known in the art, including but not limited to AAV1 to AAV12, to enable expression of the antibody or antibody fragment or antibody derivative in cells or in brain parenchyma.

Methods of Cell therapy to deliver antibodies or antibody fragments or antibody derivatives of the invention across the blood brain barrier include, but are not limited to, using the homing ability of endothelial progenitor cells (Endothelial Progenitor Cell, EPC) transfected ex vivo with a vector, and secreting antibodies or antibody fragments by these cells and delivering them to the brain to overcome the powerful filtering activity of the BBB (see, e.g., HELLER AND al., J Cell Mol med.00:1-7 (2020)), or using a polymer Cell implantation device loaded with genetically engineered cells to secrete antibodies or antibody fragments (see, e.g., marroquin Belaunzaran et al. Plos ONE 6 (4): e18268 (2011)).

Pharmaceutically acceptable carriers, diluents, excipients and excipients are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, 15 th or 18 th edition .(Alfonso R.Gennaro,ed.;Mack Publishing Company,Easton,PA,1990);Remington:the Science and Practice of Pharmacy, 19 th edition (Lippincott, williams & Wilkins, 1995), handbook of Pharmaceutical Excipients, 3 rd edition .(Arthur H.Kibbe,ed.;Amer.Pharmaceutical Assoc,1999);Pharmaceutical Codex:Principles and Practice ofPharmaceutics, 12 th edition .(Walter Lund ed.;Pharmaceutical Press,London,1994);The United States Pharmacopeia:The National Formulary(United States Pharmacopeial Convention);Fiedler's"Lexikon der Hilfstoffe", 5 th edition Edition Cantor Verlag Aulendorf 2002; "The Handbook of Pharmaceutical Excipients", 4 th 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.

The carrier, diluent, adjuvant and pharmaceutically acceptable excipient may be selected with regard to the intended route of administration and standard pharmaceutical practice. These compounds must be acceptable in the sense of being harmless to their recipients. See Remington's Pharmaceutical Sciences, 15 th or 18 th edition .(Alfonso R.Gennaro,ed.;Mack Publishing Company,Easton,PA,1990);Remington:the Science and Practice of Pharmacy, 19 th edition (Lippincott, williams & Wilkins, 1995), handbook ofPharmaceutical Excipients, 3 rd edition .(Arthur H.Kibbe,ed.;Amer.Pharmaceutical Assoc,1999);Pharmaceutical Codex:Principles and Practice of Pharmaceutics, 12 th edition .(Walter Lund ed.;Pharmaceutical Press,London,1994);The United States Pharmacopeia:The National Formulary(United States Pharmacopeial Convention);Fiedler's"Lexikon der Hilfstoffe", 5 th edition, edition Cantor Verlag Aulendorf 2002; "The Handbook of Pharmaceutical Excipients", 4 th 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.

An "effective amount" of a compound to be administered to a subject is a dosage suitable for treating, preventing or alleviating a disease, disorder or abnormality, according to sound medical judgment. The particular dosage level and frequency of dosage may depend on a variety of factors including, for example, the activity of the particular compound employed, the metabolic stability and length of action of that compound, the mode of administration, and the time of administration. An "effective amount" of a compound to be administered to a subject is a dosage suitable for treating, preventing or alleviating a condition, disease, disorder or abnormality, according to sound medical judgment. The particular dosage level and frequency of dosage may depend on a variety of factors including, for example, the activity of the particular compound employed, the metabolic stability and length of action of that compound, the mode and time of administration, the rate of excretion, and the drug combination. Patient-specific factors such as age, weight, general health, sex, diet, and severity of the particular condition can also affect the amount to be administered.

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. The clearance of a substance (in this case a binding molecule of the invention) is the sum of urine clearance and extra-renal clearance, and for substances cleared by the renal and extra-renal routes, plasma clearance exceeds urine clearance. The PK profile of a mAb is a function of its large size (150 kDa), relative polarity, fc-receptor binding and specific binding to a target antigen. The primary elimination pathway for mabs is cellular uptake followed by proteolytic degradation. The low clearance of mabs from the systemic circulation results in less frequent dosing than peptides or small molecules, which is generally more convenient for patients (Betts et al, mabs.2018).

Some inventive embodiments of the TDP-43 specific binding molecules

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

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

B. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 41, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 42 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 43, or

C. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 31, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 32 and VH-CDR3 comprising the amino acid sequence PC (Pro-Cys), or

D. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 21, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 22 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 23, or

E. VH-CDR1 comprising the amino acid sequence of SEQ ID No. 11, VH-CDR2 comprising the amino acid sequence of SEQ ID No. 12 and VH-CDR3 comprising the amino acid sequence of SEQ ID No. 13.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

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

B. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 45, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 46 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 47, or

C. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 35, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 36 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 37, or

D. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 26 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17, or

E. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 16 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

a) A heavy chain variable region (VH) comprising:

i. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 51, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 52 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 53, or

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 41, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 42 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 43, or

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 31, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 32 and VH-CDR3 comprising the amino acid sequence PC (Pro-Cys), or

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 21, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 22 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 23, or

V. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 11, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 12 and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 13, and

B) A light chain variable region (VL) comprising:

i. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 55, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 56 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 57, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 45, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 46 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 47, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 35, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 36 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 37, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 26 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 16 and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

a) A heavy chain variable region (VH) comprising:

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

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 41 or VH-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 41, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 42 or VH-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 42, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 43 or VH-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 43, or

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 31 or VH-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 31, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 32 or VH-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 32, and VH-CDR3 comprising the amino acid sequence PC (Pro-Cys), or

VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 21 or VH-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 21, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 22 or VH-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 22, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 23 or VH-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 23, or

V. VH-CDR1 comprising the amino acid sequence of SEQ ID NO. 11 or VH-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 11, VH-CDR2 comprising the amino acid sequence of SEQ ID NO. 12 or VH-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 12, and VH-CDR3 comprising the amino acid sequence of SEQ ID NO. 13 or VH-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 13, and

B) A light chain variable region (VL) comprising:

i. VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 55 or VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 55, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 56 or VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 56, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 57 or VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 57, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 45 or VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 45, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 46 or VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 46, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 47 or VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 47, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 35 or VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 35, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 36 or VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 36, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 37 or VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 37, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 26 or VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 26, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17 or VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 17, or

VL-CDR1 comprising the amino acid sequence of SEQ ID NO. 15 or VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 15, VL-CDR2 comprising the amino acid sequence of SEQ ID NO. 16 or VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 16, and VL-CDR3 comprising the amino acid sequence of SEQ ID NO. 17 or VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95% or 100% sequence identity to SEQ ID NO. 17.

In some embodiments, there is provided a TDP-43 binding molecule, particularly 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, or

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.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, 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, 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, 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, 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, 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.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

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

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

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

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

E. a light chain variable region (VL) comprising the sequence of SEQ ID NO. 14.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising:

a) A heavy chain variable region (VH) selected from:

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

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

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

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

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

B) A light chain variable region (VL) selected from:

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

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

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

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

V. a light chain variable region (VL) comprising the sequence of SEQ ID NO. 14.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, 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.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, 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.

In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen binding fragments thereof, that bind misfolded aggregated TDP-43 and non-aggregated physiological TDP-43 are provided.

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 monomeric and/or oligomeric and/or aggregated and/or post-translationally modified and/or truncated TDP-43, preferably human TDP-43.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, that binds misfolded aggregated human TDP-43 and non-aggregated physiological human TDP-43.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, which exhibits one or more, up to all, of the following characteristics:

a. Inhibit aggregation of the TDP-43 protein or fragment thereof,

B. Blocking TDP-43 intercellular transmission;

c. Disaggregating TDP-43 aggregates;

d. Blocking TDP-43 sowing;

e. neutralizing TDP-43 with seeding capability;

f. blocking TDP-43 diffusion;

g. enhanced TDP-43 clearance, and

H. The levels of phosphorylated TDP-43 were reduced in vivo.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, which exhibits one or more, up to all, of the following characteristics:

a. Inhibit aggregation of the TDP-43 protein or fragment thereof,

B. Blocking TDP-43 intercellular transmission;

c. blocking TDP-43 sowing;

d. blocking TDP-43 diffusion;

e. enhanced TDP-43 clearance, and

F. the levels of phosphorylated TDP-43 were reduced in vivo.

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

In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen binding fragments thereof, are provided that alleviate TDP-43 pathological conditions in vivo.

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

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

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

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

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, which binds to an epitope consisting of amino acid residues 304 to 313 of human TDP-43 (SEQ ID NO: 1).

