Anti-TDP-43 binding molecule and its use

Abstract

The present invention relates to a trans-active, responsive DNA-binding protein (also known as TARDB or TDP-43) with a molecular weight of 43 kDa. The present invention relates to TDP-43-specific binding molecules, particularly anti-TDP-43 antibodies or their antigen-binding fragments or derivatives, and their use. The present invention provides means and methods for diagnosing, preventing, improving, 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

[Technical Field]

[0001] The present invention relates to a trans-active, responsive DNA-binding protein (also known as TARDB or TDP-43) with a molecular weight of 43 kDa. The present invention relates to TDP-43-specific binding molecules, particularly anti-TDP-43 antibodies or their antigen-binding fragments or derivatives, and their use. The present invention provides means and methods for diagnosing, preventing, improving, and / or treating diseases, disorders, and / or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or TDP-43 proteinopathy, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-dominant age-related TDP-43 encephalopathy (LATE). [Background technology]

[0002] Age-related brain disorders (proteinopathy), characterized by pathological aggregation of proteins in the central nervous system (CNS) and peripheral organs, represent one of the leading causes of disability and mortality worldwide. The most well-characterized protein that forms aggregates is amyloid-beta in Alzheimer's disease and related disorders. Other disease-related aggregate-forming proteins that lead to neurodegeneration include, but are not limited to, tau, alpha-synuclein (aSyn, a-syn), Huntington's protein, fused in sarcoma (FUS), dipeptide repeat proteins (DPRs) produced by unconventional translation of C9orf72 repeat elongation, superoxide dismutase 1 (SOD1), and TDP-43. Diseases involving TDP-43 aggregates are generally listed as TDP-43 proteinopathy, including, but not limited to, ALS and FTD.

[0003] I.TDP-43 Introduction The trans-active response (TAR) DNA-binding protein 43kDa (TDP-43) is a 414-amino acid protein encoded by the TARDBP gene located on chromosome 1p36.2 (ALS10). TARDBP consists of six exons (exon 1 is non-coding, and exons 2-6 encode proteins). TDP-43 belongs to the heterogeneous ribonucleoprotein (hnRNP) family of RNA-binding proteins (Wang et al., Trends in Molecular Medicine Vol.14 No.11, 2008, 479-485; Lagier-Tourenne et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64). TDP-43 contains five functional domains (Figure 1 in Warraich et al., The International Journal of Biochemistry & Cell Biology 42 (2010) 1606-1609), namely two RNA recognition motifs (RRM1 and RRM2) with two highly conserved hexamer ribonucleoprotein 2 (RNP2) regions and octamer ribonucleoprotein 1 (RNP1) regions, a nuclear export signal (NES) and a nuclear localization signal (NLS) that enable the transport of bound mRAN back and forth between the nucleus and cytoplasm, and a C-terminal glycine-rich domain that mediates protein-protein interactions. TDP-43 is involved in various aspects of RNA processing, including transcription, splicing, transport, and stabilization (Buratti and Baralle, FEBS Journal 277 (2010) 2268-2281). This is a highly conserved and ubiquitously expressed protein with a tightly self-regulated expression level, which continuously traverses between the nucleus and cytoplasm but is primarily localized in the nucleus.In 2006, TDP-43 was identified as a protein that accumulates in the vast majority of cases of frontotemporal lobar degeneration (FTLD) with tau-negative, ubiquitin-positive inclusions (at the time referred to as FTLD-TDP) and in the majority of cases of amyotrophic lateral sclerosis (ALS) (Arai et al., Biochemical and Biophysical Research Communications 351 (2006) 602-611; Neumann et al., Science 314, (2006), 130-133).

[0004] Thirty-eight negative-dominant mutations in TDP-43 were identified in patients with sporadic and familial ALS, as well as hereditary FTD, primarily located in glycine-rich domains (Figure 1, Lagier-Tourenne and Cleveland, Cell 136, 2009, 1001-1004). As demonstrated by precipitation assays, TDP-43 is genetically prone to aggregation, a tendency further amplified by some ALS-associated TARDBP mutations (Ticozzi et al., CNS Neurol. Disord. Drug Targets. 2010, 9(3), 285-296), and TDP-43 aggregation is associated with clinical signs of the disease.

[0005] II. TDP-43 in neurodegeneration TDP-43 aggregates are associated with frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), corticobasal degeneration, and frontotemporal lobe degeneration with ubiquitin-positive TDP-43 inclusions), such as frontotemporal dementia (e.g., FTD linked to chromosome 9p, with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with balocin-containing protein (VCP) mutations, with or without motor neuron disease (MND), corticobasal degeneration, and frontotemporal lobe degeneration with ubiquitin-positive TDP-43 inclusions). Sexual (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-dominant age-related TDP-43 encephalopathy ( The growing list of neurodegenerative conditions (Lagier-Tourenne) includes, but is not limited to, LATE, chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerotic dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with balocin-containing protein (VCP) mutations; associated with Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy with myotilin (MYOT) gene mutations or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD). This has been identified in et al., Human Molecular Genetics, 2010, Vol. 19, Review Issue 1 R46-R64).The term LATE is intended to encompass several previously used disease names related to TDP-43 proteinopathy, including hippocampal sclerosis, age-related hippocampal sclerosis, hippocampal sclerotic dementia, age-related brain TDP-43 with sclerosis (CARTS), and TDP-43 lesions in the elderly that may involve cognitive impairment (see reviews of Kuslansky et al., 2004; Lippa and Dickson, 2004; Nelson et al., 2013, 2016b; Dutra et al., 2015).

[0006] Aggregated TDP-43 derived from patient brains exhibits several abnormal modifications, including hyperphosphorylation, ubiquitination, acetylation, and proteolytic cleavage of the C-terminal fragment (Arai et al., Biochemical and Biophysical Research Communications 351 (2006) 602-611; Neumann et al., Science 314, (2006), 130-133; Neumann et al., Acta Neuropathol. (2009) 117: 137-149; Hasegawa et al., (2008) Annals of Neurology Vol 64 No 1, 60-70; Cohen et al., Nat Commun. 6: 5845, 2015). The aggregation mechanism of TDP-43 and the species of TDP-43 involved in aggregation are not fully understood. However, there is evidence pointing to the importance of the C-terminal region of TDP-43 in pathological mechanisms. The C-terminal domain, known as the low-complexity domain (LCD), is inherently disordered and contains regions rich in glycine, hydrophobic residues, glutamine, and asparagine (Afroz et al., 2019). While this region of TDP-43 has the inherent property of forming higher-order physiological assemblies such as stress granules in disease (Gasset-Rosa et al., 2019), irreversible intermolecular and intramolecular interactions within this region can lead to pathological aggregation (Gasset-Rosa et al., 2019). In fact, recent structural and biochemical studies have demonstrated that the region of TDP-43 consisting of amino acids 272-360 within the C-terminal region adopts a stable protease-resistant amyloid core structure in the brains of ALS and FTD patients (Arseni et al., 2021; Arseni et al., 2023). Furthermore, it has been shown that disease-specific proteolytic cleavage exposing this amyloid core further enhances the seeding activity crucial for templated aggregation (Kumar et al., 2023).Such proteolytic processing and enrichment of TDP-43 in the patient's brain is a disease-specific etiological feature that adds to the post-translational modifications described above.

[0007] Another characteristic of TDP-43 pathogenesis is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. Characteristic lesions of FTLD-TDP are intracytoplasmic inclusions (NCIs and GCIs, respectively) and dystrophic neurites (DNs) in neurons and glia that are immunoreactive to TDP-43, as well as ubiquitin and p62, but negative to other neurodegenerative disease-associated proteins. Differences in the morphology and tissue distribution of these inclusions are associated with specific mutations and / or clinical manifestations. To date, four types of TDP-43 pathogenesis have been described by histological classification (Mackenzie and Neumann, J. Neurochem. (2016) 138 (Suppl. 1), 54-70). Cases of type A FTLD-TDP are characterized primarily by abundant short dystrophic neurites (DNs) and compact, oval or crescent-shaped NCIs in laminaria II of the neocortex (Figure 2f, Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70). Cases with this etiology typically present clinically as either behavioral subtype frontotemporal dementia (bvFTD) or non-fluent / agrammatic subtype of primary progressive aphasia (nfvPPA) and are associated with progranulin (GRN) mutations. Cases of type B show a moderate number of compact or granular NCIs with relatively few DNs and NIIs (neuronal intranuclear inclusions, Figure 2g, Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70) in the superficial and deep cortical layers. Most cases with co-occurring symptoms of FTD and ALS are found to have a type B FTLD-TDP etiology. Type C cases are characterized by abundant, long, and winding neurites, primarily in the thin lamina superficialis, with or without minimal NCI (Figure 2j in Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70). This etiology is particularly found in cases presenting with a semantic subtype of primary progressive aphasia (svPPA).Type D FTLD-TDP presents with abundant lenticular neuronal intranuclear inclusions (NIIs) and short DNs in the neocortex, accompanied by only slight NCIs (Figure 2k in Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70). Type E is characterized by curvilinear oligodendrocyte inclusions in the white matter, as well as granular filamentous neuronal inclusions (GFNIs) affecting all neocortical layers, and extremely fine, dot-like neural network aggregates (Edward B. Lee et al., Acta Neuropathol. 2017 July; 134(1): 65-78). This pattern of etiology is found only in cases of VCP associated with inclusion body myositis.

[0008] III. TDP-43 in FTD Frontotemporal dementia (FTD) is a clinical term encompassing a broad spectrum of disorders based on degeneration of the frontal and temporal lobes, with the pathological features named frontotemporal lobar degeneration (FTLD). FTD is the second leading cause of early degenerative dementia in the under-65 age group (Le Ber, Revue Neurologique 169 (2013) 811-819). FTD is represented by several syndromes, including bvFTD, characterized by changes in personality and behavior; semantic dementia (SD) and progressive non-fluent aphasia (PNFA), characterized by changes in language function; corticobasal syndrome (CBS); and progressive supranuclear palsy syndrome and motor neuron disease (FTD-MND), characterized by motor dysfunction. The clinical diagnosis of these syndromes is complex, and final conclusions can only be achieved through postmortem histopathological analysis, which detects aggregated proteins and delineates the affected brain regions. Regarding pathological protein inclusions, approximately 45% of cases showed pathological accumulation of misfolded tau, 45% of cases had pathological TDP-43, and a smaller subgroup had FUS and other protein aggregates.

[0009] IV. TDP-43 in ALS Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the premature loss of upper and lower motor neurons. The progression of ALS is characterized by fatal paralysis and respiratory failure with a disease course of 1 to 5 years from diagnosis to death. In most cases of sporadic ALS, the neuropathology is characterized by abnormal cytoplasmic accumulation of TDP-43 in neurons and glia of the primary motor cortex, brainstem motor nuclei, spinal cord, and associated white matter. In ALS with dementia, TDP-43 accumulation in the extramotor neocortex and hippocampus is involved. The role of TDP-43 phosphorylation in ALS patients has been investigated with the help of antibodies that specifically bind to phosphorylated TDP-43 in nuclear and cytoplasmic inclusions at amino acids S379, S403, S404, S409, and S410 as the main sites of TDP-43 phosphorylation (Hasegawa et al., Ann Neurol 2008; 64: 60-70; Neumann et al., Acta Neuropathol (2009) 117: 137-149).

[0010] V. TDP-43 in AD and other diseases The pathogenesis of TDP-43 occurs in up to 57% of the brains of Alzheimer's disease patients (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450; McAleese et al., Brain Pathol. 2017 Jul; 27(4): 472-479). TDP-43 aggregation is age-related in patients and correlates with cognitive decline, memory loss, and medial temporal lobe atrophy in AD. In AD, TDP-43 appears to represent a secondary or independent pathogenesis that shares a brain distribution overlapping with amyloid-beta and tau lesions in the medial temporal lobe. Pathogenic TDP-43 follows a typical pattern of progressive deposition described by the so-called "TDP-43 in AD" (TAD) staging scheme, where TDP-43 is first deposited in the amygdala (Stage I), followed by the hippocampus, limbic system, temporal region, and finally the striatum frontofrontal cortex (Stage V) (Josephs KA et al., Acta Neuropathol. 2014;127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450).

[0011] VI. Spread of TDP-43 While the onset and initial symptoms of ALS and FTD vary significantly among patients, a common characteristic of disease progression is the diffusion of lesions from the initial lesion area to anatomically connected brain regions. The continued worsening of symptoms can be explained by the continuous diffusion of TDP-43 lesions. TDP-43 lesions in the brains of ALS patients appear to spread through a four-step process, and the propagation is thought to occur transsynaptically via cortical descending axonal projections mediated by anterograde intraaxonal transport (Brettschneider et al., Ann Neurol. 2013 July; 74(1): 20-38). Recent experimental evidence supports the hypothesis of protein propagation of amyloid beta, tau, alpha-synuclein, and TDP-43 in neuronal tissue via a prion-like mechanism (Hasegawa et al., 2017), with clearly distinguishable origins and geomorphic diffusion patterns for these four proteins (Brettschneider J et al., Nature Rev. Neuroscience, 2015, 109). A common mechanism unified across diseases is thought to be based on intercellular diffusion of pathogenic protein aggregates. This mechanism consists of the release of aggregates from diseased cells, uptake by naive cells, and seeding of conformations of pathogenic proteins through templated conformational changes of endogenous proteins. Pathogenic TDP-43, which can induce aggregation of physiological (i.e., non-pathogenic) TDP-43, is defined as TDP-43 with seeding ability. Indeed, TDP-43 has been found to misfold, aggregate, and become a seed, a transmissible agent capable of inducing de novo misfolding. This "prion-like" paradigm is thought to be one of the key factors in disease progression.

[0012] Intercellular diffusion of TDP-43 has been studied at the molecular level in one of the few in vitro models in which preparations of insoluble TDP-43 derived from patient brains can induce intracellular aggregation in receptor cells (Nonaka et al., Cell Reports 4 (2013), 124-134; Feiler et al., 2015; Porta et al., Nat. Comm., 2018). Furthermore, it has been observed that intracellular TDP-43 aggregates are released in cooperation with exosomes prior to diffusion to subsequent cells (Nonaka et al., Cell Reports 4 (2013, 124-134)). Similarly, expression of adenovirus-transduced TDP-43 leads to cytoplasmic aggregation, which is then phosphorylated and ubiquitinated, and more importantly, acts as a seed to initiate intercellular diffusion (Ishii et al., PLoS ONE 12(6): e0179375, 2017). Patient-derived pathogenic TDP-43 can lead to widespread deposition of endogenous TDP-43 following inoculation into the brains of transgenic and wild-type mice (Porta et al., Nat. Comm., 2018). Recently, the presence of certain TDP-43 seeding capabilities in patients with CSF or ALS has been confirmed using a TDP-43 seed amplification assay (Audrain et al., 2023). TDP-43 mAbs targeting the C-terminal domain were able to neutralize these seeding species (Audrain et al., 2023).

[0013] VII. Prevention and Management of TDP-43 Proteinosis TDP-43 aggregation and pathogenic spread are key features of ALS and FTD, which are currently incurable and fatal diseases. Therefore, there is a need for new methods for the treatment and prevention of TDP-43 proteinopathy. Mutations in TDP-43 are associated with familial cases of ALS and FTD, providing a causal link between TDP-43 misfolding and disease progression.

[0014] VIII. Diagnosis of TDP-43 Proteinosis Because clinical signs can overlap with those of other diseases, especially in the early stages, diagnosing FTD based on clinical signs is insufficient.

[0015] Several approaches aim to develop biochemical biomarkers to identify various types of FTD pathogenesis. Developing antibodies against various conformations of TDP-43 could enable the creation of more sensitive and specific diagnostic tools. In parallel with biochemical biomarkers, the development of imaging biomarkers could enable early and specific detection of pathogenesis in TDP-43 proteinopathy. The ability to image TDP-43 deposition in the brain could be a substantial achievement for the development of diagnostics and drugs for TDP-43 proteinopathy. Such detection could be made possible by using antibody fragments that can penetrate cells.

[0016] The earliest event in neurodegenerative diseases based on misfolding of various proteins is the acquisition of an alternative conformation that makes the protein toxic. Furthermore, this misfolded conformation can self-replicate by recruiting endogenous normal proteins to the misfolded conformation as the mechanical basis for the observed diffusion through the affected tissue.

[0017] To develop antibodies against various conformational states of a given protein, we designed supramolecular antigenic constructs (WO2012 / 055933 and WO2012 / 020124) that control the conformation of a presented antigen to produce conformation-specific antibodies against a given target in a specific conformational state. Conformation-specific antibodies offer many advantages because they can distinguish between disease-related conformations and functional endogenous conformations of these proteins. Such antibodies target misfolded isoforms of disease-related proteins while being less likely to adsorb to proteins in normal conformations, thus offering many advantages in therapeutic applications. Similarly, for diagnostic applications, such antibodies recognize only the structural state of disease-related proteins, which is crucial for the development of sensitive and specific diagnoses.

[0018] The use of TDP-43-based biomarkers in TDP-43 protein disorders remains in a state of needing establishment. Such evaluations have been hindered, in part, by the lack of high-affinity antibodies that can be used in suitable immunoassays for the quantification of pathogenic TDP-43 in bodily fluids (Feneberg et al., Molecular Neurobiology, 2018).

[0019] Therefore, there is a clear need for biomarkers that can detect misfolded aggregated TDP-43 and non-aggregated physiological TDP-43, particularly in human samples, in order to diagnose various types of TDP-43 proteinosis and / or monitor the effectiveness of therapeutic agents used for treating diseases, disorders, and abnormalities related to TDP-43, especially TDP-43 aggregates, or TDP-43 proteinosis.

[0020] TDP-43 proteinosis is defined as a set of neurodegenerative disorders characterized by pathogenic TDP-43.

[0021] IX. Prior art Patent Application WO2008 / 151055 discloses methods and materials for determining whether a mammal has a neurodegenerative disease by using the levels of TDP-43 polypeptide and / or TDP-43 polypeptide cleavage products (such as 25kD and 35kD TDP-43 polypeptide cleavage products) in biological fluids.

[0022] Patent Application WO2013 / 061163 discloses TDP-43 specific binding molecules comprising polypeptides such as human antibodies and their fragments, derivatives, and mutants.

[0023] Patent Application WO2020 / 234473 discloses specific binding molecules comprising polypeptides such as mouse antibodies or their antigen-binding fragments.

[0024] Patent Application WO2022 / 034228 discloses TDP-43 specific binding molecules comprising polypeptides such as humanized antibodies or their antigen-binding fragments.

