Humanized anti-TDP-43 binding molecules and uses thereof

Abstract

The present invention is in the field of transactivation response DNA binding protein with molecular weight of 43 kDa (also TARDB or TDP-43). The present invention relates to a humanized TDP-43 specific binding molecule, in particular a humanized anti-TDP-43 antibody or an antigen-binding fragment or derivative thereof, and uses thereof. The present invention provides means and methods for diagnosing, preventing, ameliorating, and / or treating diseases, disorders, and / or disorders 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-predominant age-related TDP-43 encephalopathy (LATE).

Description

[Technical Field]

[0001] The present invention is in the field of 43 kDa transactivation response DNA-binding protein (also known as TARDB or TDP-43). The present invention relates to humanized TDP-43-specific binding molecules, particularly humanized anti-TDP-43 antibodies or antigen-binding fragments or derivatives thereof, and uses thereof. The present invention provides means and methods for diagnosing, preventing, ameliorating, and / or treating diseases, disorders, and / or disorders associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies, including, but not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE). [Background technology]

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

[0003] I.TDP-43 Introduction The transactivation response (TAR) DNA-binding protein 43 kDa (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 noncoding, and exons 2–6 are protein-coding). TDP-43 belongs to the heterogeneous family of hnRNP (hnRNP) RNA-binding proteins (Wang et al., Trends in Molecular Medicine Vol. 14 No. 11, 2008, pp. 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): two RNA recognition motifs (RRM1 and RRM2) with highly conserved hexameric ribonucleoprotein 2 (RNP2) and octameric ribonucleoprotein 1 (RNP1) domains; a nuclear export signal (NES) and nuclear localization signal (NLS) that enable transport of bound mRNA between the nucleus and cytoplasm; and a C-terminal glycine-rich domain that mediates protein-protein interactions. TDP-43 is involved in multiple aspects of RNA processing, including transcription, splicing, transport, and stabilization (Buratti and Baralle, FEBS Journal 277 (2010) 2268-2281). It is a highly conserved, ubiquitously expressed protein with tightly self-regulated expression levels that continuously shuttles 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 (then termed 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 dominant-negative mutations in TDP-43, primarily located in the glycine-rich domain, have been identified in patients with sporadic and familial ALS and hereditary FTD (Fig. 1 in Lagier-Tourenne and Cleveland, Cell 136, 2009, 1001-1004). As demonstrated by sedimentation assays, TDP-43 is genetically prone to aggregation, and this tendency is further enhanced by several ALS-associated TARDBP mutations (Ticozzi et al., CNS Neurol. Disord. Drug Targets. 2010, 9(3), 285-296). TDP-43 aggregation is associated with clinical disease symptoms.

[0005] II. TDP-43 in Neurodegeneration TDP-43 aggregates have been shown to be 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) linked to chromosome 9p-linked dementias (e.g., those with progranulin (GRN) mutations, those with C9orf72 mutations, those with TARDBP mutations, those with valosin-containing protein (VCP) mutations, and those with valosin-containing protein (VCP) mutations). (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of 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-predominant age-related TDP-43 encephalopathy A growing list of neurodegenerative conditions, including but 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 sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofiber myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD) (Lagier-Tourenne 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 proteinopathies that may be associated with cognitive impairment, including hippocampal sclerosis, age-related hippocampal sclerosis, hippocampal sclerosis dementia, age-related brain TDP-43 with sclerosis (CARTS), and TDP-43 pathology in older adults (for reviews, see 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 C-terminal fragments (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). Another hallmark of TDP-43 pathogenesis is the redistribution and accumulation of TDP-43 from the nucleus to the cytoplasm. The hallmark lesions of FTLD-TDP are neuronal and glial cytoplasmic inclusions (NCIs and GCIs, respectively) and dystrophic neurites (DNs) that are immunoreactive for TDP-43, ubiquitin, and p62, but negative for other neurodegenerative disease-associated proteins. Differences in the morphology of the inclusions and their tissue distribution are associated with specific mutations and / or clinical manifestations. Four types of TDP-43 pathogenesis have been described so far, based on histological classification (Mackenzie and Neumann, J. Neurochem. (2016) 138 (Suppl. 1), 54-70). Type A FTLD-TDP cases are characterized by abundant short dystrophic neurites (DNs) and compact oval or crescent-shaped NCIs, primarily in layer II of the neocortex (Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70, Figure 2f). Cases with this etiology typically present clinically as either behavioral variant frontotemporal dementia (bvFTD) or non-fluent / agrammatic variant primary progressive aphasia (nfvPPA) and are associated with mutations in progranulin (GRN).Type B cases show a moderate number of compact or granular NCIs with relatively few DNs and NIIs (neuronal intranuclear inclusions, Figure 2g in Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70) in superficial and deep cortical layers. Most cases with co-occurrence of FTD and ALS symptoms are found to have type B FTLD-TDP pathology. Type C cases have abundant, long, and tortuous neurites, primarily in superficial cortical layers, with few or no NCIs (Figure 2j in Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70). This pathology is particularly found in cases presenting with the semantic variant of primary progressive aphasia (svPPA). Type D FTLD-TDP presents with abundant lenticular neuronal intranuclear inclusions (NIIs) and short DNs in the neocortex with only a few NCIs (Mackenzie et al., 2016 J. Neurochem. 138 (Suppl. 1), 54-70, Figure 2k). Type E is characterized by curvilinear oligodendrocyte inclusions in the white matter, as well as granular filamentous neuronal inclusions (GFNIs) and extremely fine, dot-like neuropil aggregates affecting all neocortical layers (Edward B. Lee et al., Acta Neuropathol. 2017 July ; 134(1): 65-78). This pattern of pathogenesis is found exclusively in VCP cases associated with inclusion body myositis.