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

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, which binds to an epitope consisting of amino acid residues 356 to 361 of human TDP-43 (SEQ ID NO: 1).

In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen binding fragments thereof, are provided that bind to an epitope within amino acid residues 397 to 407 of human TDP-43 (SEQ ID NO: 1).

In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen binding fragments thereof, are provided that bind to an epitope consisting of amino acid residues 397 to 407 of human TDP-43 (SEQ ID NO: 1).

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

In some embodiments, there is provided a TDP-43 binding molecule, particularly 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).

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, that 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.

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

In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen binding fragments thereof, are provided that have a dissociation constant (dissociation constant, KD) of 1nM or less, preferably 750pM or less, 500pM or less, 380pM or less, 230pM or less, 200pM or less, or 110pM or less, for binding to soluble TDP-43 (SEQ ID NO: 1). For further details of suitable assays reference is made to example 3 for determination of KD.

In some embodiments, the TDP-43 binding molecule is a IgA, igD, igE, igM, igG1, igG2, igG3, or IgG4 antibody or antigen-binding fragment thereof.

In a preferred embodiment, the TDP-43 binding molecule is an IgG1 or IgG4 antibody or antigen binding fragment thereof.

In some embodiments, there is provided a TDP-43 binding molecule, particularly a TDP-43 antibody or antigen binding fragment thereof, comprising an Fc mutation, preferably an S228P mutation.

In some embodiments, there is provided an (isolated) nucleic acid, wherein the (isolated) nucleic acid encodes a TDP-43 binding molecule described herein, particularly a TDP-43 antibody and fragments thereof.

In some embodiments, an (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 described herein.

In some embodiments, an (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 described herein.

In some embodiments, an (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 described herein.

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

In some embodiments, an (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 described herein.

In some embodiments, an (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 described herein.

In some embodiments, an (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 described herein.

In some embodiments, an (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.

In some embodiments, an (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.

In some embodiments, an (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.

XII composition and method

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

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

In some embodiments, conjugated binding molecules, particularly antibodies or antigen binding fragments thereof, comprising the binding molecules, particularly antibodies or antigen binding fragments thereof, described herein, and conjugated molecules are provided. The conjugates of the invention may be referred to as immunoconjugates. Any suitable conjugation molecule may be used according to the 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., radioconjugates), nucleic acid molecules, detectable labels, therapeutic agents, toxins, and blood-brain barrier penetrating moieties. Conjugation methods are well known in the art, and several techniques for conjugating antibodies to labels or other molecules are commercially available. Conjugation is typically performed by amino acid residues (e.g., lysine, histidine, or cysteine) contained within the binding molecules of the present invention. They may depend on methods such as the NHS (succinimidyl) ester method, isothiocyanate method, carbodiimide method and periodate method. Conjugation can be achieved, for example, by producing a fusion protein. This is suitable in the case of a binding molecule conjugated to another protein molecule. Thus, suitable genetic constructs may be formed which allow expression of fusions of the binding molecules of the invention with labels or other molecules. Conjugation may be performed through a suitable linker moiety to ensure proper spatial separation of the antibody from the conjugated molecule (e.g., a detectable label). However, a joint is not required in all cases. In some embodiments, the TDP-43 specific binding molecules of the invention are linked to a detectable label.

The invention also relates to immunoconjugates comprising the TDP-43 binding molecules provided herein conjugated to one or more therapeutic agents, e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant, or animal-derived protein toxins, enzymatically active toxins, or fragments thereof), radioisotopes (i.e., radioconjugates), blood-brain barrier penetrating moieties, or detectable labels. There are a variety of techniques for improving drug delivery across the Blood Brain Barrier (BBB) as discussed herein, with the necessary modifications to the discussion. Non-invasive techniques include the so-called "trojan horse method" in which conjugated molecules deliver the binding molecules of the invention by binding to the BBB receptor and mediating transport. Suitable molecules may comprise endogenous ligands or antibodies, particularly monoclonal antibodies, which bind to specific epitopes on the BBB receptor.

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

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.

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

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 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, in combination with a pharmaceutically acceptable carrier and/or excipient and/or diluent.

In some embodiments, immunoconjugates comprising the TDP-43 binding molecules of the invention cross the blood brain barrier using a delivery vehicle or 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 small molecule, preferably a peptide, receptor ligand, single domain antibody (VHH), scFv, or Fab fragment. In some embodiments, the blood brain barrier moiety binds to a blood brain barrier receptor, which may comprise a transferrin receptor, an insulin receptor, or a low density lipoprotein receptor.

In some embodiments, the TDP-43 specific binding molecule is part of a detection and/or diagnostic kit comprising a 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 a TDP-43 specific binding molecule.

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

a. Incubating the sample with a capture antibody and a detection antibody;

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

c. Measuring the signal emitted by the detection antibody;

Wherein the capture antibody is a TDP-43 binding molecule of the invention.

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

a. Incubating the sample with a capture antibody and a detection antibody;

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

c. Measuring the signal emitted by the detection antibody;

Wherein the detection antibody is a TDP-43 binding molecule of the invention.

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

a. Incubating the sample with a capture antibody and a detection antibody;

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

c. Measuring the signal emitted by the detection antibody;

wherein the capture antibody and the detection antibody are TDP-43 binding molecules of the invention.

In some embodiments, the present invention provides a method of detecting TDP-43 in a sample comprising the steps of:

a. Incubating the sample with a capture antibody and a detection antibody to produce a mixture;

b. incubating the mixture obtained in step a with a reagent suitable for detecting TDP-43 by detecting antibodies, and

C. the signal emitted by the detection antibody is measured.

In one embodiment of the method of detecting TDP-43 in a sample, the capture antibody is a TDP-43 binding molecule of the invention. In another embodiment of the method of detecting TDP-43 in a sample, the detection antibody is a TDP-43 binding molecule of the invention. 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 invention. The capture antibody and the detection antibody may be the same antibody or different antibodies of the invention.

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 invention. The kit may be an Enzyme-linked immunosorbent assay (ELISA) kit. The kit can be a single molecule array (Single Molecule Array)) A 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 invention may be provided in a kit as a capture agent (e.g., a capture antibody) and/or as a detection agent (e.g., a detection antibody).

In some embodiments, one or more of the 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.

Kits comprising the binding molecules of the invention are also provided. In particular, such kits are useful for performing the diagnostic methods of the invention (which include 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 TDP-43 proteinopathies or for use in the methods of the invention is provided, comprising a TDP-43 specific binding molecule of the invention. Such kits may comprise all of the necessary components for performing the methods provided herein. Typically, each component is stored separately in a single unitary package. Suitable additional components contained in the kit are, for example, buffers, detectable dyes, laboratory equipment, reaction vessels, instructions, and the like. The instructions may be tailored to the specific method for which the kit is to be used. Appropriately labeled TDP-43 binding molecules of the invention are also provided, which may be included in such kits.

In some embodiments, the TDP-43 specific binding molecule is used in an immunodiagnostic method for preventing, diagnosing or treating TDP-43 proteinopathies.

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 diagnose, prevent, alleviate or treat a disease, disorder and/or abnormality associated with TDP-43, particularly associated 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), edge-dominant age-related TDP-43 encephalopathy (LATE).

In some embodiments, the TDP-43 specific binding molecule, or an immunoconjugate wherein the TDP-43 specific binding molecule is covalently attached to another suitable therapeutic agent, or a composition comprising the TDP-43 specific binding molecule, is administered to a subject in need thereof, or in a method for diagnosing or monitoring a disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteopathy selected from the group consisting of: frontotemporal dementia (FTD), such as sporadic or familial, with or without Motor Neuron Disease (MND), with granule protein precursor (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valcasein-containing protein (VCP) mutations, with 9p chromosome linkage, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusion bodies (FTLD-TDP), silver-philic granulosis, pick's disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, such as sporadic ALS, with TARDBP mutations, with angiogenic protein (ANG) mutations), amyotrophic lateral disease (AxD), edge-dominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (calendar), pecies, alzheimer's Disease (AD), alzheimer's disease (including down-and familial forms of dementia, down's disease, familial forms of dementia, down's disease, etc, polyglutamine disease (huntington's disease and spinocerebellar ataxia type 3 (SCA 3; also known as maduo-joseph disease)), hippocampal sclerotic dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathies with mutations containing valcasein ((VCP); associated with paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophies with borderline vacuoles, myofibrillar myopathies with mutations in the gene of myocontracture protein (MYOT) or in the gene encoding Desmin (DES)), traumatic Brain Injury (TBI), lewy body Dementia (DLB) or Parkinson's Disease (PD).