[0025] In view of the above, there is a need for anti-TDP-43 binding molecules that bind to misfolded aggregated TDP-43 and non-aggregated physiological TDP-43, particularly human TDP-43 (SEQ ID NO: 1). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0026] Currently, there are no approved therapies on the market to treat and / or prevent TDP-43-related diseases. Therefore, there is an urgent need to identify novel therapies that can treat and / or prevent these diseases. Accordingly, the present invention relates to binding molecules, particularly antibodies and their antigen-binding fragments, that specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43. In the present invention, misfolded TDP-43 includes misfolded monomeric, and / or misfolded oligomeric, and / or misfolded aggregates, and / or post-translationally modified, and / or misfolded cleaved TDP-43. Post-translationally modified TDP-43 includes phosphorylated, ubiquitinated, acetylated, smoylated, and / or methylated TDP-43. Physiological TDP-43 includes soluble nuclear TDP-43. It is demonstrated herein that the binding molecules of the present invention can bind to pathogenic TDP-43, including TDP-43 aggregates and phosphorylated TDP-43. That is, the present invention provides binding molecules, in particular antibodies or antigen-binding fragments thereof, that specifically recognize misfolded aggregated TDP-43 and non-aggregated physiological TDP-43. Such binding molecules are referred herein to as “pan-TDP-43” binding molecules, in particular pan-TDP-43 antibodies. As described herein, the TDP-43 binding molecules of the present invention may bind equally to misfolded aggregated TDP-43 and non-aggregated physiological TDP-43, or may specifically bind to both categories of TDP-43, but preferentially to one over the other. The present invention also provides binding molecules, in particular antibodies or antigen-binding fragments thereof, for the prevention, improvement, treatment, and / or diagnosis of diseases, disorders, and abnormalities, or TDP-43 proteinosis, related to TDP-43, in particular to TDP-43 aggregates. The present invention also provides binding molecules, particularly antibodies or their antigen-binding fragments, for detecting and / or understanding (i.e., identifying) specific types of pathogens that induce neurodegeneration.It is intended to enable more efficient and accurate selection of subjects for long-term monitoring in clinical research and to support its use as a diagnostic biomarker to support the development of novel therapies for TDP-43 proteinosis. [Means for solving the problem]

[0027] The present invention also provides TDP-43 conjugating molecules, particularly antibodies or their antigen-binding fragments, as pharmaceuticals (therapeutic agents).

[0028] While we do not wish to be bound by any particular theory, the present invention was developed on the premise that modified conformation-specific antigenic peptides and peptide fragments or the whole TDP-43 protein derived from the TDP-43 protein, as well as antibodies obtained by using the peptide or fragment or the whole TDP-43 protein as an antigen, block the intercellular propagation of TDP-43, and / or deaggregate TDP-43 aggregates, and / or block the seeding of TDP-43, and / or neutralize seeding-capable TDP-43, and / or inhibit the aggregation of TDP-43 protein or its fragments, and / or promote the clearance of TDP-43. The binding molecules of the present invention, in particular polypeptides, more specifically antibodies or their antigen-binding fragments, bind to misfolded aggregated TDP-43, in particular cytoplasmic and extracellular misfolded TDP-43. The humanized binding molecule of the present invention, particularly a polypeptide, more specifically an antibody or its antigen-binding fragment, binds to full-length TDP-43 and / or cleaved TDP-43. In one embodiment, the binding molecule of the present invention, particularly a polypeptide, more specifically an antibody or its antigen-binding fragment, specifically binds to misfolded TDP-43 in the cytoplasm. In one embodiment, the TDP-43 binding molecule of the present invention, particularly an antibody or its antigen-binding fragment, binds to and neutralizes seeding-capable TDP-43. In one embodiment, the TDP-43 binding molecule of the present invention, particularly an antibody or its antigen-binding fragment, binds to extracellular and / or aggregated TDP-43, promoting TDP-43 clearance by immune cells such as microglia via antibody-dependent cellular phagocytosis (ADCP).

[0029] Misfolded aggregated TDP-43, or pathogen-associated TDP-43, consists of TDP-43 proteins that have lost their normal folding and localization (i.e., are misfolded). Misfolded aggregated TDP-43 can be found in pre-inclusions, as well as in cytoplasmic inclusions (NCIs and GCIs, respectively) of immunoreactive neurons and glia, intranuclear inclusions (NIIs), and dystrophic neurites (DNs) of TDP-43.

[0030] Non-aggregated physiological TDP-43 is a physiologically functional TDP-43 protein that is primarily located in the nucleus and circulates back and forth within the cytoplasm, and is in a state where it can exhibit its desired function in the in vivo cellular environment.

[0031] The TDP-43 binding molecule of the present invention, particularly the anti-TDP-43 antibody or its antigen-binding fragment, surprisingly possesses at least one, preferably two, more preferably three, even more preferably four, even more preferably five, even more preferably six, even more preferably seven, and most preferably all eight of the following properties: - Blocking intercellular transmission of TDP-43; - Deaggregating TDP-43 aggregates; - Inhibiting the aggregation of TDP-43 protein or its fragments; - Block the sowing of TDP-43; - To neutralize TDP-43, which has the ability to sow seeds; - To block the spread of TDP-43; - To facilitate clearance of TDP-43; and - To reduce the level of phosphorylated TDP-43 in vivo.

[0032] Regardless of the combination of one, two, three, four, five, six, seven, or eight properties listed above, the anti-TDP-43 binding molecule of the present invention, preferably an anti-TDP-43 antibody or its antigen-binding fragment, can reduce the level of phosphorylated TDP-43 in the brain and / or improve / inhibit / reduce the development of TDP-43 lesions in an in vivo model of TDP-43 proteinopathy, more importantly in patients with TDP-43 lesions.

[0033] The anti-TDP-43 binding molecule binds to an epitope in amino acids 304-414 of human TDP-43 (SEQ ID NO: 1). The anti-TDP-43 binding molecule binds to an epitope that is essentially composed of, or consists of, amino acid residues 304-313, 356-361, or 397-407 of human TDP-43 (SEQ ID NO: 1). Alternatively, the anti-TDP-43 binding molecule binds to an epitope in amino acid residues 396-414 of human TDP-43 (SEQ ID NO: 1).

[0034] The present invention provides a TDP-43 binding molecule, particularly a TDP-43 antibody or its antigen-binding fragment, comprising the following: a. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57; or b. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47; or c. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37; or d. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17; or e. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17.

[0035] The present invention relates, in particular, to (i) an immunoconjugate containing a TDP-43 conjugate molecule, (ii) a labeled antibody containing a TDP-43 conjugate molecule, (iii) a pharmaceutical composition comprising a TDP-43 conjugate molecule and a pharmaceutically acceptable carrier and / or excipient and / or diluent, (iv) a TDP-43 conjugate molecule for human or veterinary use, (v) a TDP-43 conjugate molecule for use in the prevention, improvement, or treatment of TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinopathy, (vi) a TDP-43 conjugate molecule for diagnostic use (especially for in vivo diagnosis, but also for in vitro testing), (vii) a TDP-43 conjugate molecule for research use, especially as an analytical tool or reference molecule, (viii) a TDP-43 conjugate molecule for use as a diagnostic tool for monitoring TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinopathy, and (ix) treatment of an individual using a TDP-43 conjugate molecule The present invention further aims to provide a method for maintaining or increasing cognitive memory ability or delaying memory loss in individuals having TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinopathy; (x) a method for reducing the levels of aggregated TDP-43 and / or phosphorylated TDP-43 in an individual by treating the individual with a TDP-43 binding molecule; (xi) a nucleic acid molecule encoding a TDP-43 binding molecule; (xii) a recombinant expression vector containing the nucleic acid molecule of the present invention; (xiii) a host cell containing the nucleic acid and / or vector of the present invention; (xiv) a cell-free expression system containing the recombinant expression vector of the present 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 the TDP-43 binding molecule of the present invention and / or a nucleic acid, expression vector, host cell, and / or cell-free expression system for producing it.

[0036] The TDP-43 binding molecule of the present invention, particularly an anti-TDP-43 antibody or its antigen-binding fragment, can recruit and / or activate microglia. More specifically, the TDP-43 binding molecule of the present invention can influence the morphology of microglia with respect to cell size and activation state. This may contribute to the reduction of TDP-43 pathogenicity demonstrated by the TDP-43 binding molecule of the present invention.

[0037] In the present invention, binding molecules, particularly antibodies or their antigen-binding fragments, specifically recognize TDP-43. The binding molecules of the present invention include polypeptides and / or antibodies and / or their antigen-binding fragments that are specific to the TDP-43 protein. “Specifically recognizes TDP-43” means that the binding molecules of the present invention bind specifically, generally, and collectively to TDP-43, particularly to several epitopes within TDP-43, especially to exposed / accessible epitopes in one or more pathogenic conformations of the TDP-43 protein, with greater affinity to other epitopes. The binding molecules of the present invention that specifically bind to TDP-43, particularly polypeptides, more specifically antibodies or their antigen-binding fragments, specifically recognize misfolded agglutinated TDP-43 and non-agglutinated physiological TDP-43.

[0038] The TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, bind to both non-aggregated physiological TDP-43 and agglutinated TDP-43. That is, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, can bind to soluble and agglutinated TDP-43 with almost equal goodness. The TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, can bind to agglutinated TDP-43 with almost equal goodness compared to non-aggregated TDP-43. More specifically, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, can bind to agglutinated TDP-43 in the cytoplasm with almost equal goodness compared to non-aggregated TDP-43 in the nucleus. In other embodiments, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, can bind to both species, although they may preferentially bind to agglutinated TDP-43 compared to non-aggregated TDP-43. More specifically, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, may preferentially bind to aggregated TDP-43 in the cytoplasm compared to unaggregated TDP-43 in the nucleus, but may bind to both species. Alternatively, in other embodiments, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, may preferentially bind to unaggregated TDP-43 compared to aggregated TDP-43, but may bind to both species. More specifically, the TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, may preferentially bind to unaggregated TDP-43 in the nucleus compared to aggregated TDP-43 in the cytoplasm, but may bind to both species. These binding properties can be demonstrated, for example, using immunohistochemistry.

[0039] In some embodiments, the present invention encompasses the use of binding molecules, particularly the antibodies of the present invention described herein that specifically bind to TDP-43, and their antigen-binding fragments, as well as the use of these binding molecules for diagnosing, preventing, improving, and / or treating diseases, disorders, and / or abnormalities related to TDP-43, particularly those related to TDP-43 aggregates, or TDP-43 proteinopathy, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-dominant age-related TDP-43 encephalopathy (LATE). The methods and compositions disclosed herein have applications in the diagnosis, prevention, improvement, and / or treatment of diseases, disorders, and / or abnormalities related to TDP-43, particularly those related to TDP-43 aggregates, or TDP-43 proteinopathy, including but not limited to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Preferably, the use of these conjugating molecules for diagnosing, preventing, improving, and / or treating diseases, disorders, and / or abnormalities related to TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy is directed towards amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), or frontotemporal dementia (FTD). More preferably, the use is directed towards amyotrophic lateral sclerosis (ALS). More preferably, the use is directed towards Alzheimer's disease (AD). More preferably, the use is directed towards frontotemporal dementia (FTD).

[0040] In another embodiment, a TDP-43 binding molecule, particularly a TDP-43-specific anti-TDP-43 antibody of the present invention as described herein, or its antigen-binding fragment, is used to treat frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), corticobasal ganglia mutations, etc.) associated with chromosome 9p-linked progranulin (GRN) mutations, C9orf72 mutations, TARDBP mutations, balocin-containing protein (VCP) mutations, and motor neuron disease (MND). Sexual disorders, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-dominant age-related TDP-43 encephalopathy (L ATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerotic dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with balocin-containing protein (VCP) mutations; associated with Paget's disease of bone and frontotemporal dementia), marginal vesicles Contact with a sample to detect, diagnose, and / or monitor diseases, disorders, and / or abnormalities related to TDP-43, particularly those related to TDP-43 aggregates, or TDP-43 proteinosis, selected from oculopharyngeal muscular dystrophy with myotirin (MYOT) gene mutations or myofibrillar myopathy with mutations in genes encoding desmin (DES), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0041] In one embodiment, the present invention encompasses the use of binding molecules, particularly the antibodies or antigen-binding fragments of the present invention described herein that specifically bind to TDP-43, and these binding molecules, in particular these antibodies, for detecting the presence of TDP-43 in a sample. Thus, the TDP-43 binding molecules of the present invention, such as the anti-TDP43 antibodies described herein, can be used, for example, by using an ELISA assay or a surface adaptation assay, to screen for the presence of TDP-43 in a sample, particularly clinical samples, especially human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF), and / or urine. In some situations, tissue samples, such as brain tissue samples, may be used. The methods and compositions of the present invention also have applications in the diagnosis of pre-symptomatic diseases, and / or in monitoring disease progression and / or therapeutic effectiveness.According to some embodiments, an antibody specific to TDP-43 (e.g., a full-length antibody or a fragment or derivative of an antibody that binds to TDP-43) is used to treat frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), with or without mutations in progranulin (GRN), C9orf72, TARDBP, balocin-containing protein (VCP), linked to chromosome 9p, cerebral sclera Basal ganglia degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander disease (AxD), limbic system dominant Age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in balocin-containing protein (VCP); bone pallidum The TDP-43 binding molecule of the present invention can be used to detect, diagnose, and / or monitor samples (e.g., blood, urine, cerebrospinal fluid (CSF), interstitial fluid (ISF), or brain tissue) of suitable samples, particularly clinical samples such as blood, brain tissue, CSF, ISF, or urine with relatively high TDP-43 levels, compared to suitable controls, to indicate disease and / or more advanced disease. Many suitable immunoassay formats are known.That is, methods (e.g., ELISA, MSD (mesoscale discovery), HTRF (Homogeneous Time Resolved Fluorescence), SIMOA® (single-molecule array), and AlphaLISA®) may be performed for the purpose of diagnosing diseases exhibiting high levels of TDP-43. Alternatively, methods may be performed for monitoring purposes. Levels that increase over time may indicate disease progression. Levels that decrease over time may indicate disease regression. Methods may also be performed to monitor treatment, particularly the effectiveness of specific treatments. Treatment success can be measured in relation to the stabilization or decrease in TDP-43 levels following treatment. Example 12 of WO2020 / 234473 demonstrated that TDP-43 levels are higher in CSF samples from patients with TDP-43 proteinopathy than in control samples taken from healthy subjects (healthy controls). Control samples may be processed in parallel with or independently of test samples. In some embodiments, the control level is determined from a series of control samples taken from healthy subjects in a similar manner or under the same experimental conditions and used as a comparative reference to the level determined in the test sample. The method for quantifying TDP-43 in a suitable sample using the binding molecule of the present invention may also be used to select a therapy (for further treatment of the subject). That is, an individualized treatment method is envisioned. Samples are taken before and after the treatment. If the treatment using the therapy results in a stable, or preferably reduced, level of TDP-43 after the treatment, the therapy may be selected for the subject. If the therapy does not result in a stable, or preferably reduced, level of TDP-43 after the treatment, the therapy is not selected for the subject. The therapy may be any suitable candidate therapeutic agent for the treatment of TDP-43 proteinopathy. In a preferred embodiment, the therapy comprises the TDP-43 binding molecule of the present invention, typically in the form of a pharmaceutical composition as described herein.

[0042] The TDP-43 binding molecule of the present invention can also be used for classifying diseases into specific types or subtypes. That is, a. To carry out the method of the present invention in which the level of TDP-43 is quantified in comparison with a suitable control. b. Identify mutations in the sample derived from the target, including, but not limited to, mutations in progranulin (GRN), C9orf72, TARDBP, angiogenin (ANG), balocin-containing protein (VCP), myotilin (MYOT) gene, and the gene encoding desmin (DES), as appropriate. c. Classifying diseases, disorders, and / or abnormalities, or TDP-43 protein disorders, that are related to TDP-43, particularly TDP-43 aggregates. Methods are provided for classifying diseases, disorders, and / or abnormalities related to TDP-43, particularly those related to TDP-43 aggregates, or for classifying TDP-43 protein disorders.

[0043] Similarly, the present invention provides a method for classifying TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinosis, comprising: performing the method for quantifying the level of TDP-43 in a sample obtained from a subject having a TDP-43-related disease, disorder, and / or abnormality, or TDP-43 proteinosis, wherein the level is compared with a control sample taken from a subject having a different type or subtype of TDP-43-related disease, disorder, and / or abnormality, or TDP-43 proteinosis (i.e., a representative set of control levels is determined for the type or subtype of interest); and classifying the TDP-43-related disease, disorder, and / or abnormality, or TDP-43 proteinosis, based on the comparison. That is, the classification is based on determining the closest match between the test sample and one or more control samples. These methods may further include identifying mutations in a sample, including but not limited to mutations in progranulin (GRN), C9orf72, TARDBP, angiogenin (ANG), balocin-containing protein (VCP), myotirin (MYOT) gene, and desmin (DES) gene. The identified mutations may also be used to classify diseases, disorders, and / or abnormalities, or TDP-43 proteinopathy, related to TDP-43, particularly related to TDP-43 aggregates. To avoid doubt, the identification of mutations in a sample may be carried out by any preferred method, for example, based on nucleic acid sequencing of nucleic acid molecules in the sample. The sample may be separated and distinguished from the sample in which the TDP-43 level was determined, but it is from the same subject.

[0044] In other embodiments, the present invention provides methods for preventing, improving, and / or treating diseases, disorders, and / or abnormalities related to TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy. According to one embodiment, the method of the present invention involves administering an effective concentration of a conjugating molecule, particularly the TDP-43-specific antibody of the present invention described herein (e.g., a full-length antibody or a fragment or derivative of an antibody that binds to TDP-43), to a target. In another embodiment, the present invention provides methods for preventing, improving, and / or treating TDP-43 proteinopathy. According to some embodiments, a conjugating molecule, particularly the TDP-43-specific antibody of the present invention described herein or its antigen-binding fragment, is administered to treat, improve, and / or prevent frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). In another embodiment, binding molecules, particularly TDP-43-specific antibodies of the present invention as described herein, or antigen-binding fragments thereof, are administered to prevent, improve, and / or treat neurodegenerative diseases 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 encephalopathy (CTE), and limbic-dominant age-related TDP-43 encephalopathy (LATE).