In other embodiments, the invention 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 proteinopathies selected from frontotemporal dementia (FTD), amyotrophic Lateral Sclerosis (ALS), alzheimer's Disease (AD), parkinson's Disease (PD), chronic Traumatic Encephalopathy (CTE) and edge-dominated age-related TDP-43 encephalopathy (LATE).

Preferably, the disease, disorder and/or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or TDP-43 proteinopathies are selected from Amyotrophic Lateral Sclerosis (ALS), alzheimer's Disease (AD) and frontotemporal dementia (FTD). More preferably, the disease, disorder and/or abnormality associated with TDP-43, particularly with TDP-43 aggregates, or TDP-43 proteinopathies is Amyotrophic Lateral Sclerosis (ALS). More preferably, the disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteinopathy is Alzheimer's Disease (AD). More preferably, the disease, disorder and/or abnormality associated with TDP-43, in particular with TDP-43 aggregates, or TDP-43 proteinopathy is frontotemporal dementia (FTD).

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 therapeutic efficacy, or for predicting responsiveness, or for selecting a subject likely to respond to treatment with the 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.

In some embodiments, the TDP-43 specific binding molecules are used in methods wherein the TDP-43 specific binding molecules of the invention are contacted with a sample (e.g., blood, urine, cerebrospinal fluid, interstitial fluid (ISF), or brain tissue) to detect, diagnose, or monitor frontotemporal degeneration (FTD) or Amyotrophic Lateral Sclerosis (ALS), alzheimer's Disease (AD), chronic traumatic brain disease (CTE), peltier's syndrome, edge-dominated age-related TDP-43 encephalopathy (LATE), and/or Parkinson's Disease (PD).

In some embodiments, the TDP-43 specific binding molecules are used in methods wherein the TDP-43 specific binding molecules of the invention are 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 the group consisting of: frontotemporal dementia (FTD), e.g., sporadic or familial, with or without Motor Neuron Disease (MND), with granulin precursor (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valcasein-containing protein (VCP) mutations, with 9p chromosome linkages, corticobasal degeneration, with ubiquitin-positive TDP-43 inclusion bodies, frontotemporal lobar degeneration (FTLD-TDP), silverphilic granulosis, pick's disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), non-fluentic variant primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (ALS, e.g., sporadic ALS, with TARDBP mutations, with angiogenic protein (ANG) mutations), alemter disease (AxD), edge-dominant age-related TDP-43 brain disease (LATE), chronic traumatic encephalopathy (FTLD), paget syndrome, alzheimer's disease (AD, including down and familial forms of huntington's disease, huntington's disease (huntington's disease), familial form 3, spinocerebral dementia (huntington's disease), and the like; also known as Marchado-Joseph disease), sea horse sclerotic dementia and myopathy (sporadic inclusion body myositis, there are inclusion body myopathies containing mutations in valcasein ((VCP); associated with Paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophy with bordering vacuoles, myocontractile protein (MYOT) gene mutations or myofibrillar myopathies with mutations in the gene encoding Desmin (DES), traumatic Brain Injury (TBI), dementia with lewy bodies (DLB) or Parkinson's Disease (PD).

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 for preventing, alleviating or treating a disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteinopathies, or frontotemporal degeneration (FTD) or Amyotrophic Lateral Sclerosis (ALS), alzheimer's disease (AD, including sporadic and familial forms of AD), chronic Traumatic Encephalopathy (CTE), petri syndrome, and edge-dominant age-related TDP-43 encephalopathy (LATE), and/or Parkinson's Disease (PD).

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 treat a disorder selected from frontotemporal dementia (FTD), e.g., sporadic or familial, with or without Motor Neuron Disease (MND), with a granulin precursor (GRN) mutation, with a C9orf72 mutation, with a TARDBP mutation, with a valcasein-containing protein (VCP) mutation, a linkage with chromosome 9p, corticobasal degeneration, a frontotemporal leaf degeneration with ubiquitin-positive TDP-43 inclusion body (FTLD-TDP), silverfish, pick disease, semantic mutational primary progressive aphasia (svPPA), behavioural variant d (bvd), non-invasive primary ftp (nfvPPA), etc.), amyotrophic lateral sclerosis (e.g., with a granulin precursor (GRN), a pattern of atherosclerosis, ALS, a pattern of vascular disorder (e.g., amygdalin), a pattern of atherosclerosis, a pattern of focal brain-related disorder (ataxia, a pattern of brain-related to the brain-aging, a focal brain-affliction (e), a pattern of the brain-prestige, a pattern of the brain-related disorder (sepia, a pattern of the brain-affliction, down-brain-related disorder (ataxia), and the brain-related disorder (pattern of the brain-affliction, the brain-aging; also known as Marchado-Joseph disease), sea horse sclerotic dementia and myopathy (sporadic inclusion body myositis, there are inclusion body myopathies containing mutations in valcasein ((VCP); associated with Paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophy with bordering vacuoles, myocontractile protein (MYOT) gene mutations or myofibrillar myopathies with mutations in the gene encoding Desmin (DES), traumatic Brain Injury (TBI), dementia with lewy bodies (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.

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 for the manufacture of a medicament for the treatment of a disease, disorder and/or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or TDP-43 proteinopathies, or frontotemporal degeneration (FTD) or Amyotrophic Lateral Sclerosis (ALS), alzheimer's disease (AD, including sporadic and familial forms of AD), chronic Traumatic Encephalopathy (CTE), peli syndrome, and edge-dominated age-related TDP-43 encephalopathy (LATE) and/or Parkinson's Disease (PD).

Pharmaceutical formulations of anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates as described herein are prepared by mixing such antibodies or immunoconjugates of the desired purity with one or more optional pharmaceutically acceptable carriers and/or excipients and/or diluents (Remington's Pharmaceutical Sciences th edition, osol, a.ed. (1980)). Typically, the antibodies or fragments thereof are prepared as lyophilized formulations or as aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic 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, hexamethyl diammonium chloride, benzalkonium chloride, benzethonium chloride, phenols, butanols or benzyl alcohols, alkyl p-hydroxybenzoates such as methyl or propyl p-hydroxybenzoate, 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 such as Zn protein complexes, and/or non-ionic surfactants such as polyethylene glycol(s). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 #Baxter International, inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases (glycosaminoglycanase) such as a chondroitinase. Pharmaceutically acceptable excipients that may be used to formulate the composition 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, polyvinylpyrrolidone, cellulose-based substances (such as sodium carboxymethyl cellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and lanolin. The diluent may be a buffer. It may comprise a salt selected from the group consisting of phosphate, acetate, citrate, succinate and tartrate, and/or wherein the buffer comprises histidine, glycine, TRIS or mixtures thereof. It is further contemplated in the context of the present invention that the diluent is a buffer selected from potassium phosphate, acetic acid/sodium acetate, citric acid/sodium citrate, succinic acid/sodium succinate, tartaric acid/sodium tartrate, and histidine/histidine HCl or mixtures thereof.

Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Pat. No.6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient, if necessary, for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.

The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions (macroemulsion). Such techniques are disclosed in Remington' sPharmaceutical Sciences, 16 th edition, osol, a.ed. (1980).

Can be prepared into sustained release preparation. Some suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody or immunoconjugate, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

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

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, anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates for use in the prevention, diagnosis and/or treatment of TDP-43 proteinopathies are provided. In a preferred embodiment of the invention, anti-TDP-43 antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates are provided for the prevention, diagnosis 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/or edge-dominated age-related TDP-43 encephalopathy (LATE).

In another aspect, the invention 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.

According to any of some embodiments, the "subject" or "individual" may be an animal, a mammal, preferably a human.

In another aspect, the invention provides pharmaceutical formulations comprising any anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate provided herein, e.g., for use in any one of the methods of treatment. In one embodiment, the pharmaceutical formulation comprises any anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate 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 anti-TDP-43 antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate provided herein, and at least one additional therapeutic agent, e.g., as described below.

The antibodies or immunoconjugates of the invention can be used alone or in combination with other agents in therapy. For example, an antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent that targets alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or neuroinflammatory proteins.

For example, an antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent selected from, but not limited to, a neurological agent, 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-BACE 1 antibody, a BACE1 inhibitor, an anti-alpha-synuclein antibody, and a neuroinflammation inhibitor.

Such combination therapies described above encompass combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which case administration of the antibodies (preferred type of TDP-43 specific binding molecule) or immunoconjugates of the invention may occur before, simultaneously with and/or after administration of additional therapeutic agents and/or adjuvants. The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the invention may also be used in combination with radiation therapy.

The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates (and any additional therapeutic agents) of the invention may be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and, if desired, topical, intralesional, intrauterine or intravesical administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example by injection, for example intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. Various dosing regimens are contemplated herein, including, but not limited to, single administration or multiple administrations over different points in time, bolus administration (bolus administration), and pulsed infusion.

The antibodies (preferred types of TDP-43 specific binding molecules) or immunoconjugates of the invention can be formulated, administered and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disease, disorder and/or abnormality associated with TDP-43, particularly TDP-43 aggregate, or TDP-43 proteopathy being treated, the particular mammal being treated, the clinical condition of the subject, the cause of the disease, disorder and/or abnormality associated with TDP-43, particularly TDP-43 aggregate, or TDP-43 proteopathy, the site of delivery of the agent, the method of administration, the regimen of administration, and other factors known to medical practitioners. The antibody or immunoconjugate need not be, but is optionally formulated with one or more agents currently used for the prevention or treatment of the diseases, disorders and/or abnormalities associated with TDP-43, in particular TDP-43 aggregates, or TDP-43 proteinopathies in question. The effective amount of such other agents will depend on the amount of antibody or immunoconjugate present in the formulation, the type of disease, disorder and/or abnormality associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathic, or treatment, among other factors as described above. These are typically used at the same dosages and routes of administration as described herein, or at about 1% to 99% of the dosages described herein, or at any dosages and any routes that are empirically/clinically determined to be appropriate.

For preventing or treating a disease, the appropriate dosage of the antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the invention (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 prophylactic or therapeutic purposes, previous treatments, the clinical history of the subject and the response to the antibody or immunoconjugate, and the discretion of the attending physician. The antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate is suitably administered to the subject once or through a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1mg/kg to 10 mg/kg) of antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate may be the initial candidate dose for administration to a subject, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dose may be about 1 μg/kg to 100mg/kg or higher, depending on the factors described above. For repeated administration over days or longer, depending on the condition, treatment will generally be continued until the desired inhibition of disease symptoms occurs. An exemplary dose of antibody or immunoconjugate is about 0.05mg/kg to about 10mg/kg. Thus, one or more doses (or any combination thereof) of about 0.5mg/kg, 2.0mg/kg, 4.0mg/kg, or 10mg/kg may be administered to a subject. Such doses may be administered intermittently, e.g., weekly or every three weeks (e.g., subjects are allowed to receive about 2 to about 20, or e.g., about 6 doses of antibody). A higher initial loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be used. The progress of the treatment is readily monitored by conventional techniques and assays.

It will be appreciated that any of the above formulations or methods of treatment may be performed using both the immunoconjugate of the invention and an anti-TDP-43 antibody (the preferred type of TDP-43 specific binding molecule).

In another aspect of the invention, there is provided an article of manufacture comprising a material as described above useful for the treatment, prevention and/or diagnosis of a disease, disorder or abnormality associated with TDP-43, particularly an aggregate of TDP-43, or a TDP-43 proteinopathies. The article comprises a container and a label or package insert on or coupled to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition effective alone or in combination with another composition to treat, prevent and/or diagnose a disease, disorder and/or abnormality associated with TDP-43, particularly TDP-43 aggregate, or TDP-43 proteinopathies, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or immunoconjugate of the invention. A label or package insert indicates that the composition is to be used to treat a selected condition.

Furthermore, the article of manufacture may 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 invention, and (b) a second container comprising a composition wherein the composition comprises an additional therapeutic agent. The article in this embodiment of the invention may further comprise a packaging insert indicating that the composition is useful for treating a particular disorder. Alternatively or additionally, the article of manufacture may further 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 may also contain other materials as desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

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

In another embodiment, the invention relates to a method of reducing the level of TDP-43 comprising administering a binding molecule of the invention, an immunoconjugate of the invention, a composition of the invention or a pharmaceutical composition of the invention.

The methods of the invention may 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, particularly neuropharmaceuticals, anti-beta-amyloid antibodies, anti-tau antibodies, tau aggregation inhibitors, beta-amyloid aggregation inhibitors, anti-BACE 1 antibodies, BACE1 inhibitors, anti-alpha-synuclein antibodies, and neuroinflammatory inhibitors.

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

In other embodiments, the invention relates to a method for detecting and/or measuring TDP-43 levels comprising using a single molecule array) Techniques a sample is contacted with a binding molecule of the invention, preferably an antibody of the invention, 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.

As described herein, the binding molecules (preferably antibodies) of the present invention target (i.e., bind to) a particular domain or fragment of TDP-43. For example, the ACI-7071-810H12-Ab1 antibody binds to the protease resistant amyloid core of TDP-43. Thus, the method may be based on detecting and/or measuring the level of a specific domain or fragment of TDP-43. For example, the method may be based on detecting and/or measuring the level of protease resistant amyloid cores of TDP-43 in the C-terminal fragment. This may provide an indication of the disease or disease state, which is important for templated aggregation in view of the observation that disease-specific proteolytic cleavage exposing the amyloid core further enhances its seeding activity.

In certain embodiments, the dissociation constant (KD) of a TDP-43 binding molecule, particularly a TDP-43 antibody and fragments thereof, as provided herein is ∈1 μM, +.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 ^-13 M), particularly with respect to binding of TDP-43, particularly soluble TDP-43. For example, the TDP-43 binding molecules of the invention can have a KD for binding to soluble full-length TDP-43 of 2nM or less, in some embodiments 1nM or less, and in some more embodiments 750pM or less, 500pM or less, 380pM or less, 230pM or less, 200pM or less, or 110pM or less. Referring to Table 4, this is demonstrated in example 3 for the TDP-43 binding molecule of the present invention. In one embodiment, the binding affinity for Full Length (FL) TDP-43 can be assessed by determining the dissociation constant (KD) using surface plasmon resonance (surface plasmon resonance, SPR; biacore 8K,GE Healthcare Life Sciences). For a detailed description of a suitable SPR method that may be employed, reference is made to example 3.

In one embodiment, the TDP-43 binding molecule, particularly the TDP-43 antibody and fragments thereof, can reduce the level of pathological TDP-43 in the brain and improve/inhibit/reduce the formation of pathological conditions of TDP-43 in vivo.

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

In yet another embodiment, the TDP-43 binding molecule, particularly the TDP-43 antibody and fragments thereof, can reduce the level of phosphorylated TDP-43 in the hippocampus and improve/inhibit/reduce the formation of TDP-43 pathological conditions in vivo.

The TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, typically bind TDP-43 with high affinity. For example, it may display EC50 values of 200pM or less, 40pM or less, 20pM or less, or 10pM or less, as determined by Luminex assay. For further details of suitable assays reference is made to example 2.

The TDP-43 binding molecules of the invention, particularly antibodies or antigen binding fragments thereof, can have a half-life in mice of at least 10 days or at least 16 days. For further details on a suitable method of measuring half-life in mice reference is made to example 9.

Drawings

FIG. 1 is a graphical representation of quantification of phosphorylated TDP-43 (pTDP-43) from ipsilateral (FIG. 1 a) or contralateral (FIG. 1B) hippocampus in a mouse model of TDP-43 proteinopathies treated with ACI-7071-806H5-Ab1 (B), ACI-7071-810H12-Ab1 (C) or mAb negative control antibody (D). (E) non-vaccinated mice. (F) Monogenic CamK a mice (WT-tTA) lacking the human TDP-43 transgene. Mean ± SD.

FIG. 2 is a graphical representation of antibody plasma exposure at the end of the study after weekly ACI-7071-810H12-Ab1 i.p. administrations of 60mg/kg 13 times each in CamKIIa-hTDP-43NLSm mice. Mean ± SD.

FIG. 3 is a graphical representation of the linear relationship between pTDP-43 levels and ACI-7071-810H12-Ab1 plasma concentrations in the ipsilateral (black line and dots) and contralateral (light grey line and triangles) at the end of the study.

FIG. 4 immunoblots with (A) ACI-7071-810H12-Ab1, (B) anti-Total TDP-43 antibody or (C) anti-pTDP-43 (S409/S410) antibody for detection of TDP-43 in sarkosyl insoluble brain extracts prepared from frontal cortex of FTLD-TDP type A cases, either before (-) or after (+). The top arrows indicate the expected molecular weights of full length TDP-43 and pTDP-43. The bottom arrow indicates the expected molecular weight of the protease resistant fragment. Brackets indicate the expected molecular weight of the TDP-43C-terminal fragment (C-TERMINAL FRAGMENT, CTF). The bottom part of the blot provides an enhanced contrast image of the immunoblot at the expected molecular weight of the protease resistant fragment (about 8 to 15 kDa).