[0045] In another embodiment, the binding molecule, in particular TDP-43-specific antibodies of the present invention described herein or their antigen-binding fragments, are used to treat frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), with mutations in progranulin (GRN) linked to chromosome 9p, mutations in C9orf72, mutations in TARDBP, mutations in balocin-containing protein (VCP)), and large Corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander Diseases (AxD), limbic-dominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, balocin-containing protein (VCP) It is administered to prevent, improve, and / or treat selected diseases from among inclusion body myopathy (associated with Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy (associated with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES)), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD). [Modes for carrying out the invention]

[0046] X.Definition As used herein, an "antigen-binding molecule" is any molecule capable of specifically or selectively binding to an antigen, particularly TDP-43. The binding molecule may include or be an antibody or a fragment thereof. An anti-TDP-43 binding molecule is a molecule that binds to the TDP-43 protein at a specific recognition site, i.e., an epitope, such as an anti-TDP-43 antibody or a fragment thereof. That is, the antigen-binding molecule of the present invention binds to an epitope in the amino acid sequence of SEQ ID NO: 1. The antigen-binding molecules provided herein, particularly antibodies or their antigen-binding fragments, recognize full-length TDP-43. Other anti-TDP-43 binding molecules may also include polyvalent molecules, polyspecific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly produced molecules. Antigen-binding molecules useful examples in the present invention include antibody-like molecules. Antibody-like molecules are molecules that can exert their function by binding to target molecules (see, for example, Current Opinion in Biotechnology 2006, 17:653-658; Current Opinion in Biotechnology 2007, 18:1-10; Current Opinion in Structural Biology 1997, 7:463-469; Protein Science 2006, 15:14-27), including, for example, DARPins (WO2002 / 020565), Affibody (WO1995 / 001937), Avimer (WO2004 / 044011; WO2005 / 040229), Adnectin (WO2002 / 032925), and fynomers (WO2013 / 135588).

[0047] As used herein, the terms “anti-TDP-43 antibody” and “antibody that binds to TDP-43,” or simply “antibody,” mean an antibody that can bind to TDP-43 with sufficient affinity to be considered subject to further evaluation as a promising diagnostic and / or therapeutic agent targeting TDP-43. Generally, the term “antibody” is used herein in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or biparatope antibodies), fully human antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity. The antibodies of the present invention may be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies, or antibodies presented on the surface of phages or on the surface of chimeric antigen receptor (CAR) T cells.

[0048] An antibody's "antigen-binding fragment" or "functional fragment" refers to a molecule other than the intact or full-length antibody that contains a portion of the intact or full-length antibody and binds (completely or partially) to the antigen to which the intact or full-length antibody binds. Examples of antigen 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 polyspecific antibodies formed from antibody fragments. Antigen-binding fragments are also called "functional fragments" because they retain the binding function of the original antibody from which they were induced.

[0049] An "epitope-binding antibody" within a defined region of a protein is an antibody that requires the presence of one or more amino acids within that region to bind to that protein.

[0050] In certain embodiments, “epitope-binding antibodies” within a defined region of a protein are identified by mutation analysis, which determines that the amino acids in the protein are mutated, and the binding of the antibody to the resulting modified protein (e.g., the modified protein containing the epitope) is at least 20% of the binding to the unmodified protein. In some embodiments, “epitope-binding antibodies” within a defined region of a protein are identified by mutation analysis, which determines that the amino acids in the protein are mutated, and the binding of the antibody to the resulting modified protein (e.g., the modified protein containing the epitope) is 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 unmodified protein. In certain embodiments, antibody binding is determined by FACS, WB, or a preferred binding assay, such as ELISA.

[0051] As used in the context of this invention, the term "to bind to" defines the binding (interaction) of at least two "antigen interaction sites." The term "antigen interaction site," as used in this invention, defines a motif of a polypeptide, i.e., a part of the antibody or antigen-binding fragment of this invention that exhibits the ability to have a specific interaction with a particular antigen of TDP-43 or a particular group of antigens. The binding / interaction is also understood to define "specific recognition." The term "specifically recognizes," as used in this invention, means that the antibody can specifically interact / bind to at least two amino acids of TDP-43 as defined herein, in particular to at least two amino acids within amino acid residues 304-414 of human TDP-43 (SEQ ID NO: 1), and more specifically to at least two amino acids within amino acid residues 304-313, 356-361, 397-407, or 396-414 of human TDP-43 (SEQ ID NO: 1).

[0052] The term "pan-TDP-43 antibody" refers to an antibody that binds to misfolded agglutinated TDP-43 and non-agglutinated physiological TDP-43, including monomeric TDP-43, oligomeric TDP-43, post-translational modified TDP-43 (e.g., phosphorylated, ubiquitinated, acetylated, smoylated, and / or methylated), agglutinated TDP-43, and cleaved TDP-43.

[0053] The term "specific interaction" as used in accordance with the present invention means that the antibody or its antigen-binding fragment of the present invention does not cross-react with (poly)peptides of similar structure, or does not essentially cross-react with them. Therefore, the antibody or its antigen-binding fragment of the present invention specifically binds to / interacts with the structure of TDP-43 formed by specific amino acid sequences within amino acid residues 304-414 of human TDP-43 (SEQ ID NO: 1), and more specifically, with the structure of TDP-43 formed by specific amino acid sequences within amino acid residues 304-313, 356-361, 397-407, or 396-414 of human TDP-43 (SEQ ID NO: 1).

[0054] The cross-reactivity of antigen-binding molecules, particularly a panel of antibodies or their antigen-binding fragments under consideration, can be tested, for example, by evaluating the binding of the panel of antibodies or their antigen-binding fragments to the (poly)peptide of interest and several (poly)peptides that are somewhat (structurally and / or functionally) closely related, under conventional conditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). Only constructs (i.e., antibodies, their antigen-binding fragments, etc.) that bind to a specific epitope or (poly)peptide / protein of TDP-43 as defined herein, but do not bind to or are essentially not bound to any other epitopes or (poly)peptides of the same TDP-43, are considered specific to the epitope or (poly)peptide / protein of interest and are selected for further study using the methods provided herein. These methods may, among other things, include binding, blocking, and competition studies with structurally and / or functionally closely related molecules. These binding studies may also include FACS analysis, surface plasmon resonance (SPR, e.g., by BIACORE®), analytical centrifugation, isothermal titration calorimetering, fluorescence anisotropy, fluorescence spectroscopy, or radiolabeled ligand binding assays.

[0055] Therefore, specificity can be experimentally determined by methods known in the art and methods described herein. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, and peptide scanning.

[0056] As used herein, the term “monoclonal antibody” means an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies constituting that population are identical except for any naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed toward a single antigen site. Monoclonal antibodies are advantageous in that they can be synthesized by culturing hybridomas and are essentially uncontaminated by other immunoglobulins. The modified “monoclonal” indicates the nature of the antibody, that it lies within a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. As described above, monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method described in Kohler, Nature 256 (1975), 495.

[0057] As used herein, the term "polyclonal antibody" refers to an antibody produced in the presence of or in the presence of one or more other non-identical antibodies. Generally, polyclonal antibodies are produced from B lymphocytes in the presence of several other B lymphocytes that produce non-identical antibodies. Polyclonal antibodies are usually obtained directly from immunized animals.

[0058] As used herein, the term “fully human antibody” means an antibody containing only human immunoglobulin protein sequences. Fully human antibodies may also contain mouse carbohydrate chains if produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, “mouse antibody” or “murin antibody” means an antibody containing only mouse / murin immunoglobulin protein sequences. Alternatively, “fully human antibody” may also contain rat carbohydrate chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Similarly, the term “rat antibody” means an antibody containing only rat immunoglobulin protein sequences. Fully human antibodies may also be produced, for example, by phage display, a widely used screening technique that enables the production and screening of fully human antibodies. Phage antibodies can also be used in the context of the present invention. Phage display methods are described, for example, in U.S. Patents 5,403,484, 5,969,108, and 5,885,793. Another technique that enables the development of fully human antibodies involves modifications of mouse hybridoma techniques. Mice are transgenicized to contain the human immunoglobulin locus instead of their own genes (see, for example, U.S. Patent No. 5,877,397).

[0059] The term "chimeric antibody" refers to an antibody comprising a variable region of the present invention that has been fused or chimeric with an antibody region (e.g., a constant region) derived from another human or non-human species (e.g., mouse, horse, rabbit, dog, cattle, chicken).

[0060] The term “antibody” also refers 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, such as antibodies isolated from transgenic animals (e.g., mice) 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, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if present) derived from human germline immunoglobulin sequences. However, such antibodies are subject to in vitro mutagenesis (or in vivo somatic mutagenesis if transgenic animals are used for the human Ig sequence), and therefore the amino acid sequences of the VH and VL regions of recombinant antibodies are derived from and related to the VH and VL sequences of the human germline, but are sequences that do not naturally exist in the repertoire of human antibody germline in vivo.

[0061] "Heterogeneous antibodies" are defined in relation to transgenic non-human organisms that produce such antibodies. This term refers to antibodies that have an amino acid sequence or nucleic acid sequence that corresponds to antibodies found in organisms that are not composed of transgenic non-human animals, and generally originate from species other than transgenic non-human animal species.

[0062] The term "heterohybrid antibody" refers to an antibody that possesses light and heavy chains of different organismal origins. For example, an antibody that has both a mouse light chain and a human heavy chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric antibodies and humanized antibodies.

[0063] The term "antibody" also applies to humanized antibodies. The "humanized" form of a non-human (e.g., mouse or rabbit) antibody is a chimeric immunoglobulin, i.e., an immunoglobulin chain or fragment containing the smallest sequence derived from the non-human immunoglobulin (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody). Humanized antibodies are often human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, possessing the desired specificity, affinity, and capability. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain residues not found in either the recipient antibody or the imported CDR or framework sequence. These modifications are made to further improve and optimize the performance of the antibody. Generally, humanized antibodies will contain at least one, typically two, substantially all of the variable domains, where all or substantially all of the CDR region corresponds to those regions of non-human immunoglobulins, and all or substantially all of the FR region is the consensus sequence of human immunoglobulin. Humanized antibodies may also contain at least part of the immunoglobulin constant region (Fc), typically the constant region of human immunoglobulin. For further 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.

[0064] A well-known method for humanizing antibodies involves CDR grafting, in which a functional antigen-binding site derived from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are known in the art and are described, for example, in U.S. Patents 5,225,539, 5,693,761, and 6,407,213. Another related method is the production of humanized antibodies from transgenic animals genetically engineered to contain one or more humanized immunoglobulin loci that can undergo gene rearrangement and gene conversion (see, for example, U.S. Patent 7,129,084).

[0065] Therefore, in the context of the present invention, the term “antibody” refers to a complete immunoglobulin molecule as well as a portion of such an immunoglobulin molecule (i.e., “its antigen-binding fragment”). Furthermore, the term refers to modified and / or altered antibody molecules, as discussed above. The term also refers to antibodies produced / synthesized by recombination or synthesis. The term also refers to intact antibodies as well as their antibody fragments, e.g., separated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term “antibody” also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs, e.g., single-chain Fv(scFv) or antibody fusion proteins.

[0066] In the context of this invention, a "single-chain Fv" or "scFv" antibody fragment has a VH domain and a VL domain of the antibody, and these domains exist on a single polypeptide chain. Generally, the scFv polypeptide further contains a polypeptide linker between the VH domain and the VL domain, thereby allowing the scFv to form a desirable structure for antigen binding. The methods described for the production of single-chain antibodies are, for example, described in Plueckthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, NY (1994), 269-315.

[0067] As used herein, a “Fab fragment” consists of a CH1 region and a variable region of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0068] The "Fc" region contains two heavy chain fragments, each containing the CH2 and CH3 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0069] A "Fab fragment" contains one light chain and a portion of a heavy chain that includes a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0070] The "F(ab')2 fragment" consists of two light chains and C H 1 domain and C H It contains two heavy chains, each containing a portion of the constant region between the two domains, thereby forming an interchain disulfide bond between the two heavy chains. That is, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0071] The "Fv region" includes variable regions from both the heavy and light chains, but lacks a steady region.

[0072] Antibodies, antibody constructs, antibody fragments, antibody derivatives (all Ig-derived) or their corresponding immunoglobulin chains employed in accordance with the present invention may be further modified, either alone or in combination, by conventional methods known in the art, such as amino acid deletions, insertions, substitutions, additions, and / or recombinations and / or any other modifications known in the art. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of the immunoglobulin chain are well known to those skilled in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd edition (2001). The term “Ig-derived domain” refers particularly to a (poly)peptide construct containing at least one CDR. The cited Ig-derived domain fragments or derivatives define the (poly)peptides that are parts of the antibody molecules and / or modified by chemical / biochemical or molecular biological methods. The corresponding methods are known in the art and are described in particular in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 2nd edition (1989) and 3rd 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: A Molecular Cloning Manual Cold Spring Harbor Laboratory Press (2002)).

[0073] The term "CDR" as used herein refers to a "complementarity-determining region," which is well known in the art. A CDR is the portion of an immunoglobulin that determines the specificity of the molecule and contacts a specific ligand. CDRs are the most variable parts of a molecule and contribute to the diversity of these molecules. Each V domain contains three CDR regions: CDR1, CDR2, and CDR3. CDR-H represents the CDR region of the variable heavy chain, and CDR-L relates to the CDR region of the variable light chain. VH signifies the variable heavy chain, and VL signifies the variable light chain. The CDR region of the Ig-derived region can be determined as described in Kabat, “Sequences of Proteins of Immunological Interest”, 5th edit. NIH Publication no. 91-3242 US Department of Health and Human Services (1991). The CDR sequences provided herein are defined according to Kabat. However, it will be understood by those skilled in the art that the present invention intends for the CDR sequence to encompass binding molecules defined according to any useful identification / numbering scheme.For example, to define CDR, Chothia (Canonical structures for the hypervariable regions of immunoglobulins. Chothia C, Lesk AM. J Mol Biol. 1987 Aug 20; 196(4):901-17), IMGT (IMGT, the international ImMunoGeneTics database. Giudicelli V, Chaume D, Bodmer J, Mueller W, Busin C, Marsh S, Bontrop R, Marc L, Malik A, Lefranc MP. Nucleic Acids Res. 1997 Jan 1; 25(1):206-11 and Unique database numbering system for immunogenetic analysis. Lefranc MP. Immunol Today. 1997 Nov; 18(11):509), MacCallum(MacCallum RM, Martin AC, Thornton JM, J Mol Biol. 1996 Oct 11; The numbering schemes of 262(5):732-45) and Martin (Abhinandan KR, Martin ACR. Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains. Mol Immunol. (2008) 45:3832-9. 10.1016 / j.molimm.2008.05.022) may be adopted.

[0074] Accordingly, in the context of the present invention, the antibody molecules described above herein are selected from the group consisting of complete antibodies (immunoglobulins, IgG1, IgG2, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE, etc.), 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 polyvalent single-chain antibodies.

[0075] The “humanization approach” is well known in the art and has been described in particular for antibody molecules, e.g., Ig-derived molecules. The term “humanization” means a humanized form of a non-human (e.g., mouse) antibody or fragment thereof (Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding subsequence of the antibody) that contains some portion of a sequence derived from a non-human antibody. Humanized antibodies include human immunoglobulins in which residues derived from the complementarity-determining region (CDR) of human immunoglobulin are replaced with CDR-derived residues from non-human species such as mouse, rat, or rabbit, which have the desired binding specificity, affinity, and ability. Generally, humanized antibodies will substantially contain all of at least one, generally two, variable domains, where all or substantially all of the CDR region corresponds to the CDR region of the non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin consensus sequence. Humanized antibodies will optimally contain at least a portion of the immunoglobulin constant region (Fc), typically a portion of human immunoglobulin. See, in particular, Jones et al., Nature 321 (1986), 522-525, and Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596. Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acids introduced from a non-human source that retains the binding activity of the original antibody. Methods for humanizing antibodies / antibody molecules are described in more detail in Jones et al., Nature 321 (1986), 522-525; Reichmann et al., Nature 332 (1988), 323-327; and Verhoeyen et al., Science 239 (1988), 1534-1536.Specific examples of humanized antibodies, such as antibodies targeting EpCAM, are well known in this art (see, for example, 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).

[0076] Therefore, in the context of the present invention, an antibody molecule or its antigen-binding fragment that can be humanized and successfully incorporated into a pharmaceutical composition is provided.

[0077] Those skilled in the art will understand that epitopes are contained in the TDP-43 protein, but may also be contained in its degradation products or may be chemically synthesized peptides. The amino acid positions are indicated only to show the position of the corresponding amino acid sequence in the TDP-43 protein sequence. The present invention encompasses all peptides containing epitopes. Peptides may be part of polypeptides longer than 100 amino acids, or may be small peptides shorter than 100, preferably less than 50, more preferably less than 25, and even more preferably less than 16 amino acids. The amino acids of such peptides may be natural amino acids or non-natural amino acids (e.g., beta-amino acids, gamma-amino acids, D-amino acids), or combinations thereof. Furthermore, the present invention may encompass each retroinversopeptide of an epitope. Peptides may be unbound or bound. They may be bound, for example, to small molecules (e.g., drugs or fluorescent substances), to high molecular weight polymers (e.g., polyethylene glycol (PEG), polyethyleneimine (PEI), hydroxypropyl methacrylate (HPMA), etc.), or to proteins, fatty acids, sugar moieties, or inserted into membranes.

[0078] To test whether the antibody in question and the antibody of the present invention recognize the same epitope, the following competitive study is performed: Vero cells infected with 3 MOI (multiple degrees of infection) are incubated for 20 hours with various concentrations of the antibody in question as a competitor for 1 hour. In a second incubation step, the antibody of the present invention is added at a constant concentration of 100 nM, and its binding is detected by flow cytometry using a fluorescently labeled antibody against the constant domain of the antibody of the present invention. Binding that is inversely proportional to the concentration of the antibody in question indicates that both antibodies recognize the same epitope. However, many other assays are known in the art that can be used.

[0079] The present invention also relates to the production of specific antibodies against the natural and recombinant polypeptides of TDP-43. This production is based on the immunization of animals, such as mice. However, other animals for antibody / antiserum production are also envisioned in the present invention. For example, monoclonal and polyclonal antibodies can be produced by rabbits, mice, goats, donkeys, and others. Polynucleotides encoding the correspondingly selected TDP-43 polypeptide can be subcloned into a suitable vector, and the recombinant polypeptide is expressed in an expressible organism, such as bacteria. That is, the expressed recombinant protein can be injected intraperitoneally into mice, and the resulting specific antibody can be obtained from mouse serum provided, for example, by intracardiac blood puncture. The present invention also envisions the production of specific antibodies against the natural and recombinant polypeptides by using DNA / RNA vaccine strategies illustrated in the attached examples. DNA vaccine strategies are well known in the art and include gene gun or jet injection, and liposome-mediated delivery by intramuscular or intradermal injection. That is, antibodies against a polypeptide, protein, or epitope of TDP-43, in particular the epitope of the antibody provided herein, can be obtained by directly immunizing an animal by directly injecting a vector expressing the epitope of the antibody of the present invention, which is located in amino acid residues 304-414 of SEQ ID NO: 1, more specifically in amino acid residues 304-313, 356-361, 397-407, or 396-414 of SEQ ID NO: 1, into the muscle. The amount of specific antibody obtained can be quantified using the ELISA described herein and below. Further methods for antibody production are well known in the art. See, for example, Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988.