Examples

EXAMPLE 1 preparation of TDP-43 vaccine composition

Liposome-based vaccines were prepared according to the protocol disclosed in WO 2012/055933. Vaccines comprising full-length TDP-43 (FL TDP-43) protein as antigen (Table 2, SEQ ID NO: 1) were used for antibody production.

TABLE 2 description of TDP-43 protein and peptide antigens

Example 2 production of anti-TDP-43 antibodies

A. Immunization of mice

Female C57BL/6JOlaHsd (C57 BL/6) and BALB/C OlaHsd (BALB/C) wild-type mice (Harlan, USA) were received at 9 weeks of age. Vaccination began 10 weeks. In the presence of monophosphoryl hexaacyl lipid A, 3-deacylation (Synthesis) (3D- (6-acyl)) In the case of adjuvant, mice were vaccinated with full-length TDP-43 protein displayed on the liposome surface.

Mice were vaccinated by 200 μl subcutaneous injection (s.c.) on days 0, 4, 8, 21, 35 and 70. Mice were bled 7 days prior to immunization (preimmune plasma) and heparinized plasma was prepared at days 15, 28, 42, 77 and 136 after the first immunization. Mice for myeloma fusion were additionally vaccinated with three once daily booster injections of TDP-43 protein following i.p. injections 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 indicates high titers of antibodies against TDP-43.

B. hybridoma production and subcloning selection

Mice were euthanized and spleen cells from four individual mice were used for fusion with myeloma cells. The selection of antibodies from successfully fused hybridoma cell lines proceeds as follows. The diluted (1:32) cell culture supernatants were analyzed using a Luminex bead-based multiplex assay (Luminex, THE NETHERLANDS). Luminex beads were conjugated to FL TDP-43 and IgG captured with anti-mouse IgG-Fc antibodies (Jackson Immunoresearch, USA) specific for IgG1, igG2a, igG2b, igG2c and IgG3 subclasses. Binding to the beads conjugated to FL TDP-43 identified 386 hits (hit) from mice immunized with FL TDP-43 liposome vaccine.

Viable hybridomas are cultured using a selection medium containing serum. Clones that preferentially bound to the inclusion bodies of TDP-43 in human FTD brains and clones that bound to the C-terminus of TDP-43 were selected for further subcloning. After limiting dilution, the cloned hybridomas are cultured in low immunoglobulin-containing medium and stable colonies are selected for antibody selection and selection. Antibodies shown in table 3 were identified from this screen.

TABLE 3 EC50 values determined by Luminex assay

Example 3 characterization of antibodies by Surface Plasmon Resonance (SPR)

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).

KD determination by SPR for soluble TDP-43

The instrument was prepared with running buffer PBS-P+ and flow cells (Fc) 1 and 2 of channels 1 to 8 were activated with fresh solution of EDC/NHS (amine coupling kit, ratio of two reagents 1:1,GE Healthcare Life Sciences,BR-1006-33) at 10. Mu.L/min for 420 seconds. Soluble TDP-43 (Selvita) was diluted to a final concentration of 5. Mu.g/mL in sodium acetate pH 4.5 and injected onto Fc 2 at a flow rate of 10. Mu.L/min for 80 seconds. All flow-through cells were quenched with 1M ethanolamine (GE HEALTHCARE LIFE SCIENCES, BR-1006-33) at 10. Mu.L/min for 420 seconds. The fixed level after ethanolamine quenching was about 370RU on all eight channels. Three start-up cycles were run prior to analysis. Elevated mAb concentrations of 1.2 to 100nM were injected with single cycle kinetics, prepared by 3-fold serial dilutions in running buffer, contact time 300 seconds, and dissociation time 3600 seconds, flow rate 30 μl/min. Each cycle was followed by a regeneration using 10mM glycine-HCl pH 1.7 at 10 μl/min at 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 Biacore 8K evaluation software using a 1:1 kinetic fitting model with RI and overall Rmax. Kinetic parameters (Table 4) were obtained, binding rate constant (ka), dissociation rate constant (KD), affinity constant (KD).

TABLE 4 ka, KD, KD values of antibodies to soluble TDP-43

Example 4 determination of binding regions

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

Epitope mapping was determined using a custom peptide array library (Pepscan, netherlands). Briefly, overlapping linear peptide arrays covering the entire TDP-43 were used to define epitopes.

Peptide synthesis

To reconstruct the epitopes of the target molecules, libraries of peptide-based mimetics were synthesized using Fmoc-based solid phase peptide synthesis. Amino-functionalized polypropylene supports (supports) were obtained by grafting with proprietary hydrophilic polymer formulations, followed by reaction with tert-butoxycarbonyl-hexamethylenediamine (t-butyloxycarbonyl-hexamethylenediamine, bocHMDA) using dicyclohexylcarbodiimide (dicyclohexylcarbodiimide, DCC) and N-hydroxybenzotriazole (N-hydroxybenzotriazole, HOBt), and subsequent cleavage of the Boc-group using trifluoroacetic acid (trifluoroacetic acid, TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on amino-functionalized solid supports by custom-made modified JANUS liquid treatment station (PERKIN ELMER).

ELISA screening

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

TABLE 5 epitope for test antibodies

B. determination of binding regions using ELISA assays for ACI-7071-806H5-Ab1

ACI-7071-806H5-Ab 1 was screened by ELISA assay to determine binding regions using a library of N-terminally biotinylated peptides. Peptide sequences are provided in table 6.

96-Well streptavidin-coated ELISA plates were incubated with 5. Mu.g/mL biotinylated peptide. Plates were washed 4 times with 0.05% tween-20/PBS and then blocked with 1% bovine serum albumin (bovine serum albumin BSA) in 0.05% tween-20/PBS for 1 hour at 37 ℃. Antibodies purified from hybridoma supernatants were then added at 1 μg/ml and incubated at 37 ℃ for 2 hours, after which the plates were washed. AP conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories) was added at 1/1000 dilution in 0.05% Tween-20/PBS for 1 hour at 37 ℃. After the final wash, the plates were incubated with pNPP (Sigma-Aldrich, switzerland) AP substrate solution and read at 405nm using an ELISA reader (Tecan). The identified binding regions are provided in table 7. The test antibodies were found to bind to TP-52, TP-95, TP-96, TP-102 corresponding to regions 402 to 414, 396 to 414 (pS 403/404) and 402 to 414 (pS 409/410), respectively.

TABLE 6 peptides for determination of binding regions by ELISA

Peptide numbering	The a.a. position in SEQ ID NO. 1
		TP-52	402 To 414
TP-95	396 To 414
		TP-96	396 To 414 (S403/404 with phosphorylation)
TP-102	402 To 414 (S409/410 with phosphorylation)

TABLE 7 binding regions for test antibodies

Example 5 detection of TDP-43 in brain tissue from FTD/ALS subject by immunohistochemistry

Target engagement was evaluated in immunohistochemical experiments on tissues from the brain of FTD subjects. Human FTD brain tissue was obtained from UCSF neurodegenerative disease think tank. All materials were collected from donors from whom think tank had obtained written informed consent regarding brain necropsy and use of materials and clinical information for research purposes. Immunohistochemistry was performed on 10 μm thick frozen sections using a fluorescently labeled secondary antibody for detection. As controls, rat monoclonal anti-phosphorylated TDP-43p409/410 antibody (Biolegend, 829901) for detection of phosphorylated TDP-43 and secondary antibody without primary antibody (no 1℃Ab) for detection of non-specific background were used.

All antibodies of the invention bind to non-aggregated physiological nucleus TDP-43 and aggregated TDP-43. A detailed evaluation of the binding characteristics is summarized in table 8.

TABLE 8 detection of TDP-43 in brain tissue from FTLD-TDP subjects

NA: data is not available; absence of; +/-is unclear; weak + + +: medium ++ +: rich

Example 6 in vitro function in recombinant TDP-43 aggregation assay

To evaluate the in vitro function of the antibodies, the antibodies were tested for their ability to inhibit TDP-43 aggregation. FL TDP-43 is fused at the C-terminus to a maltose binding protein (maltose binding protein, MBP) separated by a tobacco etch virus (Tobacco Etch Virus, TEV) protease cleavage site and recombinantly produced. Aggregation of 2.5. Mu.M TDP-43-TEV-MBP fusion protein in 30mM Tris,150mM NaCl,pH 7.4 was induced by addition of TEV protease (AcTEV, invitrogen) in the presence of 2.5. Mu.M each anti-TDP-43 antibody or negative control mAb that did not bind to TDP-43, and absorbance was monitored in a 600nm mu clear 96 well plate (Greiner) over 1.5 hours. For evaluation, the endpoints were 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 by more than 97% compared to the negative control mAb (table 9). The p-value obtained for each mAb was <0.0001 when compared to the control mAb, and was analyzed statistically using one-way ANOVA followed by Dunnett's multiple comparison test.