[0080] That is, under specified assay conditions, the identified antibody and the corresponding epitope of TDP-43 bind 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 site selected for specificity to a particular target analyte. Various immunoassay formats can be used to select antibodies that react specifically with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies that react specifically with an analyte. For a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity, see Shepherd and Dean (2000), Monoclonal Antibodies: A Practical Approach, Oxford University Press and / or Howard and Bethell. Typically, specific or selective reactions will be at least twice the background signal to noise, more typically 10 to 100 times the background. Those skilled in the art are in a position to provide and generate specific binding molecules for novel polypeptides. Specific binding assays can be readily employed to avoid undesirable cross-reactivity, and polyclonal antibodies can be easily purified and selected by known methods, for example (see Shepherd and Dean, ibid.).

[0081] The "class" of an antibody refers to the type of constant domain or constant region in its heavy chain. Antibodies have five main classes: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0082] In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it is desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions, and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Assuming that the final construct has the desired properties, such as antigen-binding ability, any combination of deletions, insertions, and substitutions can be made to reach the final construct.

[0083] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial modifications are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to the classes of amino acid side chains. The amino acid substitutions are introduced into the antibody of interest, and the product is screened for desired activity, such as retention / improvement of antigen binding, decreased immunogenicity, or improvement of antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0084] [Table 1]

[0085] Amino acids can be classified according to the common characteristics of their side chains. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basicity: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe

[0086] Non-conservative substitutions require replacing one member of one of these classes with one of another.

[0087] One type of substitution mutant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting mutants selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. An exemplary substitution mutant is an affinity-mature antibody, which can be conveniently generated using phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, the mutant antibody is presented on a phage, and it is screened for specific biological activities (e.g., binding affinity).

[0088] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications are made in the CDR's "hot spots," i.e., residues encoded by codons that frequently undergo mutations during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in the SDR (a-CDR), and the resulting VH or VL variants are tested for binding affinity. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the mutable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-oriented approach, where several CDR residues (e.g., 4-6 residues simultaneously) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and especially CDR-L3 are often targeted.

[0089] In certain embodiments, substitutions, insertions, or deletions may occur in one or more CDRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made in the CDR. Such modifications may be in the "hot spots" of the CDR, i.e., outside the SDR. In certain embodiments of the VH and VL sequences of the variants provided above, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions.

[0090] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a group of residues or 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 antibody-antigen interaction is affected. Further substitutions are introduced at the amino acid positions, and the functional sensitivity to the initial substitutions is demonstrated. Alternatively, or in addition, the crystal structure of the antigen-antibody complex is used to identify contact sites between the antibody and antigen. Such contact residues and neighboring residues can be targeted or excluded as candidate substitutions. Mutants can be screened to determine whether they contain desired properties.

[0091] Amino acid sequence insertions include the fusion of amino and / or carboxyl terminals over lengths ranging from one residue to polypeptides containing 100 or more residues, as well as the insertion of single or multiple amino acid residues into a sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusion to the N-terminus or C-terminus of an antibody with an enzyme (e.g., for ADEPT), or polypeptides that extend the serum half-life of an antibody.

[0092] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree to which they are glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0093] If the antibody contains an Fc region, the carbohydrate bound to it may change. Natural antibodies produced by mammalian cells typically contain branched, bifurcated oligosaccharides bound to Asn297 of the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bifurcated oligosaccharide structure. In some embodiments, the oligosaccharide in the antibody of the present invention may be modified to produce antibody variants with improved properties.

[0094] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may 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 glycans at Asn297 relative to the total of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region (Eu numbering of Fc region residues; see Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)). However, due to minor sequence variations in the antibody, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated mutants may have improved ADCC function. See, for example, U.S. Patent Publication 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications concerning "defucosylated" or "fucose-deleted" antibody variants include US2003 / 0157108;WO2000 / 61739;WO2001 / 29246;US2003 / 0115614;US2002 / 0164328;US2004 / 0093621;US2004 / 013214 This includes 0;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); and Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Publication 2003 / 0157108Al, Presta, L; and WO2004 / 056312Al, Adams et al., particularly Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki et al., Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085l07).

[0095] Further antibody variants are provided that have fragmented oligosaccharides, for example, antibody variants in which a bifurcated oligosaccharide bound to the Fc region of the antibody is fragmented by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and U.S.2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0096] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) that includes an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0097] In certain embodiments, the antibodies provided herein bind to pathogenic TDP-43, forming an immune complex that is removed by antibody-dependent cell phagocytosis (ADCP). This consequently promotes TDP-43 clearance. ADCP is mediated by the interaction between the Fc fragment of the antibody and an Fc receptor, such as an Fc gamma receptor, expressed on the surface of embryonic immune cells, such as microglia or dendritic cells. By modifying the Fc portion of the antibody, the Fc-mediated function can be tuned to achieve the desired effect.

[0098] In certain embodiments, the present invention intends to present antibody variants with some, but not all, effector functions, making them desirable candidates for applications where the antibody's half-life in vivo is important but certain effector functions (e.g., complement activation and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that an antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes and microglia express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); and No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0099] Alternatively, non-radioactive assay methods may be employed (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.

[0100] Alternatively, the ADCC activity of the molecule of interest can be evaluated in vivo using animal models, such as those disclosed in Clynes et al., Proc. Nat'l Acad. sci. USA 95:652-656 (1998).

[0101] A C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. A CDC assay may be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). The binding of FcRn and the determination of its in vivo clearance / half-life can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0102] Antibodies with reduced effector function include antibodies with one or more substitutions of Fc region residues 234, 235, 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Certain antibody variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). Such Fc mutants include the so-called "DANA" Fc mutant (U.S. Patent No. 7,332,581) with alanine substitutions at residues 265 and 297, or the so-called "DANG" Fc mutant with alanine substitutions at residue 265 and glycine substitutions at residue 297, as well as Fc mutants with two or more substitutions at amino acid positions 265, 269, 270, 297, and 327. Alternatively, antibodies with reduced effector function include antibodies with one or more substitutions at Fc region residues 234, 235, and 329, and the so-called "PG-LALA" Fc mutant (Lo, M. et al., Journal of Biochemistry, 292, 3900-3908) with alanine substitutions at residues 234 and 235 and glycine substitution at residue 329. Other known mutations at positions 234, 235, and 321, as well as the L234F / L235E / P331S mutation in the CH2 domain, including so-called TM mutants, can be used (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies derived from human IgG4 isotypes include the S228P / L235E mutation, which stabilizes the hinge and reduces FgR binding (Schlothauer et al, PEDS, 29 (10):457-466). Constant domain numbering follows the EU numbering system.

[0103] Other Fc variants include those with substitutions of one or more Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, a substitution of Fc region residue 434 (US Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); US Patent Nos. 5,648,260; and US Patent Nos. 5,624,821.

[0104] In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0, resulting in an extended antibody half-life. Antibodies with enhanced affinity for FcRn include antibodies having one or more substitutions at Fc region residues 252, 253, 254, 256, 428, and 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)).

[0105] In certain embodiments, it is desirable to produce a cysteine-modified antibody, for example, a "thioMAb" in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is thereby located at an accessible site on the antibody, which can then be used to conjugate the antibody to other parts, such as a drug part or a linker-drug part, to produce an immunoconjugate, as further described herein. In certain embodiments, any one or more of the following residues, namely V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region, may be substituted with cysteine. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0106] In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinoid moieties known and readily available in the art. Moieties suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in production due to its water stability. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers that bind to the antibody varies, and if two or more polymers bind, they may be the same molecule or different molecules. Generally, the number and / or type of polymers used in derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapeutic purposes under defined conditions, and others.

[0107] In another embodiment, a conjugate of an antibody and a non-proteinaceous moiety that is selectively heated by exposure to radiation is 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, but is not limited to any wavelength, including wavelengths that heat the non-proteinaceous moiety to a temperature that does not harm normal cells but kills cells in close proximity to the antibody and non-proteinaceous moiety.

[0108] Antibodies may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody against misfolded TDP-43 as described herein is provided. Such nucleic acid may encode an amino acid sequence containing VL and / or VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In further embodiments, one or more vectors containing such nucleic acid (e.g., recombinant expression vectors) are provided. In further embodiments, host cells containing such nucleic acid are provided. In one such embodiment, the host cell comprises (1) a vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and an amino acid sequence containing VH of the antibody, or (2) a first vector containing nucleic acid encoding an amino acid sequence containing VL of the antibody and a second vector containing nucleic acid encoding an amino acid sequence containing VH of the antibody (e.g., transformed by these). In one embodiment, the host cell is a eukaryote, e.g., a cell from a Chinese hamster ovary (CHO) or a lymphocyte-like cell (e.g., YO, NSO, Sp20). In one embodiment, a method is provided for producing an antibody against misfolded TDP-43, which comprises culturing host cells containing the nucleic acid encoding the antibody as provided above under conditions suitable for antibody expression, and may also comprise recovering the antibody from the host cells (or the culture medium of the host cells).

[0109] For recombinant production of antibodies against misfolded TDP-43, the nucleic acid encoding the antibody is isolated, for example, as described above, and inserted into one or more vectors for further cloning and / or expression in host cells or cell-free expression systems. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody).

[0110] In one embodiment, a method is provided for producing a TDP-43 binding molecule, particularly an antibody or its antigen-binding fragment, the method comprising culturing a host cell or cell-free expression system containing a nucleic acid encoding the TDP-43 binding molecule as provided above, under conditions suitable for the expression of the TDP-43 binding molecule, and isolating the TDP-43 binding molecule.

[0111] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, especially when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Val. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli)). After expression, antibodies can be isolated from the bacterial cell paste into a soluble fraction and further purified.

[0112] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts, including strains of fungi and yeast whose glycosylation pathways have been "humanized," resulting in antibody production in a partially or completely human glycosylation pattern, are also suitable hosts for cloning or expression of antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0113] Suitable host cells for the expression of glycosylated antibodies can also be induced from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0114] Plant cell cultures can also be used as hosts. See, for example, U.S. Patents 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTBODIES® technology for antibody production in transgenic plants).

[0115] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the SV40-transformed macaque kidney CV1 line (COS-7); human embryonic kidney line (e.g., 293 or 293 cells described in Graham et al., J. Gen Viral. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)); macaque kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HeLa); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (WI38); human hepatocytes (Hep G2); mouse mammary tumor cells (MMT 060562); TRI cells described in Mather et al., Annals N. Y Aead. Sei. 383:44-68 (1982); MRC These include 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. cii. USA 77:4216 (1980)), as well as myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Val. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0116] To deliver molecules across the blood-brain barrier (BBB), several approaches are known in the art, including altering the administration route, disrupting the BBB and altering its permeability, nanoparticle delivery, the Trojan horse approach, receptor-mediated transport, and cell and gene therapies.

[0117] 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 into the brain (see, e.g., Gillet al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers®, Guildford Pharmaceutical), or intranasal administration to bypass the blood-brain barrier (see Mittal et al., Drug Deliv. 21(2):75-86. (2014)).

[0118] Methods of barrier disruption include, but are not limited to, ultrasound (see, e.g., U.S. Patent Publication 2002 / 0038086), osmotic pressure (e.g., by administration of hypertonic mannitol (Neuwelt, EA, Implication of the Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press, NY (1989))), and permeability enhancement with, for example, bradykinin or permeability enhancer A-7 (see, e.g., U.S. Patents 5,112,596, 5,268,164, 5,506,206, and 5,686,416).

[0119] Methods for 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 Publications 2002 / 0065259, 2003 / 0162695, and 2005 / 0124533); activated potassium channels (see, e.g., U.S. Patent Publication 2005 / 0089473); and inhibition of ABC drug transporters (see, e.g., U.S. Patent Publication 2003 / 0073713).

[0120] Trojan horse delivery methods for delivering antibodies or antibody fragments across the blood-brain barrier include, but are not limited to, antibody cationization (see, e.g., U.S. Patent No. 5,004,697) and the use of cell-penetrating peptides such as Tat peptides to enable entry into the CNS (e.g., Dietz et al., J. Neurochem. 104:757-765 (2008)).

[0121] Nanoparticle delivery methods for delivering antibodies or antigen-binding fragments across the blood-brain barrier include, but are not limited to, encapsulating antibodies or antigen-binding fragments in extracellular vesicles such as liposomes or exosomes linked to antibodies or antigen-binding fragments or, instead, peptides that bind to receptors on the vascular endothelium of the blood-brain barrier (see, e.g., U.S. Patent Publication 20020025313), and coating antibodies or antigen-binding fragments with low-density lipoprotein particles (see, e.g., U.S. Patent Publication 20040204354) or apolipoprotein E (see, e.g., U.S. Patent Publication 20040131692).

[0122] The antibodies of the present invention can be further modified to enhance penetration across the blood-brain barrier. The antibodies of the present invention or their antigen-binding fragments can be fused with polypeptides that bind to blood-brain barrier receptors. BBB receptors include, but are not limited to, transferrin receptors, insulin receptors, or low-density lipoprotein receptors. The polypeptide may be a peptide, a receptor ligand, a single-domain antibody (VHH), an scFv, or a Fab fragment.

[0123] The antibodies of the present invention can also be delivered as 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. A non-limiting example is a viral vector containing a nucleic acid molecule encoding the antibody of the present invention for targeted delivery to endothelial cells of the BBB, pericytes of the BBB, or astrocytes. In some embodiments, endothelial cells of the BBB, pericytes of the BBB, or astrocytes express the antibody and secrete it into the brain parenchyma. A preferred example is a viral vector containing a nucleic acid molecule encoding the antibody of the present invention for targeted delivery in endothelial cells of the BBB, where the endothelial cells express the antibody and secrete it into the brain parenchyma. The viral vector may be a recombinant adeno-associated virus vector (rAAV) selected from any AAV serotype known in the art, including but not limited to AAV1 to AAV12, which allows the antibody or antibody fragment or antibody derivative to be expressed intracellularly or within the brain parenchyma.

[0124] Cell therapy methods for delivering antibodies, antibody fragments, or antibody derivatives of the present invention across the blood-brain barrier include, but are not limited to, the use of homing capabilities of endothelial progenitor cells (EPCs) transfected ex vivo by a vector and their secretion and delivery of antibodies or antibody fragments to the brain, or the use of polymeric cell-embedded devices carrying genetically engineered cells for secreting antibodies or antibody fragments (see, for example, Marroquin Belaunzaran et al. PLoS ONE 6(4): e18268 (2011)), in order to overcome the potent filtering activity of the BBB (see, e.g., Heller and al., J Cell Mol Med. 00:1-7 (2020)), or the use of polymeric cell-embedded devices carrying genetically engineered cells for secreting antibodies or antibody fragments.

[0125] Pharmaceutically acceptable carriers, diluents, adjuvants, and excipients are well known in pharmaceutical technology, for example, in Remington's Pharmaceutical Sciences, 15th or 18th Ed. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th Ed. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd Ed. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th Ed. (Walter Lund ed.; Pharmaceutical Press, London, 1994); United States Pharmacopeia: National Collection of Pharmaceuticals (American Pharmaceutical Society); Fiedler's “Lexikon der Hilfstoffe” 5th Ed., Edition Cantor Verlag Aulendorf This information is found in “The Handbook of Pharmaceutical Excipients”, 4th Ed., American Pharmaceuticals Association, 2002; and in Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992). These disclosures are thus incorporated herein by reference.

[0126] Carriers, diluents, adjuvants, and pharmaceutical excipients can be selected in relation to the intended route of administration and standard pharmaceutical practices. These compounds must be acceptable in the sense that they are not harmful to their recipients. Remington's Pharmaceutical Sciences, 15th or 18th Ed. (Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990); Remington: the Science and Practice of Pharmacy 19th Ed. (Lippincott, Williams & Wilkins, 1995); Handbook of Pharmaceutical Excipients, 3rd Ed. (Arthur H. Kibbe, ed.; Amer. Pharmaceutical Assoc, 1999); Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12th Ed. (Walter Lund ed.; Pharmaceutical Press, London, 1994); United States Pharmacopoeia: National Formulary (American Pharmaceutical Council); Fiedler's “Lexikon der Hilfstoffe” 5th Ed., Edition Cantor Verlag Aulendorf 2002; “The Handbook of Pharmaceutical Excipients”, 4th Ed., American Pharmaceuticals Association, See also Goodman and Gilman's: The Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992). These disclosures are thus incorporated herein by reference.

[0127] The "effective dose" of a compound to be administered to a patient is the appropriate dosage to treat, prevent, or improve a disease, disorder, or abnormality, according to reasonable medical judgment. The specific dose level and frequency of administration may depend on various factors, including, for example, the activity of the particular compound used, its metabolic stability and duration of action, and the mode and timing of administration. Patient-specific factors, such as age, weight, general health, sex, diet, and the severity of a particular condition, may also influence the amount to be administered.

[0128] The term "clearance" (also referred to as "clearance value," "CL," or "systemic clearance") relates to the efficiency of eliminating substances from the body. The clearance of a substance (in this case, the bound molecule of the present invention) is the sum of urinary and extrarenal clearance. For substances eliminated by renal and extrarenal pathways, plasma clearance exceeds urinary clearance. The PK properties of mAbs are a function of their large size (150 kDa), relative polarity, Fc receptor binding, and specific binding to target antigens. The primary elimination pathway for mAbs is cellular uptake followed by proteolysis. Because mAbs have low clearance from systemic circulation, they can be administered less frequently than peptides or small molecules, which is often more advantageous for patients (Betts et al., MABs. 2018).