TABLE 9 inhibition of TDP-43 aggregation

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

Example 7 in vitro Functions of FTLD-TDP brain derived TDP-43 seed in immune depletion

Sarkosyl insoluble brain fraction (Sarko-spin) was prepared according to the published protocol (LAFERRIERE ET al, 2019). UsingProtein G magnetic beads were immunodepleted. For a single reaction, 20 μl of beads were used with 3 μg of antibody for 10 μ g sarkosyl insoluble brain extract (total protein). Prior to antibody addition, 500. Mu.L PBS-0.05% was used firstThe beads were washed twice with-20 and then with 100. Mu.L PBS-0.05%-20 Washes. 100. Mu.L of PBS-0.05% was usedThe beads were resuspended in 30. Mu.g/mL of antibody in-20. The beads/antibody reactions were incubated at room temperature for 30 minutes with constant rotation and shaking (HulaMixer ^TM Sample Mixer 159420 d, thermofisher). The bead/antibody complex was incubated with PBS-0.05%-20 Washes twice and once with PBS, followed by the addition of sarkosyl insoluble brain extract. The FTLD-TDP type A brain extract pool was diluted to 100 μg/mL. The bead/antibody complex was resuspended using 100 μl of extract and incubated at room temperature for 30 minutes under constant rotation and shaking. Supernatants were collected as immunodepleted fractions using magnetic supports and characterized by western blotting. ACI-7071-806H5-Abl was effective in immunodepleting TDP-43 seed from FTLD-TDP brain extract (Table 10).

TABLE 10 immune depletion of TDP-43 seeds in brain extracts of patients

Antibody name	Immune depletion
		ACI-7071-806H5-Ab1	+++

NA: data is not available; absence of; +/-is unclear; weak + + +: medium ++ +: rich

Example 8 in vitro function of uptake of TDP-43 aggregates by microglia

For the preparation of mouse primary microglial cells, the cortex separated from CD1 mice (CHARLES RIVER, france) was enzymatically and mechanically dissociated on day 5 (P5) after birth of CD1 mice as described in the nervous tissue dissociation kit (Neural Tissue Dissociation Kit) (P) (Miltenyi, 130-092-628). From the obtained cell suspension, microglia were purified using CD11b/c microbeads according to the manufacturer's instructions (Miltenyi, 130-093-634). Microglia cells were seeded into 60 wells of a 96-well tissue culture plate (Falcon, 353219) at a density of 3×10 ⁵ cells per well and maintained in complete growth medium adapted from (5). The growth medium consisted of DMEM/F12 (Gibco, 31331-093) supplemented with 2.5% heat-inactivated FBS, 1% PS, 200ng/mL tumor growth factor beta 2 (TGF-. Beta.2; peprotech, 100-35B), 100ng/mL interleukin 34 (IL-34; R & D Systems), 5. Mu.g/mL N-acetylcysteine (Sigma, A9165), 5. Mu.g/mL insulin (Sigma, I6634), 100. Mu.g/mL apotransferrin (Sigma, T1147), 100ng/mL sodium selenite (Sigma, S-5261), and wool cholesterol (ovine wool cholesterol) (1.5. Mu.g/mL, avanti Polar Lipids). During the experiment, basal medium, i.e., supplemented with 1% penicillin/streptomycin, 5. Mu.g/mL N-acetylcysteine (Sigma, A9165), 5. Mu.g/mL insulin (Sigma, I6634), 100. Mu.g/mL apotransferrin (Sigma, T1147) and 100ng/mL sodium selenite (Sigma, S-5261) was used.

Microglia cells were plated in growth medium at 30,000 cells per well and incubated for 48 hours. Immunocomplexes were prepared at 2X final concentration in basal medium by mixing pHrodo ^TM -labeled TDP-43 aggregates and ACI-7071-806H5-Ab1, ACI-7071-810H12-Ab1 or negative control mAb in dilution plates (Eppendorf 96 well sterile) and incubating overnight at 4 ℃. The dilution plate was equilibrated at room temperature while the cells were washed three times with basal medium. After the last wash, 100. Mu.L of basal medium was kept on the cells, to which 100. Mu.L of pHrodo ^TM -labeled TDP-43 aggregates from the dilution plate were added. Cells were immediately placed within Incucyte for continuous 24 hour real-time imaging of phase contrast (to delineate microglia) and green fluorescence (to quantify labeled TDP-43 within microglia). The uptake of TDP-43 aggregates by microglia was significantly improved in the presence of both mAbs ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1 (Table 11). When compared to negative control mabs, p-values <0.001 were obtained for each mAb for statistical analysis using one-way ANOVA followed by Tukey multiple comparisons.

TABLE 11 percent enhancement of immune complex uptake by microglia

Example 9: pharmacokinetics in mice

9 Female mice (C57 BL/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. The free plasma concentrations of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1 were determined after single intraperitoneal (i.p.) administration of 60mg/kg of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1, respectively. Plasma samples were collected from the tail veins of 3 mice at each time point, except at time point 72 hours, after dosing, at 0.25 hours, 1 hour, 8 hours, 24 hours, 72 hours and 7 days, 10 days, 14 days, 21 days and 28 days. Table 12 shows half-lives obtained for both antibodies.

TABLE 12 half-life of antibodies in mice

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

Both antibodies exhibit good and similar PK parameters, which makes them desirable candidates for applications where the in vivo half-life of the antibodies is important (e.g., therapeutic use in humans).

Example 10 in vivo functional efficacy

The purpose of this study was to evaluate the therapeutic effect of intraperitoneally (i.p.) delivered antibodies in a mouse model of TDP-43 proteinopathic disease.

Method of

Lifetime (in-LIFE PHASE)

Double transgenic CamKIIa-hTDP43NLSm animals were produced by crossing hemizygous females (JAX Stock #14650: B6; C3-Tg (tetO-TARDBP) 4 Vle/J) with hemizygous males (JAX Stock # 0070004: B6.Cg-Tg (CamKIIa-tTA) 1 Mmay/DboJ). Breeders (breeders) and mice maintain a 200mg/kg doxycycline diet until 12.5≥2 weeks of age. At 13.5≥2 weeks of age, camKIIa-hTDP43NLSm mice were deeply anesthetized with 1mg/kg buprenorphine and fixed in a stereotactic frame. Sarkosyl insoluble extracts from brains of FTLD-TDP cases were sonicated prior to injection into the dorsal hippocampus. Each injection site (needle was introduced into the left hemisphere according to bregma coordinates: 2.0mm and 1.3mm left from the midline; three dorsal hippocampal sites with an initial depth of-1.95 mm below the dura and then needle portions were withdrawn to-1.55 mm for the second injection and again to-1.15 mm for the last injection) received 1 μl Sarkosyl insoluble extract at a rate of 0.3 μl/min with a rest period of 4 minutes after injection. One day later, intraperitoneal injections of mAb (60 mg/kg) were started weekly for 13 consecutive weeks. Three different antibodies, ACI-7071-806H5-Ab1, ACI-7071-810H12-Ab 1 and negative control mAb were tested. Final tissue collection was performed 3 months after injection.

Tissue sealing, sectioning, immunofluorescent staining and quantification

Frozen tissue pieces were sectioned at a thickness of 20 μm per piece. Staining was performed on a Leica BOND-RX. Coronary sections from 4 levels covering the hippocampus were processed for immunolabeling. For triple pTDP43/NeuN/Iba1 immunofluorescence (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 at 100 ℃ for 10 min in Leica ER1 buffer pH 6 (AR 9640) followed by incubation with protein blockers (PowerVision IHC/ISH Super Blocking, leica, ref.pv6122). Slides were then incubated with primary antibodies in two steps, first with phospho-TDP 43 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, the secondary antibodies were incubated in two steps, first with a mixture of three antibodies against mouse-Cy 3 (Jackson, goat Ab, 1/200), rabbit-Alexa 488 (Jackson, goat Ab, 1/200) and rat-biotin (Jackson, goat Ab, 1/250), and second with streptavidin-Cy 5 (Jackson, 1/300). Finally, the slides were incubated with DAPI (1/300). All antibodies were diluted in BOND antibody dilutions (accession number AR 9352) and slides were mounted in anti-fade and coverslipped.