[0129] XI. Embodiment of an Inventive Molecule for Specific Binding of TDP-43 In some embodiments, a. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53; or b. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43; or c. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); or d. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23; or e. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13 TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0130] In some embodiments, a. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57; or b. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47; or c. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37; or d. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17; or e. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17 TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0131] In some embodiments, a)i. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53; or ii. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43; or iii. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); or iv. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23; or VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13. Heavy chain variable region (VH) including and b)i. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57; or ii. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47; or iii. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37; or iv. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17; or VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17. Light chain variable region (VL) including TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0132] In some embodiments, a)i. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51 or VH-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 51; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52 or VH-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 52; and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53 or VH-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 53; or ii. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41 or VH-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 41; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42 or VH-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 42; and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43 or VH-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 43; or iii. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31 or VH-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 31; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32 or VH-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 32; and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); or iv. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21 or VH-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 21; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22 or VH-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 22; and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23 or VH-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 23; or v. VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11 or VH-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 11; VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12 or VH-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 12; and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13 or VH-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 13. Heavy chain variable region (VH) including and b)i. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55 or VL-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 55; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56 or VL-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 56; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57 or VL-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 57; or ii. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45 or VL-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 45; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46 or VL-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 46; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47 or VL-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 47; or iii. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35 or VL-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 35; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36 or VL-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 36; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37 or VL-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 37; or iv. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15 or VL-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 15; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26 or VL-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 26; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17 or VL-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 17; or v. VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15 or VL-CDR1 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 15; VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16 or VL-CDR2 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17 or VL-CDR3 containing an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 17. Light chain variable region (VL) including TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0133] In some embodiments, a. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57; or b. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47; or c. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37; or d. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17; or e. Heavy chain variable region (VH) containing VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and light chain variable region (VL) containing VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17. TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0134] In some embodiments, a. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 50; or b. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 40; or c. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 30; or d. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 20; or e. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 10. TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0135] In some embodiments, a. A light chain variable region (VL) containing the sequence of SEQ ID NO: 54, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 54; or b. A light chain variable region (VL) containing the sequence of SEQ ID NO: 44, or a light chain variable region (VL) having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. A light chain variable region (VL) containing 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 with the amino acid sequence of SEQ ID NO: 34; or d. A light chain variable region (VL) containing the sequence of SEQ ID NO: 24, or a light chain variable region (VL) having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or e. Light chain variable region (VL) containing the sequence of sequence number 14. TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0136] In some embodiments, a)i. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 50; or ii. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 40; or iii. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 30; or iv. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 20; or v. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 10. The heavy chain variable region (VH) selected from and b)i. A light chain variable region (VL) containing the sequence of SEQ ID NO: 54, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 54; or ii. A light chain variable region (VL) containing the sequence of SEQ ID NO: 44, or a light chain variable region (VL) having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 44; or iii. A light chain variable region (VL) containing 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 with the amino acid sequence of SEQ ID NO: 34; or iv. A light chain variable region (VL) containing the sequence of SEQ ID NO: 24, or a light chain variable region (VL) having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or v. Light chain variable region (VL) containing the sequence of sequence number 14 Light chain variable region (VL) selected from TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0137] In some embodiments, a. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 50; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 54; or b. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 40; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 44 or a light chain variable region (VL) having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 30; and a light chain variable region (VL) containing 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 with the amino acid sequence of SEQ ID NO: 34; or d. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 20; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 24 or a light chain variable region (VL) having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or e. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 10; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 14. TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0138] In some embodiments, a. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 50 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 54; or b. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 44; or c. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 30 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 34; or d. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 20 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 24; or e. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 10 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 14 TDP-43 binding molecules containing the TDP-43 antibody, particularly a TDP-43 antibody or its antigen-binding fragment, are provided.

[0139] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or their antigen-binding fragments, that bind to misfolded aggregated TDP-43 and non-aggregated physiological TDP-43.

[0140] In some embodiments, TDP-43-binding molecules that bind monomeric and / or oligomeric and / or aggregated and / or post-translationally modified and / or cleaved TDP-43, preferably human TDP-43, are provided, particularly TDP-43 antibodies or antigen-binding fragments thereof.

[0141] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, that bind to misfolded agglutinated human TDP-43 and non-agglutinated physiological human TDP-43 are provided.

[0142] In some embodiments, the following characteristics are observed: a. Inhibiting the aggregation of TDP-43 protein or its fragments, b. Blocking intercellular propagation of TDP-43, c. Deaggregating TDP-43 aggregates, d. Block the sowing of TDP-43. e. Neutralizing TDP-43, which has seed-sowing capabilities. f. Blocking the spread of TDP-43, g. To promote the clearance of TDP-43, and h. Reduce the level of phosphorylated TDP-43 in vivo. A TDP-43 binding molecule exhibiting one or more, or at most all, of these characteristics, particularly a TDP-43 antibody or its antigen-binding fragment, is provided.

[0143] In some embodiments, the following characteristics are observed: a. Inhibiting the aggregation of TDP-43 protein or its fragments, b. Blocking intercellular propagation of TDP-43, c. Block the sowing of TDP-43. d. Blocking the spread of TDP-43, e. To promote the clearance of TDP-43, and f. Reduce the level of phosphorylated TDP-43 in vivo. A TDP-43 binding molecule exhibiting one or more, or at most all, of these characteristics, particularly a TDP-43 antibody or its antigen-binding fragment, is provided.

[0144] In some embodiments, TDP-43-binding molecules that promote TDP-43 clearance are provided, particularly TDP-43 antibodies or antigen-binding fragments thereof.

[0145] In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that reduce TDP-43 lesions in vivo.

[0146] In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or their antigen-binding fragments, are provided that reduce the levels of misfolded aggregated TDP-43 and / or phosphorylated TDP-43 in vivo.

[0147] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or their antigen-binding fragments, are provided that reduce the level of phosphorylated TDP-43 in the hippocampus.

[0148] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to epitopes within amino acid residues 304-414 of human TDP-43 (SEQ ID NO: 1).

[0149] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to epitopes within amino acid residues 304-313 of human TDP-43 (SEQ ID NO: 1).

[0150] 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 304-313 of human TDP-43 (SEQ ID NO: 1).

[0151] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to epitopes within amino acid residues 356-361 of human TDP-43 (SEQ ID NO: 1).

[0152] In some embodiments, a TDP-43 binding molecule, particularly a TDP-43 antibody or its antigen-binding fragment, is provided that binds to an epitope consisting of amino acid residues 356-361 of human TDP-43 (SEQ ID NO: 1).

[0153] In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to epitopes within amino acid residues 397-407 of human TDP-43 (SEQ ID NO: 1).

[0154] 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 397-407 of human TDP-43 (SEQ ID NO: 1).

[0155] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to epitopes within amino acid residues 396-414 of human TDP-43 (SEQ ID NO: 1).

[0156] 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 396-414 of human TDP-43 (SEQ ID NO: 1).

[0157] In some embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided that bind to the protease-resistant amyloid core of TDP-43. The protease-resistant amyloid core of TDP-43 consists of amino acids 272-360 of TDP-43.

[0158] In some embodiments, the TDP-43 binding molecule is an antibody or its antigen-binding fragment.

[0159] In some embodiments, TDP-43-binding molecules, particularly TDP-43 antibodies or antigen-binding fragments thereof, are provided, having a dissociation constant (KD) of 1 nM or less, preferably 750 pM or less, 500 pM or less, 380 pM or less, 230 pM or less, 200 pM or less, or 110 pM or less for binding to soluble TDP-43 (SEQ ID NO: 1). Further details of a suitable assay for determining the KD can be found in Example 3.

[0160] In some embodiments, the TDP-43 binding molecule is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody or its antigen-binding fragment.

[0161] In preferred embodiments, the TDP-43 binding molecule is an IgG1 or IgG4 antibody, or an antigen-binding fragment thereof.

[0162] In some embodiments, TDP-43 binding molecules containing Fc mutations, preferably S228P mutations, are provided, particularly TDP-43 antibodies or antigen-binding fragments thereof.

[0163] In some embodiments, TDP-43 binding molecules, particularly (isolated) nucleic acids encoding the TDP-43 antibody described herein and its fragments, are provided.

[0164] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 18, which encodes the heavy chain variable region (VH) of the anti-TPD-43 antibody described herein.

[0165] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 19, which encodes the light chain variable region (VL) of the anti-TPD-43 antibody described herein.

[0166] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 28, which encodes the heavy chain variable region (VH) of the anti-TPD-43 antibody described herein.

[0167] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 29, which encodes the light chain variable region (VL) of the anti-TPD-43 antibody described herein.

[0168] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 38, which encodes the heavy chain variable region (VH) of the anti-TPD-43 antibody described herein.

[0169] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 39, which encodes the light chain variable region (VL) of the anti-TPD-43 antibody described herein.

[0170] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 48, which encodes the heavy chain variable region (VH) of the anti-TPD-43 antibody described herein.

[0171] In some embodiments, an isolated nucleic acid is provided, comprising Sequence ID No. 49, which encodes the light chain variable region (VL) of the anti-TPD-43 antibody described herein.

[0172] In some embodiments, an isolated nucleic acid is provided containing Sequence ID No. 58, which encodes the heavy chain variable region (VH) of the anti-TPD-43 antibody described herein.

[0173] In some embodiments, an isolated nucleic acid is provided containing Sequence ID No. 59, which encodes the light chain variable region (VL) of the anti-TPD-43 antibody described herein.

[0174] XII. Compositions and Methods The present invention also relates to a pharmaceutical composition comprising a TDP-43 conjugating molecule, in particular the antibody of the present invention as described herein or its antigen-binding fragment, and a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0175] In some embodiments, a pharmaceutical composition is provided comprising the (isolated) antibody described herein and a pharmaceutically acceptable carrier.

[0176] In some embodiments, conjugated conjugated molecules, particularly antibodies or their antigen-binding fragments, are provided, including the conjugated molecules described herein, in particular antibodies or their antigen-binding fragments. The conjugates of the present invention are referred to as immunoconjugates. Any suitable conjugated molecule may be employed in accordance with the present invention. Suitable examples include, but are not limited to, enzymes (e.g., alkaline phosphatase or horseradish peroxidase), avidin, streptavidin, biotin, protein A / G, magnetic beads, fluorescent dyes, radioisotopes (i.e., radioconjugates), nucleic acid molecules, detectable labels, therapeutic agents, toxins, and blood-brain barrier penetration portions. Conjugation methods are well known in the art, and several techniques for conjugating antibodies to labels or other molecules are commercially available. Conjugation is typically mediated by amino acid residues (e.g., lysine, histidine, or cysteine) contained in the conjugated molecules of the present invention. These methods may rely on methods such as the NHS (succinimidyl) ester method, isothiocyanate method, carbodiimide method, and periodic acid method. Conjugation can be achieved, for example, through the creation of a fusion protein. This is appropriate when the binding molecule conjugates with another protein molecule. That is, a suitable gene construct can be formed that enables the expression of a fusion of the binding molecule of the present invention with a label or other molecule. Conjugation may be via a suitable linker moiety to ensure suitable spatial separation of the antibody and the conjugate molecule, for example, a detectable label. However, a linker is not required in all examples. In some embodiments, the TDP-43 specific binding molecule of the present invention is linked to a detectable label.

[0177] The present invention also relates to immunoconjugates comprising one or more therapeutic agents, e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzyme-active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioisotopes (i.e., radioconjugates), blood-brain barrier permeable portions, or TDP-43 binding molecules provided herein, conjugated to a detectable label. As discussed herein, various methods exist for improving drug delivery across the blood-brain barrier (BBB), and these considerations are applied with modifications as necessary. Non-invasive methods include the so-called "Trojan horse approach," in which the conjugated molecule delivers the binding molecule of the present invention by mediating binding to and transport of the BBB receptor. Preferred molecules include endogenous ligands or antibodies, particularly monoclonal antibodies, that bind to specific epitopes on the BBB receptor.

[0178] In some embodiments, an immunoconjugate comprising the (isolated) antibody and therapeutic agent described herein is provided. In some embodiments, a labeled antibody comprising the antibody and a detectable label described herein is provided.

[0179] In some embodiments, the TDP-43 specific binding molecule is a portion of an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another preferred therapeutic agent.

[0180] In some embodiments, the TDP-43 specific binding molecule or the immunoconjugate containing the same exists as a composition containing the TDP-43 specific binding molecule.

[0181] In some embodiments, the TDP-43 specific binding molecule is a pharmaceutical composition comprising the TDP-43 specific binding molecule or an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another suitable therapeutic agent, or a portion of a composition comprising the TDP-43 specific binding molecule in combination with a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0182] In some embodiments, the immune conjugate comprising the TDP-43 binding molecule of the present invention crosses the blood-brain barrier using a delivery medium or a blood-brain barrier moiety. In some embodiments, the delivery medium comprises liposomes or extracellular vesicles. In some embodiments, the TDP-43 binding molecule is ligated to a blood-brain barrier moiety. In some embodiments, the blood-brain barrier moiety is a polypeptide or small molecule, preferably a peptide, a receptor ligand, a single-domain antibody (VHH), scFv, or a Fab fragment. In some embodiments, the blood-brain barrier moiety binds to a blood-brain barrier receptor, which may include a transferrin receptor, an insulin receptor, or a low-density lipoprotein receptor.

[0183] In some embodiments, the TDP-43 specific binding molecule is part of a detection and / or diagnostic kit comprising the TDP-43 specific binding molecule, an immunoconjugate in which the TDP-43 specific binding molecule is covalently linked to another preferred therapeutic agent, or a composition comprising the TDP-43 specific binding molecule.

[0184] In some embodiments, the TPD-43 binding molecule described herein is a. A step of incubating the sample with the capture antibody and the detection antibody; b. A step in which the mixture obtained in step a is incubated with a reagent suitable for detection by a detection antibody; c. Step of measuring the signal emitted by the detection antibody. Used in pairing assays including, The capture antibody is the TDP-43 binding molecule of the present invention.

[0185] In some embodiments, the TPD-43 binding molecule described herein is a. A step of incubating the sample with the capture antibody and the detection antibody; b. A step in which the mixture obtained in step a is incubated with a reagent suitable for detection by a detection antibody; c. Step of measuring the signal emitted by the detection antibody. Used in pairing assays including, The detection antibody is the TDP-43 binding molecule of the present invention.

[0186] In some embodiments, the TPD-43 binding molecule described herein is a. A step of incubating the sample with the capture antibody and the detection antibody; b. A step in which the mixture obtained in step a is incubated with a reagent suitable for detection by a detection antibody; c. Step of measuring the signal emitted by the detection antibody. Used in pairing assays including, The capture antibody and detection antibody are TDP-43-binding molecules of the present invention.

[0187] In some embodiments, the present invention a. A step of incubating a sample with a capture antibody and a detection antibody to produce a mixture; b. A step of incubating the mixture obtained in step a with a reagent suitable for the detection of TDP-43 by a detection antibody; and c. Step of measuring the signal emitted by the detection antibody. This invention provides a method for detecting TDP-43 in a sample, including [specific component].

[0188] In one embodiment of the method for detecting TDP-43 in a sample, the capture antibody is the TDP-43 conjugating molecule of the present invention. In another embodiment of the method for detecting TDP-43 in a sample, the detection antibody is the TDP-43 conjugating molecule of the present invention. In a further embodiment of the method for detecting TDP-43 in a sample, both the capture antibody and the detection antibody are the TDP-43 conjugating molecule of the present invention. The capture antibody and the detection antibody may be the same antibody or different antibodies of the present invention.

[0189] In some embodiments, a pairing assay kit for detecting TDP-43 in a sample is provided. The pairing assay kit comprises one or more TDP-43-binding molecules of the present invention. The kit may be an enzyme-coupled immunosorbent assay (ELISA) kit. The kit may be a single-molecule array (Simoa®) kit. The kit comprises a capture reagent and / or a detection reagent. The kit may further comprise a detection reagent. The TDP-43-binding molecules of the present invention may be provided in the kit as a capture reagent, e.g., a capture antibody, and / or a detection reagent, e.g., a detection antibody.

[0190] In some embodiments, one or more TDP-43 conjugated molecules described herein are used in a pairing assay that includes the step of incubating a sample with a capture antibody and a detection antibody, the sample being human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF), and / or urine, preferably CSF.

[0191] Kits containing the binding molecule of the present invention are also provided. In particular, such kits may be useful for carrying out the diagnostic methods of the present invention (including classification, monitoring, and treatment selection methods). That is, kits containing the TDP-43 specific binding molecule of the present invention are provided for the diagnosis of diseases, disorders, and / or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or for use in the methods of the present invention. Such kits may contain all the components necessary to carry out the methods provided herein. Typically, each component is stored separately in a single whole package. Preferred additional components to be included in the kit include, for example, buffers, detectable dyes, laboratory equipment, reaction vessels, instructions for use, etc. The instructions for use may be tailored to the specific method in which the kit is employed. Preferably labeled TDP-43 binding molecules of the present invention, to be included in such kits, are also provided.

[0192] In some embodiments, TDP-43-specific binding molecules are used in immunodiagnostic methods for use in the prevention, diagnosis, or treatment of TDP-43 proteinosis.

[0193] In some embodiments, a TDP-43-specific binding molecule, or an immunoconjugate in which the TDP-43-specific binding molecule is covalently linked to another preferred therapeutic agent, or a composition comprising the TDP-43-specific binding molecule, administered to a subject in need thereof, is used to diagnose, prevent, improve, or treat diseases, disorders, and / or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or TDP-43 proteinopathy, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-dominant age-related TDP-43 encephalopathy (LATE).

[0194] In some embodiments, 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 the TDP-43 specific binding molecule, is administered to a subject in need, or to frontotemporal dementia (e.g., with mutations in progranulin (GRN) linked to chromosome 9p, with mutations in C9orf72, with mutations in TARDBP, with mutations in balocin-containing protein (VCP), motor neuron disease ( Sporadic or familial FTD with or without MND, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander Alzheimer's disease (AxD), limbic-dominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerotic dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with balocin-containing protein (VCP) mutations; Paget's disease of bone and It is used in methods for diagnosing or monitoring diseases, disorders, and / or abnormalities, or TDP-43 protein disorders, that are associated with TDP-43, particularly TDP-43 aggregates, selected from frontotemporal dementia, oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0195] In other embodiments, the present invention relates to any method for detecting, diagnosing, or monitoring diseases, disorders, and / or abnormalities, or TDP-43 proteinopathy, related to TDP-43, particularly related to TDP-43 aggregates, selected from 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).

[0196] Preferably, the disease, disorder, and / or abnormality, or TDP-43 proteinosis associated with TDP-43, particularly associated with TDP-43 aggregates, is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and frontotemporal dementia (FTD). More preferably, the disease, disorder, and / or abnormality, or TDP-43 proteinosis associated with TDP-43, particularly associated with TDP-43 aggregates, is amyotrophic lateral sclerosis (ALS). More preferably, the disease, disorder, and / or abnormality, or TDP-43 proteinosis associated with TDP-43, particularly associated with TDP-43 aggregates, is Alzheimer's disease (AD). More preferably, the disease, disorder, and / or abnormality, or TDP-43 proteinosis associated with TDP-43, particularly associated with TDP-43 aggregates, is frontotemporal dementia (FTD).