The IF slides were digitized using an Axio scan.z1 digital full-slide scanner (Carl Zeiss, canada). The image is Quality Control (QC) reviewed and the final image is transmitted to Biopensive servers for image processing and analysis. The ROI was defined using a U-Net convolutional neural network trained on a manually drawn tissue slice dataset. The ROI was then visually QC-reviewed and manually adjusted as needed. IHC staining quantification (shown as average staining density in fig. 1) was performed on each digitized IHC slide using Biospective PERMITS ^TM software. In addition, double co-localization of pTDP-43 and NeuN and triple co-localization of pTDP-43, neuN and Iba1 were calculated from the segmented images. IHC analysis and quantification was performed in a manner that was not informed about the cohort. Any potential outliers due to technical reasons are removed before the data is blinded. Data are expressed as mean ± standard deviation.

Statistical analysis

Statistical analysis was performed in MATLAB. The data is first evaluated for normalization using a normal probability map, followed by evaluation of variance homogeneity, if applicable. Treatment groups were compared using one-way ANOVA (ANOVA a 1), post-hoc comparisons using Tukey honest significance differences (multcompare), or Kruskal-Wallis (kruskalwallis) for non-normally distributed clinical data. For weight measurement, two-way mixed ANOVA was also used to study any interactions between groups and time points. As an additional exploratory measurement, a direct t-test or Mann-Whitney comparison was performed between the two groups. (P values <0.05 are shown by asterisks).

Results

Inoculation of brain extracts in double transgenic mice (CamKIIa-hTDP 43 NLSm) resulted in pTDP-43 pathological conditions in both ipsilateral (same brain hemisphere where brain extracts were injected) and contralateral (brain hemisphere opposite the brain extract injection site) brain horses when compared to non-vaccinated mice (E in fig. 1) or (WT-tTA) single gene CamK a mice lacking the human TDP-43 transgene (F in fig. 1).

Thirteen intraperitoneal administrations of ACI-7071-810H12-Ab1 weekly resulted in a steady state antibody plasma level of 1787 μg/mL measured at the end of the study (FIG. 2). Assuming a blood to brain permeability of 0.1%, the theoretical concentration in the brain was calculated and found to be 1787ng/mL. A comparison of ACI-7071-810H12-Ab1 KD (0.38 pM or 57 ng/mL) with the theoretical brain concentration (1787 ng/mL. Apprxeq.30 times higher than KD) highlights the favorable ratio of therapeutic use of ACI-7071-810H12-Ab 1. Mice treated with ACI7071-810H12-Ab1 showed a statistically significant decrease of pTDP-43 (C in fig. 1) compared to negative control mAb (D in fig. 1) in both ipsilateral and contralateral brain horses. In addition, linear regression analysis showed a negative correlation trend between ACI-7071-810H12-Ab1 antibody exposure and pTDP-43 pathological amounts (on both ipsilateral and contralateral sides), confirming the exposure-response relationship (ipsilateral: r ² =0.37 and p=0.06; contralateral: r ² =0.23 and p=0.17; fig. 3). ACI-7071-806H5-Ab1 showed a trend of pTDP-43 pathologically decrease in both the ipsilateral and contralateral sides (B in FIG. 1). These data indicate that in this mouse model, the tested monoclonal antibodies effectively captured extracellular TDP-43, which resulted in pathological spread of TDP-43.

Example 11 antibody sequencing

The cloned hybridoma cell lysates were used for gene sequencing of the variable regions. The mouse hybridomas are harvested and lysed using a lysis buffer containing a guanidine salt to inactivate the rnases. cDNA was obtained by reverse transcription of total mRNA. The DNA fragment encoding the antibody variable region was amplified by RACE-PCR (Takara Bio, catalog # 634839) using specific primers that annealed in the antibody constant region. The 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 ofImmunological Interest,91-3242 (1991). The nucleotide sequences of the heavy (VH) and light (VL) chain variable regions are shown in table 13. The translated protein sequences of the selected heavy (VH) and light (VL) chain variable regions, and their Complementarity Determining Regions (CDRs), are shown in table 14.

EXAMPLE 12 binding of ACI-7071-810H12-Ab1 to protease resistant amyloid core of TDP-43

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

Sarkosyl insoluble brain extracts from FTLD-TDP type a patients (prepared as described previously; LAFERRIERE ET al., 2019) were immunoblotted with pronase and pronase-free treatments. Briefly, sarkosyl insoluble samples were treated with 0.4mg/mL pronase (Sigma, 10165921001) at 21℃for 1 hour and then centrifuged at 20,000g for 30 minutes at 4 ℃. The supernatant was discarded and the pellet was resuspended in PBS by sonicating 30 times with an ultrasonic probe (Q-Sonica) at an amplitude of 30.

Sarkosyl insoluble extracts were mixed with 4 Xsample loading buffer and 0.1mM dithiothreitol (Dithiothreitol, DTT) and boiled at 95℃for 10 min. Samples were loaded onto 4% to 12% Bis-Tris gels and migrated at 100 volts (V) for 90 minutes. Proteins were transferred to nitrocellulose membranes using the iBlot2 system (20 milliamp (mA), 7 minutes). At the position ofThe membrane was blocked in blocking buffer for 1 hour at room temperature with stirring. Primary antibodies (ACI-7071-810H 12-Ab1, TDP-43 (Proteintech, 60019-2-IG) or pTDP-43 (Biolegend, 829901)) were combined in PBS-0.1% Tween-20%Diluted to 1:1000 in blocking buffer (1:1) and incubated overnight on the membrane at 4 ℃ with stirring. The membrane was washed 3 times in PBS-0.1% Tween-20 with stirring. The secondary anti-donkey anti-mouse IRDye680CW and donkey anti-rat IRDye800CW) The membranes were diluted 1:10,000 in the same buffer as the primary antibody and incubated for 1 hour at room temperature with constant stirring. After washing the membrane 3 times in PBS containing 0.1% Tween-20, useThe Odyssey imager scans.

Immunoblots for FTLD-TDP type a sarkosyl insoluble brain extracts showed that (a) ACI-7071-810H12-Ab1 bound to the C-terminal fragment in addition to the 43kDa band corresponding to full length TDP-43 (fig. 4). A similar signal was obtained with (C) an antibody binding to the TDP-43 phosphorylation epitope pS409/410, indicating that the C-terminal fragment bound by ACI-7071-810H12-Ab1 retains the disease-specific phosphorylation site (FIG. 4). However, only ACI-7071-810H12-Ab1 was shown to bind to the protected core of TDP-43 after limited proteolysis of sarkosyl insoluble FTLD-TDP type A brain extracts with pronase treatment (FIG. 4). In contrast, antibodies that bound to the N-terminal ((B) TDP-43 antibody bound to RRM2 region) or C-terminal region (pS 409/410 antibody, (C)) of the amyloid core showed no signal on immunoblots of samples after limited proteolysis.

These data confirm that ACI-7071-810H12-Ab1 binds to protease resistant amyloid cores of full length TDP-43 and TDP-43 fragments (expected to be about 8 to 9kDa;Arseni et al, 2021;Arseni et al, 2023 based on reported structure). Such binding properties are valuable for use in therapy, as 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 proteolytic processing of TDP-43 and its enrichment in the brain of patients have been shown to be disease-specific pathological features. Taken together, these data further support the potential of ACI-7071-810H12-Ab1 for use as a therapeutic or diagnostic antibody.

TABLE 13 nucleotide sequences of the heavy chain (VH) and light chain (VL) variable regions

TABLE 14 amino acid sequences of heavy chain (VH) and light chain (VL) variable regions and their CDRs

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

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention 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. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those suitably derived from other aspects of the invention, including individually (inisolation).