[0197] In some embodiments, TDP-43-specific binding molecules are used in methods for diagnosing pre-symptomatic disease, monitoring disease progression and therapeutic efficacy, predicting responsiveness, or selecting subjects that may respond to treatment with TDP-43-specific binding molecules. The methods are preferably carried out using human blood or urine samples. Most preferably, the methods include ELISA-type assays or surface adaptation assays.

[0198] In some embodiments, the TDP-43-specific binding molecule is used in a method of contacting the TDP-43-specific binding molecule of the present invention 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 encephalopathy (CTE), Perry syndrome, limbic-dominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0199] In some embodiments, the TDP-43 specific binding molecule is associated with frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), corticobasal degeneration, and frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (F TLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutation, ALS with angiogenin (ANG) mutation), Alexander disease (AxD), limbic-dominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), The TDP-43 specific binding molecule of the present invention is used in a method of contacting a sample (e.g., blood, urine, cerebrospinal fluid, interstitial fluid (ISF), or brain tissue) to detect, diagnose, or monitor a disease selected from Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerotic dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in balocin-containing protein (VCP); associated with Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

[0200] In some embodiments, 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 the TDP-43 specific binding molecule, is administered to a subject in need or used to prevent, improve, or treat diseases, disorders, and / or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or TDP-43 proteinosis, or frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD, including sporadic and familial forms of AD), chronic traumatic encephalopathy (CTE), Perry syndrome, and limbic-dominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0201] In some embodiments, 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 the TDP-43 specific binding molecule, is administered to a subject in need, or to frontotemporal dementia (e.g., with mutations in progranulin (GRN), C9orf72, TARDBP, or balocin-containing protein (VCP) linked to chromosome 9p). Sporadic or familial FTD with or without motor neuron disease (MND) associated with mutations, corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc., amyotrophic lateral sclerosis (e.g., sporadic ALS, sudden change in TARDBP) ALS with mutations in angiogenin (ANG), Alexander disease (AxD), limbic-dominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy It is used to treat diseases selected from (sporadic inclusion body myositis, inclusion body myopathy with balocin-containing protein (VCP) mutations; associated with Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy with myotilin (MYOT) gene mutations or desmin (DES) gene mutations, traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD). Preferably, treatment of the disease helps preserve or enhance intellectual cognition and / or reduce brain levels of TDP-43 aggregates.

[0202] In some embodiments, a TDP-43-specific binding molecule, or an immunoconjugate in which the TDP-43-specific binding molecule is covalently linked to another preferred therapeutic agent, or a composition comprising the TDP-43-specific binding molecule, which is administered to a subject requiring it, is used to manufacture pharmaceuticals for treating diseases, disorders, and / or abnormalities related to TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinosis, or frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD, including sporadic and familial forms of AD), chronic traumatic encephalopathy (CTE), Perry syndrome, and limbic-dominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0203] Pharmaceutical formulations of anti-TDP-43 antibodies (preferred forms of TDP-43-specific binding molecules) or immunoconjugates described herein are prepared by mixing such antibodies or immunoconjugates of desired purity with any one or more pharmaceutically acceptable carriers and / or excipients and / or diluents (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Typically, the antibody or fragments thereof are prepared as lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used, and are not limited to, buffering agents such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (approximately 10 residues). Polypeptides (less than 12 units); 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 dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs containing rHuPH20 and methods of use are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase. Pharmaceutically acceptable excipients that can be used to formulate compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers 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, cellulosic substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block copolymers, polyethylene glycol, and lanolin. The diluent may be a buffer. These may include salts selected from the group consisting of phosphates, acetates, citrates, succinates, and tartrates, and / or the buffer may include histidine, glycine, TRIS-glycine, Tris, or mixtures thereof. In the context of the present invention, it is also conceivable that the diluent is a buffer selected from the group consisting of potassium phosphate, acetate / sodium acetate, citrate / sodium citrate, succinate / sodium succinate, tartaric acid / sodium tartrate, and histidine / histidine HCl, or mixtures thereof.

[0204] Exemplary lyophilized antibody or immunoconjugate formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody or immunoconjugate formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations containing a histidine acetate buffer.

[0205] The formulations described herein may contain two or more active ingredients, preferably ingredients having complementary activities that do not adversely affect each other, as necessary for the specific indication being treated.

[0206] The active ingredient may be trapped in microcapsules prepared by, for example, coacervation or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such methods are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0207] Sustained-release preparations may be prepared. A preferred example of a sustained-release preparation includes a semipermeable matrix of a solid hydrophobic polymer containing an antibody or immunoconjugate, the matrix of which is in the form of a molded article, such as a film or microcapsule. Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration with a sterile filtration membrane.

[0208] Any of the antigen-binding molecules, anti-TDP-43 antibodies, or immunoconjugates provided herein can be used in methods such as therapeutic methods.

[0209] In another embodiment, an anti-TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or immunoconjugate is provided for use as a pharmaceutical. In a further embodiment, an anti-misfold TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or immunoconjugate is provided for use in a method of treatment. In a particular embodiment, an anti-TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or immunoconjugate is provided for use in the prevention, diagnosis, and / or treatment of TDP-43 proteinosis. In preferred embodiments of the present invention, an anti-TDP-43 antibody (preferred type of TDP-43-specific binding molecule) or immunoconjugate is provided for use in the prevention, diagnosis, and / or treatment of diseases, disorders, and / or abnormalities related to TDP-43, particularly TDP-43 aggregates, or TDP-43 protein disorders, including but not limited to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and / or limbic-dominant age-related TDP-43 encephalopathy (LATE).

[0210] In further embodiments, the present invention provides the use of an anti-TDP-43 antibody (preferred form of a TDP-43 specific binding molecule) or an immunoconjugate in the manufacture or preparation of a pharmaceutical product. In one such embodiment, the method further comprises administering an effective amount of at least one further therapeutic agent, for example, described below, to an individual.

[0211] The "subject" or "individual" in any of the embodiments may be an animal, a mammal, and preferably a human.

[0212] In further embodiments, the present invention provides a pharmaceutical formulation comprising either an anti-TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or an immunoconjugate provided herein, for use in, for example, any therapeutic method. In one embodiment, the pharmaceutical formulation comprises either an anti-TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or an immunoconjugate provided herein, as well as a pharmaceutically acceptable carrier and / or excipient and / or diluent (discussed elsewhere herein). In another embodiment, the pharmaceutical formulation comprises either an anti-TDP-43 antibody (preferred form of a TDP-43-specific binding molecule) or an immunoconjugate provided herein, as well as at least one further therapeutic agent, for example, described below.

[0213] The antibodies or immunoconjugates of the present invention can be used for therapeutic purposes alone or in combination with other agents. For example, the antibodies (preferred form of the TDP-43 specific binding molecule) or immunoconjugates of the present invention may be co-administered with at least one further therapeutic agent targeting alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or neuroinflammatory proteins.

[0214] For example, the antibody (preferred type of TDP-43 specific binding molecule) or immunoconjugate of the present invention may be co-administered with at least one further therapeutic agent selected from, but not limited to, neuropharmaceuticals, anti-amyloid-beta antibodies, anti-tau antibodies, tau aggregation inhibitors (including small molecules), beta-amyloid aggregation inhibitors (including small molecules), anti-BACE1 antibodies, BACE1 inhibitors, anti-alpha-synuclein inhibitors, anti-alpha-synuclein antibodies, and neuroinflammatory inhibitors.

[0215] Such combination therapies noted above include combination administration (two or more therapeutic agents are included in the same or different formulations) and individual administration (in which case the antibody of the present invention (preferred form of the TDP-43 specific binding molecule) or immunoconjugate is administered before, simultaneously with, and / or after the administration of further therapeutic agents and / or adjuvants). The antibody of the present invention (preferred form of the TDP-43 specific binding molecule) or immunoconjugate may also be used in combination with radiotherapy.

[0216] The antibody (preferred form of the TDP-43 specific binding molecule) or immunoconjugate (and any further therapeutic agent) of the present invention may be administered by any preferred means, including parenteral, intrapulmonary, and intranasal administration, and, if desired for local treatment, intralesional, intrauterine, or intracapsular administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, for example, by injection, such as intravenous or subcutaneous injection, depending on whether the administration is short-term or chronic. Various dosing schedules, including but not limited to single or multiple doses, bolus administration, and pulse infusion, are intended herein.

[0217] The antibody (preferred form of TDP-43 specific binding molecule) or immunoconjugate of the present invention is formulated, administered, and given in a manner consistent with appropriate medical standards. Factors to be considered in this context include the specific disease, disorder, and / or abnormality or TDP-43 proteinosis being treated, related to TDP-43, particularly related to TDP-43 aggregates; the mammal being treated; the clinical condition of the individual subject; the cause of the disease, disorder, and / or abnormality or TDP-43 proteinosis related to TDP-43, particularly related to TDP-43 aggregates; the site of drug delivery; the method of administration; the scheduling of administration; and other factors known to the clinician. The antibody or immunoconjugate does not need to be formulated with one or more agents currently used to prevent or treat TDP-43 proteinosis related to TDP-43, particularly related to TDP-43 aggregates, disease, disorder, and / or abnormality or problem. The effective dose of such other agents depends on the amount of antibodies or immune conjugates present in the formulation, the type or treatment of TDP-43 proteinosis, or diseases, disorders, and / or abnormalities related to TDP-43, particularly TDP-43 aggregates, and other factors discussed above. These are generally used in the same dosages and routes of administration described herein, or at approximately 1–99% of the dosages described herein, or in any dosage and route of administration that is experimentally / clinically determined to be appropriate.

[0218] For the prevention or treatment of a disease, the appropriate dosage of the antibody (preferred form of the TDP-43-specific binding molecule) or immune conjugate of the present invention (when used alone or in combination with one or more additional other therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immune conjugate, the severity and course of the disease, whether the antibody or immune conjugate is administered for preventive or therapeutic purposes, previous treatments, the subject's medical history, and response to the antibody or immune conjugate, as well as the discretion of the attending physician. The antibody (preferred form of the TDP-43-specific binding molecule) or immune conjugate is preferably administered to the subject in a single dose or over a series of treatments. Depending on the type and severity of the disease, an antibody (preferred form of the TDP-43-specific binding molecule) or immune conjugate of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dosage to the subject, for example, in one or more individual doses or by continuous infusion. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, the treatment is generally continued, depending on the condition, until the desired suppression of disease symptoms occurs. One exemplary dose of antibody or immunoconjugate may range from approximately 0.05 mg / kg to approximately 10 mg / kg. That is, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the subject. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the subject receives approximately 2 to approximately 20 doses, or for example, approximately 6 doses of antibody). An initial high loading dose followed by one or more lower doses may be administered. However, other drug regimens may also be useful. The progress of this therapy is readily monitored by conventional methods and assays.

[0219] It is understood that any of the above formulations or treatment methods may be carried out using both the immunoconjugate and the anti-TDP-43 antibody (preferred form of the TDP-43 specific binding molecule) of the present invention.

[0220] In another aspect of the present invention, an article for manufacture is provided comprising the above-mentioned materials useful for the treatment, prevention, and / or diagnosis of diseases, disorders, or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or TDP-43 proteinopathy. The article for manufacture includes a container and a label or accompanying information on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and others. The container may be formed from a variety of materials such as glass or plastic. The container may hold a composition that is effective on its own or in combination with another composition for the treatment, prevention, and / or diagnosis of diseases, disorders, and / or abnormalities related to TDP-43, particularly related to TDP-43 aggregates, or TDP-43 proteinopathy, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one active agent in the composition is the antibody or immunoconjugate of the present invention. The label or accompanying information indicates that the composition is used to treat a selected condition.

[0221] Furthermore, the manufacturing article may include (a) a first container containing a composition comprising the antibody of the present invention (a preferred form of the TDP-43 specific binding molecule) or an immunoconjugate; and (b) a second container containing a composition comprising a further therapeutic agent. The manufacturing article in this embodiment of the present invention may further include a document indicating that the composition can be used to treat a particular condition. Alternatively, the manufacturing article may further include a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution. The manufacturing article may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0222] In further embodiments, the present invention relates to a method for maintaining or increasing cognitive memory, motor and language functions, or preventing and / or delaying the decline of cognitive memory, motor and language functions, in a subject, comprising administering a binding molecule of the present invention, an immunoconjugate of the present invention, a composition of the present invention, or a pharmaceutical composition of the present invention.

[0223] In further embodiments, the present invention relates to a method for reducing TDP-43 levels, comprising administering the conjugate molecule of the present invention, the immunoconjugate of the present invention, the composition of the present invention, or the pharmaceutical composition of the present invention.

[0224] The method of the present invention may include the administration of additional therapies, preferably selected from antibodies or small molecules targeting alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or neuroinflammatory proteins, but not limited thereto, particularly at least one additional therapy selected from neuropharmaceuticals, anti-beta-amyloid antibodies, anti-tau antibodies, tau agglutination inhibitors, beta-amyloid agglutination inhibitors, anti-BACE1 antibodies, BACE1 inhibitors, anti-alpha-synuclein antibodies, and neuroinflammatory inhibitors.

[0225] The present invention further relates to a method for detecting TDP-43, comprising contacting a sample with the binding molecule of the present invention, preferably the antibody of the present invention, wherein the sample is a brain sample, cerebrospinal fluid sample, interstitial fluid (ISF) sample, urine sample, or blood sample.

[0226] In further embodiments, the present invention relates to a method for detecting and / or measuring TDP-43 levels using a single-molecule array (SIMOA®) technique, comprising contacting a sample with the binding molecule of the present invention, preferably the antibody of the present invention, wherein the sample is a human blood sample, cerebrospinal fluid (CSF) sample, interstitial fluid (ISF) sample, or urine sample, preferably a CSF sample.

[0227] As described herein, the binding molecule (preferably an antibody) of the present invention targets (i.e., binds to) specific domains or fragments of TDP-43. For example, the ACI-7071-810H12-Ab1 antibody binds to the protease-resistant amyloid core of TDP-43. That is, 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 the protease-resistant amyloid core of TDP-43 at the C-terminal fragment. Given the observation that disease-specific proteolytic cleavage exposing this amyloid core further enhances its seeding activity, which is important for templated aggregation, this may provide an indicator of disease or disease state.

[0228] In certain embodiments, the TDP-43 binding molecule, in particular the TDP-43 antibody and its fragments provided herein, has a binding concentration of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 nM) with respect to TDP-43, particularly soluble TDP-43. -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The TDP-43 binding molecule of the present invention has a dissociation constant (KD) of M. For example, the TDP-43 binding molecule of the present invention may have a KD of 2 nM or less, 1 nM or less in certain embodiments, for binding to soluble full-length TDP-43, and in more specific embodiments, a KD of 750 pM or less, 500 pM or less, 380 pM or less, 230 pM or less, 200 pM or less, or 110 pM or less for soluble full-length TDP-43. This is demonstrated for the TDP-43 binding molecule of the present invention with reference to Table 4 in Example 3. In one embodiment, the binding affinity to full-length (FL) TDP-43 may be evaluated by determining the dissociation constant (KD) using surface plasmon resonance (SPR; Biacore 8K, GE Healthcare Life Sciences). See Example 3 for a detailed description of a suitable SPR method that may be employed.

[0229] In one embodiment, TDP-43 binding molecules, particularly TDP-43 antibodies and their fragments, reduce the levels of pathogenic TDP-43 in the brain and improve / inhibit / reduce the formation of TDP-43 lesions in vivo.

[0230] In further embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies and their fragments, reduce the levels of phosphorylated TDP-43 in the brain, thereby improving / inhibiting / reducing the formation of TDP-43 lesions in vivo.

[0231] In further embodiments, TDP-43 binding molecules, particularly TDP-43 antibodies and their fragments, reduce the levels of phosphorylated TDP-43 in the hippocampus, thereby improving / inhibiting / reducing the formation of TDP-43 lesions in vivo.

[0232] The TDP-43 binding molecules of the present invention, particularly antibodies or their antigen-binding fragments, typically bind to TDP-43 with high affinity. For example, they can demonstrate EC50 values ​​of ≤200 pM, ≤40 pM, ≤20 pM, or ≤10 pM as determined by the Luminex assay. Further details of preferred assays can be found in Example 2.

[0233] The TDP-43 binding molecule of the present invention, particularly the antibody or its antigen-binding fragment, may have a half-life of at least 10 days, or at least 16 days, in mice. Further details of preferred methods for measuring the half-life in mice can be found in Example 9. [Brief explanation of the drawing]

[0234] [Figure 1A]Graphical representation of the quantification of phosphorylated TDP-43 (pTDP-43) from the ipsilateral (Fig. 1a) or contralateral (Fig. 1b) hippocampus of a TDP-43 proteinopathy mouse model treated with ACI-7071-806H5-Ab1 (B), ACI-7071-810H12-Ab1 (C), or mAb negative control antibody (D). (E) Non-inoculated mice. (F) Single-gene CamK2a mice without the human TDP-43 transgene (WT-tTA). Mean ± SD. [Figure 1B] Same as above. [Figure 2] Graphical representation of antibody plasma exposure at the test endpoint after 13 weekly i.p. administrations of ACI-7071-810H12-Ab1 at 60 mg / kg each in CamKIIa-hTDP-43NLSm mice. Mean ± SD. [Figure 3] Graphical representation of the linear correlation between pTDP-43 levels and plasma concentration of ACI-7071-810H12-Ab1 at the ipsilateral (black line and dots) and contralateral (light gray line and triangles) test endpoints. [Figure 4] Immunoblot using (A) ACI-7071-810H12-Ab1, (B) anti-total TDP-43 antibody, or (C) anti-pTDP-43 (S409 / S410) antibody to detect TDP-43 in Sarkosyl-insoluble brain extracts prepared from the frontal cortex of cases of type A FTLD-TDP before (-) or after (+) limited proteolysis. The upper arrow indicates the predicted molecular weights of full-length TDP-43 and pTDP-43. The lower arrow indicates the predicted molecular weights of protease-resistant fragments. The brackets indicate the predicted molecular weights of the C-terminal fragments (CTFs) of TDP-43. The lower part of the blot provides an image with enhanced contrast of the immunoblot at the predicted molecular weights of protease-resistant fragments (about 8 - 15 kDa).

Example

[0235] [Example 1]

[0236] Preparation of the TDP-43 vaccine composition According to the protocol published in WO2012 / 055933, a liposomal vaccine was prepared. A vaccine containing the full-length TDP-43 (FL TDP-43) protein (Table 2, SEQ ID NO: 1) as an antigen was used for antibody production.

[0237]

Table 2

[0238] Generation of anti-TDP-43 antibodies A. Mouse immunization Female C57BL / 6JOlaHsd (C57BL / 6) and BALB / c OlaHsd (BALB / c) wild-type mice (Harlan, USA) were received at 9 weeks of age. Vaccination started at 10 weeks. Mice were vaccinated with the full-length TDP-43 protein presented on the surface of liposomes in the presence of monophosphoryl hexaacyl lipid A, 3-deacyl (Synthetic) (3D-(6-acyl)PHAD®) as an adjuvant. Mice were vaccinated by 200 μl subcutaneous injection (s.c.) on days 0, 4, 8, 21, 35, and 70. Blood was collected from the mice 7 days before immunization (pre-immune plasma) and on days 15, 28, 42, 77, and 136 after the first immunization, and heparinized plasma was prepared. Mice used for myeloma fusion were further vaccinated by three daily booster injections of the TDP-43 protein by i.p. injection without adjuvant. The vaccine response was measured in mouse plasma. Binding of antibodies derived from plasma of immunized mice to immobilized recombinant full-length (FL) TDP-43 indicated a high titer of antibodies against TDP-43.

[0239] B. Selection for generation and subcloning of hybridomas Mice were euthanized, and splenocytes from four individual mice were used for fusion with myeloma cells. Antibody screening from the successfully fused hybridoma cell lines was performed as follows: Diluted (1:32) cell culture supernatants were analyzed using a Luminex bead multiplex assay (Luminex, The Netherlands). Luminex beads were conjugated with captured IgG containing FL TDP-43 and anti-mouse IgG-Fc antibodies (Jackson Immunoresearch, USA) specific to IgG1, IgG2a, IgG2b, IgG2c, and IgG3 subclasses. Binding of FL TDP-43 to the conjugated beads identified 386 hits derived from mice immunized with the FL TDP-43 liposomal vaccine.

[0240] Viable hybridomas were grown using serum-containing selective medium. Clones that preferentially bound to TDP-43 inclusions in human FTD brain and clones that bound to the C-terminus of TDP-43 were selected for further subcloning. After limiting dilution, clonal hybridomas were grown in low immunoglobulin-containing medium, and stable colonies were selected for antibody screening and selection. The antibodies shown in Table 3 were identified from this screening.

[0241] [Table 3] [Example 3]

[0242] Antibody characterization by surface plasmon resonance (SPR) Soluble TDP-43 was immobilized on a CM5 Series S sensor chip (GE Healthcare, BR-1005-30), and measurements were performed using a Biacore 8K instrument (GE Healthcare Life Sciences).

[0243] Determination of KD of soluble TDP-43 by SPR The instrument was filled with operating buffer PBS-P+, and flow cells (Fc) 1 and 2 of channels 1-8 were activated with fresh EDC / NHS (Amine Coupling Kit, reagent ratio 1:1, GE Healthcare Life Sciences, BR-1006-33) at a flow rate of 10 μL / min for 420 seconds. Soluble TDP-43 (Selvita) was diluted with sodium acetate at pH 4.5 to a final concentration of 5 μg / mL and injected into Fc2 at a flow rate of 10 μL / min for 80 seconds. All flow cells were quenched with 1 M ethanolamine (GE Healthcare Life Sciences, BR-1006-33) at a flow rate of 10 μL / min for 420 seconds. The immobilization level after ethanolamine quenching was approximately 370 RU in all 8 channels. Three startup cycles were performed prior to analysis. In the operating buffer, increasing concentrations of mAb, prepared by 3-fold serial dilutions ranging from 1.2 nM to 100 nM, were injected using single-cycle kinetics with a contact time of 300 seconds, a dissociation time of 3600 seconds, and a flow rate of 30 μL / min. Following each cycle, one regeneration was performed using 10 mM glycine-HCl, pH 1.7, with a contact time of 30 seconds and a flow rate of 10 μL / min, followed by a stabilization time of 300 seconds. The results obtained from single-cycle kinetics were double-referenced using blank Fc1 and buffer cycles, and evaluated using Biacore 8K evaluation software with a 1:1 kinetic fit model using RI and global Rmax. The following kinetic parameters were obtained: binding rate constant (ka), dissociation rate constant (kd), and affinity constant (KD) (Table 4).

[0244] [Table 4] [Example 4]

[0245] Determination of the binding region Epitope mapping using peptide arrays for ACI-7071-704H9-Ab1, ACI-7071-707A6-Ab1, ACI-7071-801H1-Ab1, and ACI-7071-810H12-Ab1. Epitope mapping was confirmed using a custom-made peptide array library (Pepscan, Netherlands). Briefly, epitopes were defined using an array of overlapping linear peptides that cover the entirety of TDP-43.

[0246] Peptide synthesis To reconstitute the epitopes of target molecules, a library of peptide mimetic compounds was synthesized using Fmoc-based solid-phase peptide synthesis. Amino-functionalized polypropylene supports were obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonylhexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) and N-hydroxybenzotriazole (HOBt), and then cleavage of the Boc group using trifluoroacetic acid (TFA). Peptides were synthesized on the amino-functionalized solid supports using standard Fmoc peptide synthesis with a custom-modified JANUS liquid handling station (Perkin Elmer).

[0247] ELISA screening Antibody binding to each synthesized peptide was tested using a PEPSCAN ELISA. The peptide array was incubated with the primary antibody solution (overnight at 4°C). After washing, the peptide array was incubated at 25°C for 1 hour with a 1 / 1000 dilution of a suitable antibody-peroxidase conjugate. After washing, 2,2'-azino-di-3-ethylbenzthiazoline sulfonate (ABTS), a substrate for peroxidase, and 20 μl / ml of 3% H2O2 were added. After 1 hour, the color development was measured. The color development was quantified using a charge-coupled device (CCD) camera and image processing system. The determined epitopes are provided in Table 5.

[0248] [Table 5]

[0249] Determination of the Binding Region Using an ELISA Assay for B.ACI-7071-806H5-Ab1 ACI-7071-806H5-Ab1 was screened by an ELISA assay, and the binding region was determined using a library of peptides with biotinylated N-termini. The peptide sequences are provided in Table 6.

[0250] A streptavidin-coated 96-well ELISA plate was incubated with 5 μg / mL of biotinylated peptide. The plate was washed four times with 0.05% Tween-20 / PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20 / PBS at 37 °C for 1 hour. Then, the antibody purified from the hybridoma supernatant was added at 1 μg / ml and incubated at 37 °C for 2 hours, after which the plate was washed. An AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, United Kingdom) was added at a 1 / 1000 dilution in 0.05% Tween-20 / PBS at 37 °C for 1 hour. After the final wash, the plate was incubated with pNPP (Sigma-Aldrich, Switzerland), an AP substrate solution, and read at OD405 using an ELISA plate reader (Tecan). The determined binding regions are provided in Table 7. The antibodies tested were found to bind to the following peptides corresponding to regions 402 - 414, 396 - 414, 396 - 414 (pS403 / 404), and 402 - 414 (pS409 / 410), respectively: TP-52, TP-95, TP-96, TP-102.

[0251] [Table 6]

[0252] [Table 7] [Example 5]

[0253] Immunohistochemical detection of TDP-43 in brain tissue derived from FTD / ALS patients Target involvement was evaluated by immunohistochemical experiments on brain-derived tissue from FTD subjects. Human FTD brain tissue was obtained from the UCSF Neurodegenerative Disease Brain Bank. All materials were collected from donors who underwent brain autopsy for research purposes and provided written informed consent to the brain bank regarding the use of materials and clinical information. Immunohistochemistry was performed on 10 μm thick frozen sections using fluorescently labeled secondary antibodies for detection. The following antibodies were used as controls: rat monoclonal anti-phospho-TDP-43 p409 / 410 antibody (Biolegend, 829901) for detecting phosphorylated TDP-43, and secondary antibody without primary antibody (No. 1°Ab) for detecting nonspecific background.

[0254] All antibodies in this invention bound to both non-aggregated physiological nuclear TDP-43 and aggregated TDP-43. A detailed evaluation of the binding characteristics is summarized in Table 8.

[0255] [Table 8] NA: No data available; - None; + / - Unclear; + Weak; ++ Moderate; +++ Abundant [Example 6]

[0256] In vitro functionality in recombinant TDP-43 aggregation assay To evaluate the functionality of antibodies in vitro, the ability of antibodies to inhibit TDP-43 aggregation was tested. FL TDP-43 was fused at the C-terminus of maltose-binding protein (MBP), which was isolated by the tobacco etch virus (TEV) protease cleavage site and recombined. Aggregation of 2.5 μM TDP-43-TEV-MBP fusion protein was induced in 30 mM Tris, 150 mM NaCl, pH 7.4, in the presence of either 2.5 μM anti-TDP-43 antibody or a negative control mAb that does not bind to TDP-43, respectively, by the addition of TEV protease (AcTEV, Invitrogen). Absorbance was monitored at 600 nm for 1.5 hours in a MicroClear 96-well plate (Greiner). For evaluation, the endpoint was normalized to the negative control mAb, and the percentage of aggregated TDP-43 was calculated for each antibody. All antibodies significantly inhibited TDP-43 aggregation by more than 97% compared to the negative control mAb (Table 9). P-values ​​of less than 0.0001 were obtained for each mAb compared to the control mAb. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test.

[0257] [Table 9] [Example 7]

[0258] In vitro functionality of FTLD-TDP brain-derived TDP-43 seeds in immune depletion. Sarcosyl-insoluble brain fractions (sarcospins) were prepared according to a published protocol (Laferriere et al., 2019). Immunoexhaustion was performed using Dynabeads® Protein G magnetic beads. For single reactions, 20 μL of beads were used with 3 μg of antibody for 10 μg of sarcosyl-insoluble brain extract (total protein). Before adding the antibody, the beads were first rinsed twice with 500 μL of PBS-0.05% Tween®-20, and then washed with 100 μL of PBS-0.05% Tween®-20. The beads were resuspended with 100 μL of 30 μg / mL antibody in PBS-0.05% Tween®-20. The bead / antibody reaction was incubated at room temperature for 30 minutes under constant rotation and shaking (HulaMixer® Sample Mixer 15920D, Thermofisher). The bead / antibody complex was rinsed twice with PBS-0.05% Tween®-20 and once with PBS, after which sarcosyl-insoluble brain extract was added. The pool of type A FTLD-TDP brain extract was diluted to 100 μg / mL. The bead / antibody complex was resuspended using 100 μL of the extract and incubated at room temperature for 30 minutes under constant rotation and shaking. The supernatant was collected as an immunodepletion fraction using a magnetic support and characterized by Western blotting. ACI-7071-806H5-Ab1 was able to efficiently immunodeplete TDP-43 seeds derived from FTLD-TDP brain extract (Table 10).

[0259] [Table 10] NA: No data available; - None; + / - Unclear; + Weak; ++ Moderate; +++ Abundant [Example 8]

[0260] In vitro functionality of TDP-43 aggregate uptake by microglia For the preparation of mouse primary microglia, cortical tissue isolated from 5-day-old (P5) CD1 mice (Charles River, France) was enzymatically and mechanically dissociated as described in Neural Tissue Dissociation Kits (P) (Miltenyi, 130-092-628). From the resulting cell suspension, microglia were purified using CD11b / c microbeads according to the manufacturer's instructions (Miltenyi, 130-093-634). The microglia were then placed in 60 inner wells of a 96-well tissue culture plate (Falcon, 353219) at a rate of 3 × 10⁶ per well. 5 Cells were seeded at a cell density and maintained in a complete growth medium modified from (5). The growth medium consisted of DMEM / F12 (Gibco, 31331-093) supplemented with 2.5% heat-inactivated FBS, 1% PS, 200 ng / mL tumor growth factor β2 (TGF-β2; Peprotech, 100-35B), 100 ng / mL interleukin-34 (IL-34; R&D Systems), 5 μg / mL N-acetylcysteine ​​(Sigma, A9165), 5 μg / mL insulin (Sigma, I6634), 100 μg / mL apotransferrin (Sigma, T1147), 100 ng / mL sodium selenite (Sigma, S-5261), and wool cholesterol (1.5 μg / mL, Avanti Polar Lipids). During the experiment, a basic medium was used: DMEM / F12 (Gibco, 31331-093) supplemented with 1% penicillin / streptomycin, 5 μg / ml N-acetylcysteine ​​(Sigma, A9165), 5 μg / ml insulin (Sigma, I6634), 100 μg / mL apotransferrin (Sigma, T1147), and 100 ng / mL sodium selenite (Sigma, S-5261).

[0261] Microglia were seeded in growth medium at 30,000 cells per well and incubated for 48 hours. Immune complexes were prepared at 2× final concentration by mixing pHrodo®-labeled TDP-43 aggregates with ACI-7071-806H5-Ab1, ACI-7071-810H12-Ab1, or a negative control mAb in basal medium in a dilution plate (Eppendorf 96-well, sterile) and incubating overnight at 4°C. The dilution plate was equilibrated at room temperature and the cells were washed three times with basal medium. After the final wash, 100 μL of basal medium remained on the cells, to which 100 μL of pHrodo®-labeled TDP-43 aggregates from the dilution plate were added. Cells were immediately placed in an Incucyte for 24 hours for live imaging with phase contrast (to depict microglia) and green fluorescence (to quantify labeled TDP-43 in microglia). In the presence of two mAbs, ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1, microglia uptake of TDP-43 aggregates was significantly increased (Table 11). P-values ​​of less than 0.001 were obtained for each mAb compared to the negative control mAb. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons.

[0262] [Table 11] [Example 9]

[0263] Pharmacokinetics in mice In this study, nine female mice (C57BL / 6 strain, 7 - 11 weeks old) were used for each antibody tested. The animals were purchased from Lingchang / Vital River Laboratory Animal Co., Ltd. After single intraperitoneal (i.p.) administration of 60 mg / kg each of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1, the free plasma concentrations of ACI-7071-806H5-Ab1 and ACI-7071-810H12-Ab1 were determined. Plasma samples were collected from the tail vein of three mice per time point at the following times after dose administration: 0.25 hour, 1 hour, 8 hours, 24 hours, 72 hours, and on days 7, 10, 14, 21, and 28. At the 72-hour time point, samples were collected from six mice. The half-lives obtained for the two antibodies are shown in Table 12.

[0264] [Table 12]

[0265] Both antibodies exhibited good and similar PK parameters and are desirable candidates for applications where the in vivo half-life of an antibody is important, such as therapeutic use in humans. [Example 10]

[0266] In vivo functional efficacy The aim of this study was to evaluate the therapeutic effect of antibodies delivered intraperitoneally (i.p.) in a mouse model of TDP-43 proteinopathy.

[0267] Method Survival profile Hemizygous female (JAX stock #14650: B6;C3-Tg(tetO-TARDBP <000,0009>Double transgenic CamKIIa-hTDP43NLSm animals were generated by mating a hemizygote male (JAX stock #007004:B6.Cg-Tg(CamKIIa-tTA)1Mmay / DboJ) with a breeder mouse (4Vle / J). Breeders and mice were maintained on a 200 mg / kg doxycycline (DOX) diet until 12.5 ± 2 weeks of age. At 13.5 ± 2 weeks of age, CamKIIa-hTDP43NLSm mice were deeply anesthetized with 1 mg / kg buprenorphine and fixed to a stereotactic frame. Sarcosyl-insoluble extracts from the brains of FTLD-TDP cases were sonicated and injected into the dorsal hippocampus. One μl of sarcosyl-insoluble extract was injected at a rate of 0.3 μl / min into each injection site (the needle was introduced into the left hemisphere after Bregma coordinates; 2.0 mm anterior and 1.3 mm left of the midline; three dorsal hippocampal locations subdural, at an initial depth of -1.95 mm, then partially withdrawn to -1.55 mm for the second injection, and then withdrawn again to -1.15 mm for the final injection), with a 4-minute rest period after injection. One day later, weekly intraperitoneal injections of mAb (60 mg / kg) were initiated and continued for 13 consecutive weeks. Three different antibodies, ACI-7071-806H5-Ab1, ACI-7071-810H12-Ab1, and a negative control mAb were tested. Terminal tissue samples were taken three months after injection.

[0268] Tissue mounting, sectioning, immunofluorescence staining, and quantification. Frozen tissue blocks were sectioned into sections 20 μm thick. Staining was performed using Leica BOND-RX. Four levels of coronal sections covering the hippocampus were processed for immunolabeling. For triple pTDP43 / NeuN / Iba1 immunofluorescence (IF) staining, slides were first fixed / cleared with methanol / acetone (1:1) for 10 minutes and washed with PBS. Epitope collection was then performed in Leica ER1 buffer pH 6 (AR9640) at 100°C for 10 minutes, followed by incubation with Protein Block (PowerVision IHC / ISH Super Blocking, Leica, Ref. PV6122). Next, the slides were incubated with primary antibodies in two steps: first with phospho-TDP43 Ab (Biolegend, Ref. 829901, rat Ab, 1 / 500), followed by a mixture of NeuN (Millipore, Ref. MAB377(CH), mouse Ab, 1 / 500) and Iba1 (Wako, Ref. 019-19741, rabbit Ab, 1 / 1000). Then, secondary antibodies were incubated in two steps: first with a mixture of three antibodies—anti-mouse Cy3 (Jackson, goat Ab, 1 / 200), anti-rabbit Alexa488 (Jackson, goat Ab, 1 / 200), and anti-rat biotin (Jackson, goat Ab, 1 / 250), followed by streptavidin-Cy5 (Jackson, 1 / 300). Finally, the slides were incubated with DAPI (1 / 300). All antibodies were diluted with BOND antibody diluent (Cat no. AR9352), the slides were mounted in a fading inhibitor, and coverslips were applied.

[0269] IF slides were digitized using an Axio Scan.Z1 digital full-slide scanner (Carl Zeiss, Canada). Images were subjected to quality control (QC) review, and the final images were transferred to the Biospective server for image processing and analysis. ROIs were defined using a U-Net convolutional neural network trained on a dataset of manually stained tissue sections. ROIs were then subjected to visual QC review and manually adjusted if necessary. IHC staining quantification (presented in Figure 1 as mean staining density) was performed for each digitized IHC slide using Biospective's PERMITS® software. In addition, dual colocalization of pTDP-43 and NeuN, and triple colocalization of pTDP-43, NeuN, and Iba1 were calculated from segmented images. IHC analysis and quantification were performed in a blinded manner for the cohort. Any potential outliers due to technical reasons were excluded before deblinding the data. Data are expressed as mean ± standard deviation.