Claims (60)

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, or

   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. 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. 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. 4. The TDP-43 binding molecule of any one of the preceding claims which binds misfolded aggregated TDP-43 and non-aggregated physiological TDP-43.
5. 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. 6. The TDP-43 binding molecule of any one of the preceding claims which binds misfolded aggregated human TDP-43 and non-aggregated physiological human TDP-43.
7. 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. Inhibit aggregation of the TDP-43 protein or fragment thereof,

   B. Blocking TDP-43 intercellular transmission;

   c. Disaggregating TDP-43 aggregates;

   d. Blocking TDP-43 sowing;

   e. neutralizing TDP-43 with seeding capability;

   f. blocking TDP-43 diffusion;

   g. enhanced TDP-43 clearance, and

   H. The levels of phosphorylated TDP-43 were reduced in vivo.
8. 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. Inhibit aggregation of the TDP-43 protein or fragment thereof,

   B. Blocking TDP-43 intercellular transmission;

   c. blocking TDP-43 sowing;

   d. blocking TDP-43 diffusion;

   e. enhanced TDP-43 clearance, and

   F. the levels of phosphorylated TDP-43 were reduced in vivo.
9. 9. The TDP-43 binding molecule of any one of the preceding claims which enhances TDP-43 clearance.
10. 10. A TDP-43 binding molecule according to any one of the preceding claims which reduces TDP-43 pathological conditions in vivo.
11. 11. The TDP-43 binding molecule of any one of the preceding claims which reduces the level of misfolded aggregated TDP-43 and/or phosphorylated TDP-43 in vivo.
12. 12. The TDP-43 binding molecule of any one of the preceding claims which reduces the level of phosphorylated TDP-43 in the hippocampus.
13. 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. 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. 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. 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. 17. The TDP-43 binding molecule of any one of the preceding claims which is an antibody or antigen binding fragment thereof.
18. 18. The TDP-43 binding molecule of any one of the preceding claims, which has a dissociation constant (KD) of 1nM or less for binding to soluble TDP-43 (SEQ ID NO: 1), preferably 750pM or less, 500pM or less, 380pM or less, 230pM or less, 200pM or less or 110pM or less.
19. 19. The TDP-43 binding molecule of any one of the preceding claims which is a IgA, igD, igE, igM, igG, igG2, igG3 or IgG4 antibody or antigen-binding fragment thereof.
20. 20. The TDP-43 binding molecule of any one of the preceding claims which is an IgG1 or IgG4 antibody or antigen binding fragment thereof.
21. 21. The TDP-43 binding molecule of any one of the preceding claims, comprising an Fc mutation, preferably an S228P mutation.
22. 22. An immunoconjugate comprising the TDP-43 binding molecule according to any one of the preceding claims.
23. 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. 24. The immunoconjugate of claim 23, wherein the delivery vehicle comprises a liposome or an extracellular vesicle.
25. 25. The immunoconjugate of claim 23, wherein the TDP-43 binding molecule is attached to the blood brain barrier moiety.
26. 26. The immunoconjugate of claim 23 or 25, wherein the blood brain barrier moiety is a polypeptide or small molecule, preferably a peptide, receptor ligand, single domain antibody (VHH), scFv, or Fab fragment.
27. 27. The immunoconjugate of claim 23, 25, or 26, wherein the blood-brain barrier moiety binds to a blood-brain barrier receptor.
28. 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. 29. A labeled binding molecule, in particular a labeled antibody, comprising a TDP-43 binding molecule according to any one of claims 1 to 21.
30. 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. 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 pharmaceutical use.
32. 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 related disease, disorder and/or abnormality, or TDP-43 proteinopathy.
33. 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, the labeled binding molecule of claim 29 or the pharmaceutical composition of claim 30 for diagnostic use.
34. 34. A TDP-43 binding molecule or immunoconjugate, labelled binding molecule or pharmaceutical composition for use as claimed in claim 33 for the diagnosis of a disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathies.
35. 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, 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. 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 in monitoring a disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathies.
37. 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 related disease, disorder and/or abnormality or TDP-43 proteinopathies is frontotemporal dementia (FTD), e.g. sporadic or familial, with or without Motor Neuron Disease (MND), with particle protein precursor (GRN) mutation, with C9orf72 mutation, with TARDBP mutation, with valcasein protein (VCP) mutation, with 9p chromosome linkage, corticobasal degeneration, ubiquitin-positive TDP-43 inclusion body-containing frontotemporal lobar degeneration (FTLD) (FTLD-TDP), silver-philic particle disease, pick disease, semantically variable primary progressive aphorium (svPPA), behavioural variant FTD (bvd), non-rheological primary progressive aphorium (nfvPPA), etc.), amyotrophic Lateral Sclerosis (ALS), e.g. peal, ALS, TARDBP, atherosclerosis (also known as multifario-factor), polyaxial disease (atd), focal-3, diffuse focal-phase (atd), focal-related brain-related pattern (septicemia), brain-related pattern (including huntington's), brain-type (multiflower-pattern 3, and diffuse-pattern 3-phase (septicemia), brain-related pattern (septics), brain-related disorder (pattern 3, brain-related disorder (pattern) and brain-related disorder (pattern (brain-related disorder) or pattern (brain-associated disease) pattern (brain-associated with the brain pattern associated with the brain-associated symptoms There are inclusion body myopathies containing mutations in valcasein ((VCP); associated with Paget's disease and frontotemporal dementia), ocular pharyngeal muscular dystrophy with bordering vacuoles, myocontractile protein (MYOT) gene mutations or myofibrillar myopathies with mutations in the gene encoding Desmin (DES), traumatic Brain Injury (TBI), dementia with lewy bodies (DLB) or Parkinson's Disease (PD).
38. 38. The TDP-43 binding molecule or immunoconjugate or labelled binding molecule or pharmaceutical composition for use according to claim 37, wherein the TDP-43 related 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 edge-dominated age-related TDP-43 encephalopathy (LATE).
39. 39. The TDP-43 binding molecule or immunoconjugate or labelled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy is Amyotrophic Lateral Sclerosis (ALS).
40. 40. The TDP-43 binding molecule or immunoconjugate or labelled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy is Alzheimer's Disease (AD).
41. 41. The TDP-43 binding molecule or immunoconjugate or labelled binding molecule or pharmaceutical composition for use according to claim 38, wherein the TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy is frontotemporal dementia (FTD).
42. 42. A method of maintaining or improving cognitive memory capacity or slowing memory loss in an individual suffering from a TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathic disease, 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. 43. A method of reducing the level of aggregated TDP-43 and/or phosphorylated TDP-43 in a subject, comprising administering to the subject 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. 44. The method of claim 42 or 43, wherein the method comprises administering at least one additional therapeutic agent.
45. 45. The method of claim 44, wherein the additional therapeutic agent targets alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or neuroinflammatory protein.
46. 46. A nucleic acid molecule encoding the TDP-43 binding molecule of any one of claims 1 to 21.
47. 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. 48. A nucleic acid molecule encoding a TDP-43 binding molecule comprising the nucleotide sequence shown below:

    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, 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. 49. The nucleic acid molecule of claim 46, 47 or 48, wherein the nucleic acid is part of a viral vector for targeted delivery to the blood brain barrier or any other cell type in the CNS.
50. 50. The nucleic acid molecule of claim 49, wherein said targeted delivery is to endothelial cells of the blood brain barrier, pericytes or astrocytes of the blood brain barrier, preferably endothelial cells of the blood brain barrier.
51. 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 AAV 12.
52. 52. A recombinant expression vector comprising the nucleic acid of any one of claims 46 to 51.
53. 53. A host cell comprising the nucleic acid of any one of claims 46 to 51 and/or the vector of claim 52.
54. 54. A cell-free expression system comprising the recombinant expression vector of claim 52.
55. 55. A method for producing a TDP-43 binding molecule, in particular an antibody or antigen binding fragment thereof, comprising the steps of:

    a. culturing a host cell according to claim 53 or a cell-free expression system according to claim 54 under conditions suitable for the production of said TDP-43 binding molecule, in particular said antibody or antigen binding fragment thereof, and

    B. isolating the TDP-43 binding molecule, in particular the antibody or antigen binding fragment thereof.
56. 56. A method of detecting and/or quantifying TDP-43 in a sample obtained from a subject, the method comprising contacting the sample with a TDP-43 binding molecule according to 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. 57. Use of the TDP-43 binding molecule of any one of claims 1 to 21 in a pairing assay comprising the steps of:

    a. Incubating the sample with a capture antibody and a detection antibody;

    b. Incubating the mixture obtained in step a with a reagent 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 antibodies as defined in any one of claims 1 to 21.
58. 58. The use of a TDP-43 binding molecule according to claim 57, wherein the detection antibody is selected from the group of antibodies as defined in any one of claims 1 to 21.
59. 59. The method of claim 56 or the use of a TDP-43 binding molecule of claim 57 or 58, wherein the sample is human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF) and/or urine, preferably CSF.
60. 60. Kit for diagnosing a TDP-43 related disease, disorder and/or abnormality, or TDP-43 proteinopathy, or for use according to any of claims 31 to 41, or for use in a method according to any of claims 42 to 45, comprising a TDP-43 binding molecule according to any of claims 1 to 21.