[0270] statistical analysis Statistical analysis was performed using MATLAB. The data were first assessed for normality using a normal probability plot, and then, where appropriate, for uniformity of variability. Treatment groups were compared using one-way ANOVA (anova1) and Tukey's honest significance test (multcompare) for post-hoc comparisons, or Kruskal-Wallis (kruskalwallis) for non-normally distributed clinical data. For weight measurements, two-way mixed ANOVA was also used to examine any interactions between groups and time points. As a further preliminary measure, direct t-tests or Mann-Whitney comparisons were performed between the two groups (p-values ​​less than 0.05 were asterisked). * (As shown).

[0271] result Inoculation of bitransgenic mice (CamKIIa-hTDP43NLSm) with brain extract resulted in pTDP-43 lesions in both the ipsilateral (same hemisphere as injected) and contralateral (opposite hemisphere to the injection site) hippocampi compared to uninoculated mice (Figure 1E) or single-gene CamK2a mice lacking the human TDP-43 transgene (WT-tTA) (Figure 1F).

[0272] Thirteen weekly intraperitoneal administrations of ACI-7071-810H12-Ab1 resulted in a steady-state plasma antibody level measured at 1787 μg / mL, the endpoint of the study (Figure 2). Assuming a 0.1% penetration from the blood into the brain, the theoretical concentration in the brain was calculated to be 1787 ng / mL. A comparison of the KD (0.38 pM or 57 ng / mL) of ACI-7071-810H12-Ab1 with the theoretical brain concentration (1787 ng / mL, approximately 30 times greater than the KD) highlights the beneficial proportion of ACI-7071-810H12-Ab1 for therapeutic applications. Mice treated with ACI-7071-810H12-Ab1 demonstrated a statistically significant reduction in pTDP-43 in both ipsilateral and contralateral hippocampi compared to the negative control mAb (Figure 1D) (Figure 1C). Furthermore, linear regression analysis showed a trend of negative correlation (both ipsilateral and contralateral) between exposure to ACI-7071-810H12-Ab1 antibody and the amount of pTDP-43 lesions, confirming an exposure-response correlation (ipsilateral: r 2 =0.37, p=0.06; contralateral: r 2 =0.23, and p=0.17; Figure 3). ACI-7071-806H5-Ab1 demonstrated a trend toward reduction of pTDP-43 lesions in both ipsilateral and contralateral (Figure 1B). These data demonstrate that the tested monoclonal antibody can effectively capture extracellular TDP-43 involved in the diffusion of TDP-43 lesions in this mouse model. [Example 11]

[0273] Antibody sequencing Clonal hybridoma cell lysates were used for gene sequencing of the variable region. Mouse hybridomas were harvested and lysed using a lysis buffer containing guanidinium salt to inactivate RNases. cDNA was obtained by reverse transcription of total mRNA. DNA fragments encoding the variable region of the antibody were amplified by RACE-PCR (Takara Bio, cat#634839) using specific primers that anneal in the constant region of the antibody. The PCR products were gel-purified and cloned into a shuttle vector for Sanger sequencing. Sequencing was performed in both directions and duplicated at both ends. Sequences were analyzed using multiplex alignment (Clustal tool) and annotated using Kabat's algorithm as described in Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242 (1991). The nucleotide sequences of the variable regions of the heavy chain (VH) and light chain (VL) are shown in Table 13. Table 14 shows the translated protein sequences for the variable regions of the selected heavy chain (VH) and light chain (VL), as well as their complementarity-determining regions (CDRs). [Example 12]

[0274] Binding of ACI-7071-810H12-Ab1 to the 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.

[0275] Immunoblots were performed on sarcosyl-insoluble brain extracts from type A FTLD-TDP patients, with or without pronase treatment (prepared as previously described; Laferriere et al., 2019). Briefly, sarcosyl-insoluble samples were treated with 0.4 mg / mL pronase (Sigma, 10165921001) at 21°C for 1 hour, followed by centrifugation at 20,000 g at 4°C for 30 minutes. The supernatant was discarded, and the pellet was resuspended in PBS by sonication with a sonicator probe (Q-Sonica) at amplitude 30 for 30 pulses.

[0276] Sarcosyl-insoluble extracts were mixed with 4× sample loading buffer and 0.1 mM dithiothreitol (DTT) and boiled at 95°C for 10 minutes. The samples were loaded onto 4–12% Bis-Tris gels and electrophoresed at 100 volts (V) for 90 minutes. Proteins were transferred to nitrocellulose membranes using the iBlot 2 system (20 mA, 7 minutes). The membranes were blocked in LI-COR® blocking buffer at room temperature for 1 hour with shaking. Primary antibodies (ACI-7071-810H12-Ab1, TDP-43 (Proteintech, 60019-2-Ig), or pTDP-43 (Biolegend, 829901)) were diluted 1:1000 in PBS-0.1% Tween-20 / LI-COR® blocking buffer (1:1) and incubated overnight on the membrane at 4°C under shaking. The membrane was washed three times with PBS-0.1% Tween-20 under shaking. Secondary antibodies, donkey anti-mouse IRDye680CW and donkey anti-rat IRDye800CW (LI-COR®), were diluted 1:10,000 in the same buffer as the primary antibodies, and the membrane was incubated at room temperature for 1 hour under constant shaking. After washing three times with PBS containing 0.1% Tween-20, the membrane was scanned using a LI-COR® Odyssey imager.

[0277] Immunoblots of sarcosyl-insoluble brain extracts of type A FTLD-TDP revealed binding of (A)ACI-7071-810H12-Ab1 to the C-terminal fragment, in addition to a 43kDa band corresponding to full-length TDP-43 (Figure 4). Similar signals were obtained with an antibody (C) that binds to the TDP-43 phosphorylation epitope pS409 / 410, demonstrating that the C-terminal fragment to which ACI-7071-810H12-Ab1 binds retains a disease-specific phosphorylation site (Figure 4). However, after limited proteolysis of sarcosyl-insoluble type A FTLD-TDP brain extracts using pronase treatment, it was shown that only ACI-7071-810H12-Ab1 binds to the protected core of TDP-43 (Figure 4). In contrast, antibodies that bind to the N-terminus of the amyloid core ((B) TDP-43 antibody binding to the RRM2 region) or antibodies that bind to the C-terminal region (pS409 / 410 antibody (C)) did not show a signal in immunoblots of the samples after limited proteolysis.

[0278] These data confirm that ACI-7071-810H12-Ab1 binds to the protease-resistant amyloid core of full-length TDP-43 or its fragments (expected to be around 8-9 kDa based on reported structures, Arseni et al., 2021; Arseni et al., 2023). Since exposure of the amyloid core after disease-specific proteolytic cleavage has been shown to further enhance the seeding activity of TDP-43 (Kumar et al., 2023), such binding properties are valuable for therapeutic use. Furthermore, since proteolytic processing of TDP-43 and their enrichment in patient brains have been shown to be disease-specific pathological features, these binding properties are valuable for diagnostic use. In conclusion, these data further support the potential of ACI-7071-810H12-Ab1 for use as a therapeutic or diagnostic antibody.

[0279] [Table 13-1] [Table 13-2] [Table 13-3]

[0280] [Table 14-1] [Table 14-2] [Table 14-3]

[0281] [ka] [ka] [ka] [ka]

[0282] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in which this invention pertains. All publications and patents specifically described herein are incorporated by reference as a whole for all purposes relating to this invention.

[0283] The present invention should not be limited to the specific embodiments described herein. Indeed, from the foregoing description and the accompanying drawings, various modifications of the present invention, in addition to those described herein, will be obvious to those skilled in the art. Such modifications are intended to be included within the scope of the appended claims. Furthermore, it is considered that all aspects and embodiments of the present invention described herein can be broadly applied to and combined with any other consistent embodiments, including, as necessary, embodiments (including isolated ones) derived from other aspects of the present invention.

Claims

1. a. A heavy chain variable region (VH) comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 51, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 52, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 53; and a light chain variable region (VL) comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 55, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 56, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 57; or b. A heavy chain variable region (VH) comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 41, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 42, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 43; and a light chain variable region (VL) comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 45, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 46, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 47; or c. A heavy chain variable region (VH) comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 31, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 32, and VH-CDR3 containing the amino acid sequence PC (Pro-Cys); and a light chain variable region (VL) comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 35, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 36, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 37; or d. A heavy chain variable region (VH) comprising VH-CDR1 containing the amino acid sequence of SEQ ID NO: 21, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 22, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 23; and a light chain variable region (VL) comprising VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 26, and VL-CDR3 containing the amino acid sequence of SEQ ID NO: 17; or e. Heavy chain variable region (VH) including VH-CDR1 containing the amino acid sequence of SEQ ID NO: 11, VH-CDR2 containing the amino acid sequence of SEQ ID NO: 12, and VH-CDR3 containing the amino acid sequence of SEQ ID NO: 13; and light chain variable region (VL) including VL-CDR1 containing the amino acid sequence of SEQ ID NO: 15, VL-CDR2 containing the amino acid sequence of SEQ ID NO: 16, and VL-CDR3 containing the amino acid sequence of SEQ ID NO:

17. A TDP-43 binding molecule containing, particularly a TDP-43 antibody or its antigen-binding fragment.

2. a. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 50; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 54; or b. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 40; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 44 or a light chain variable region (VL) having at least 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 44; or c. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 30; and a light chain variable region (VL) containing 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 with the amino acid sequence of SEQ ID NO: 34; or d. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 20; and a light chain variable region (VL) containing the sequence of SEQ ID NO: 24 or a light chain variable region (VL) having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24; or e. A heavy chain variable region (VH) containing 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 with the amino acid sequence of SEQ ID NO: 10; and a light chain variable region (VL) containing the sequence of SEQ ID NO:

14. A TDP-43 binding molecule according to claim 1, comprising:

3. a. A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 50 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 54; or b. A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 40 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 44; or c. A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 30 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 34; or d. A heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 20 and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 24; or e. Heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 10 and light chain variable region (VL) containing the amino acid sequence of SEQ ID NO: 14 A TDP-43 binding molecule according to any one of the claims, comprising:

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

5. A TDP-43-binding molecule according to any one of the claims, which binds to monomeric and / or oligomeric and / or aggregated and / or post-translationally modified and / or cleaved TDP-43, preferably human TDP-43.

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

7. The following characteristics: a. Inhibiting the aggregation of TDP-43 protein or its fragments, b. Blocking the intercellular propagation of TDP-43. c. Deaggregating TDP-43 aggregates, d. Blocking the sowing of TDP-43. e. Neutralizing TDP-43, which has seed-sowing ability. f. Blocking the diffusion of TDP-43, g. To promote TDP-43 clearance, and h. To reduce the level of phosphorylated TDP-43 in vivo. A TDP-43 binding molecule according to any one of the claims, exhibiting one or more, or at most all of, of the above.

8. The following characteristics: a. Inhibiting the aggregation of TDP-43 protein or its fragments, b. Blocking the intercellular propagation of TDP-43. c. Blocking the sowing of TDP-43. d. Blocking the diffusion of TDP-43. e. To promote TDP-43 clearance, and f. To reduce the level of phosphorylated TDP-43 in vivo. A TDP-43 binding molecule according to any one of the claims, exhibiting one or more, or at most all of, of the above.

9. A TDP-43 binding molecule according to any one of the claims, which promotes the clearance of TDP-43.

10. A TDP-43 binding molecule according to any one of the claims, which reduces TDP-43 lesions in vivo.

11. A TDP-43 binding molecule according to any one of the claims, which reduces the level of misfolded aggregated TDP-43 and / or phosphorylated TDP-43 in vivo.

12. A TDP-43 binding molecule according to any one of the claims, which reduces the level of phosphorylated TDP-43 in the hippocampus.

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

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

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

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

17. A TDP-43 binding molecule according to any one of the claims, wherein the antibody or an antigen-binding fragment thereof.

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

19. A TDP-43 binding molecule according to any one of the claims, which is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody or an antigen-binding fragment thereof.

20. A TDP-43 binding molecule according to any one of the claims, wherein the TDP-43 binding molecule is an IgG1 or IgG4 antibody or an antigen-binding fragment thereof.

21. A TDP-43 binding molecule according to any one of the claims, comprising an Fc mutation, preferably an S228P mutation.

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

23. The immunoconjugate according to claim 22, which crosses the blood-brain barrier using a delivery medium or a blood-brain barrier portion.

24. The immunoconjugate according to claim 23, wherein the delivery medium comprises liposomes or extracellular vesicles.

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

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

27. The immunoconjugate according to claim 23, 25, or 26, wherein the blood-brain barrier portion binds to a blood-brain barrier receptor.

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

29. A labeled conjugating molecule comprising a TDP-43 conjugating molecule according to any one of claims 1 to 21, particularly a labeled antibody.

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

31. A TDP-43 conjugated molecule according to any one of claims 1 to 21, or an immunoconjugate according to any one of claims 22 to 28, or a pharmaceutical composition according to claim 30, for human or veterinary use.

32. A TDP-43 binding molecule according to any one of claims 1 to 21, an immunoconjugate according to any one of claims 22 to 28, or a pharmaceutical composition according to claim 30, for use in the prevention, improvement, or treatment of diseases, disorders, and / or abnormalities related to TDP-43, or TDP-43 proteinosis.

33. A TDP-43 binding molecule according to any one of claims 1 to 21, or an immunoconjugate according to any one of claims 22 to 28, a labeled binding molecule according to claim 29, or a pharmaceutical composition according to claim 30, for diagnostic purposes.

34. A TDP-43-binding molecule, or an immunoconjugate, labeled binding molecule, or pharmaceutical composition for use according to claim 33, for the diagnosis of TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinosis.

35. A TDP-43 conjugate molecule according to any one of claims 1 to 21, or an immunoconjugate according to any one of claims 22 to 28, a labeled conjugate molecule according to claim 29, or a pharmaceutical composition according to claim 30, particularly for research use as an analytical tool or reference molecule.

36. A TDP-43 binding molecule according to any one of claims 1 to 21, or an immunoconjugate according to any one of claims 22 to 28, a labeled binding molecule according to claim 29, or a pharmaceutical composition according to claim 30, for use as a diagnostic tool for monitoring TDP-43-related diseases, disorders, and / or abnormalities or TDP-43 proteinosis.

37. Diseases, disorders, and / or abnormalities associated with TDP-43, or TDP-43 proteinopathy, are frontotemporal dementias (e.g., those linked to chromosome 9p, those with progranulin (GRN) mutations, those with C9orf72 mutations, those with TARDBP mutations, those with balocin-containing protein (VCP) mutations, sporadic or familial FTDs with or without motor neuron disease (MND), corticobasal degeneration, and ubiquitin-positive TDP. Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD) (FTLD-TDP), argyrophilic granule disease, Pick's disease, semantic subtype primary progressive aphasia (svPPA), behavioral subtype FTD (bvFTD), non-fluent subtype primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), Alexander disease (AxD), limbic-dominant age-related TDP-43 encephalopathy (LA TE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerotic dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with balocin-containing protein (VCP) mutations; Paget's disease of bone and frontotemporal dementia) A TDP-43 conjugate molecule, or immunoconjugate, or labeled conjugate molecule, or pharmaceutical composition for use according to any one of claims 32, 34, or 36, relating to) oculopharyngeal muscular dystrophy with marginal vesicles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES), traumatic brain injury (TBI), Lewy body dementia (DLB), or Parkinson's disease (PD).

38. A TDP-43-binding molecule, immunoconjugate, labeled 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 limbic-dominant age-related TDP-43 encephalopathy (LATE).

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

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

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

42. A method for maintaining or increasing cognitive memory ability or delaying memory loss in an individual having a TDP-43-related disease, disorder, and / or abnormality or TDP-43 proteinopathy, comprising administering to the individual a TDP-43-binding molecule according to any one of claims 1 to 21, an immunoconjugate according to any one of claims 22 to 28, or a pharmaceutical composition according to claim 30.

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

44. The method according to claim 42 or 43, comprising administering at least one further therapeutic agent.

45. The method according to claim 44, wherein the further therapeutic agent targets alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or a neuroinflammatory protein.

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

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

59.

48. a. The heavy chain variable region (VH) coded by SEQ ID NO: 58 and the light chain variable region (VL) coded by SEQ ID NO: 59; or b. The heavy chain variable region (VH) coded by SEQ ID NO: 48 and the light chain variable region (VL) coded by SEQ ID NO: 49; or c. The heavy chain variable region (VH) coded by SEQ ID NO: 38 and the light chain variable region (VL) coded by SEQ ID NO: 39; or d. The heavy chain variable region (VH) coded by SEQ ID NO: 28 and the light chain variable region (VL) coded by SEQ ID NO: 29; or e. The heavy chain variable region (VH) encoded by SEQ ID NO: 18 and the light chain variable region (VL) encoded by SEQ ID NO: 19 A nucleic acid molecule encoding a TDP-43 binding molecule, containing the nucleotide sequence shown as follows.

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

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

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

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

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

54. A cell-free expression system containing the recombinant expression vector described in claim 52.

55. a. A step of culturing the host cells described in claim 53 or the cell-free expression system described in claim 54 under conditions suitable for producing a TDP-43 binding molecule, particularly an antibody or its antigen-binding fragment, and b. Steps for isolating TDP-43-binding molecules, particularly antibodies or their antigen-binding fragments. A method for producing a TDP-43-binding molecule, particularly an antibody or its antigen-binding fragment, which includes [the specified substance].

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

57. a. A step of incubating the sample with the capture antibody and the detection antibody; b. A step of incubating the mixture obtained in step a with a reagent suitable for detection by a detection antibody; c. A step to measure the signal emitted by the detected antibody. The use of a TDP-43 binding molecule according to any one of claims 1 to 21 in a pairing assay, A method wherein the capture antibody is selected from the antibodies described in any one of claims 1 to 21.

58. The use of the TDP-43 conjugating molecule according to claim 57, wherein the detection antibody is selected from the antibodies described in any one of claims 1 to 21.

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

60. A kit comprising a TDP-43 binding molecule according to any one of claims 1 to 21 for the diagnosis of TDP-43-related diseases, disorders, and / or abnormalities or TDP-43 proteinopathy, or for use according to any one of claims 31 to 41, or for use in the method according to any one of claims 42 to 45.

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