[0007] III. TDP-43 in FTD Frontotemporal dementia (FTD) is a clinical term encompassing a wide spectrum of disorders based on degeneration of the frontal and temporal lobes, and the pathological features are named frontotemporal lobar degeneration (FTLD). FTD is the second most common cause of early degenerative dementia in the age group under 65 years (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), progressive supranuclear palsy syndrome, and motor neuron disease (FTD-MND), characterized by motor dysfunction. Clinical diagnosis of these syndromes is complex, and a definitive conclusion can only be reached through postmortem histopathological analysis to detect aggregated proteins and define the affected brain regions. With regard to pathological protein inclusions, approximately 45% of cases show pathological accumulations of misfolded tau, 45% of cases have pathological TDP-43, and a smaller subgroup has aggregates of FUS and other proteins.

[0008] IV. TDP-43 in ALS Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the premature loss of upper and lower motor neurons. ALS progression is characterized by fatal paralysis and respiratory failure, with a disease course of 1–5 years from diagnosis to death. In most cases of sporadic ALS, 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. ALS with dementia involves accumulation of TDP-43 in the extramotor neocortex and hippocampus. The role of TDP-43 phosphorylation in ALS patients has been explored with the help of antibodies that specifically bind to phosphorylated TDP-43 in nuclear and cytoplasmic inclusions 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).

[0009] V. TDP-43 in AD and other diseases TDP-43 pathology occurs in up to 57% of brains of patients with Alzheimer's disease (Josephs KA et al., Acta Neuropathol. 2014; 127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014; 127(3): 441-450; McAleese et al., Brain Pathol. 2017 Jul; 27(4): 472-479). TDP-43 aggregation is associated with patient age and correlates with cognitive decline, memory loss, and medial temporal lobe atrophy in AD. In AD, TDP-43 likely represents a secondary or independent etiology, sharing an overlapping brain distribution with amyloid beta and tau pathology in the medial temporal lobe. Pathogenic TDP-43 follows a stereotypical pattern of progressive deposition described by the so-called "TDP-43 in AD" (TAD) staging scheme, in which TDP-43 is initially deposited in the amygdala (Stage I), followed by the hippocampus, limbic, temporal, and finally fronto-striatal regions (Stage V) (Josephs KA et al., Acta Neuropathol. 2014;127(6): 811-824; Josephs KA et al., Acta Neuropathol. 2014;127(3): 441-450).

[0010] VI. TDP-43 Spread Although the onset and initial symptoms of ALS and FTD vary significantly among patients, a common feature of disease progression is the spread of lesions from the initial focal area to the majority of neurons. The continued worsening of symptoms may be explained by the continued spread of TDP-43 lesions. TDP-43 lesions in the brains of ALS patients appear to spread via a four-step process, with propagation occurring transsynaptically via cortico-subduction axonal projections via anterograde axonal transport (Brettschneider et al., Ann Neurol. 2013 July; 74(1): 20-38). Recent experimental evidence supports the hypothesis of amyloid beta, tau, alpha-synuclein, and TDP-43 protein propagation in neuronal tissues via a prion-like mechanism (Hasegawa et al., 2017), with clearly distinct initiation points and topographical 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 the intercellular spread of pathogenic protein aggregates. This mechanism consists of release of aggregates from diseased cells, uptake by naive cells, and seeding of pathogenic protein conformations through templated conformational changes of endogenous proteins. Pathogenic TDP-43 that can induce aggregation of physiological (i.e., non-pathogenic) TDP-43 is defined as TDP-43 with seeding capacity. Indeed, TDP-43 has been found to misfold, aggregate, and become seeds that are propagators capable of inducing de novo misfolding, a "prion-like" paradigm that is believed to be one of the key elements in disease progression.The intercellular spread of TDP-43 has been studied at the molecular level in a few in vitro models, where preparations of insoluble TDP-43 from patient brains can induce the formation of intracellular aggregates 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 association with exosomes prior to their spread to subsequent cells (Nonaka et al., Cell Reports 4 (2013, 124-134)). Similarly, adenoviral-mediated expression of TDP-43 leads to cytoplasmic aggregates that become phosphorylated and ubiquitinated and, more importantly, act as seeds to initiate cell-to-cell spread (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).

[0011] VII. Prevention and Treatment of TDP-43 Proteinopathies TDP-43 aggregation and pathogenic spread are key features of the currently incurable and fatal diseases ALS and FTD. Therefore, there is a need for new methods for treating and preventing TDP-43 proteinopathies. Mutations in TDP-43 are associated with familial cases of ALS and FTD, providing a causal link between TDP-43 misfolding and disease progression.

[0012] VIII. Diagnosis of TDP-43 Proteinopathy Diagnosis of FTD based on clinical signs is poor because clinical manifestations can overlap with other diseases, especially in the early stages.

[0013] Several approaches aim to develop biochemical biomarkers to distinguish between various forms of FTD pathogenesis. The development of antibodies against various conformations of TDP-43 may enable the generation of more sensitive and specific diagnostic tools. In parallel with biochemical biomarkers, the development of imaging biomarkers may enable early and specific detection of pathogenesis in TDP-43 proteinopathies. The ability to image TDP-43 deposition in the brain could be a substantial advance for diagnostics and drug development for TDP-43 proteinopathies. The use of cell-permeable antibody fragments could enable such detection.

[0014] The earliest event in various protein misfolding-based neurodegenerative diseases is the acquisition of an alternative conformation that renders the protein toxic. Furthermore, this misfolded conformation can self-propagate by recruiting endogenous normal proteins to the misfolded conformation, which serves as the mechanistic basis for the observed diffusion through affected tissues.

[0015] To develop antibodies against various conformational states of a given protein, researchers have designed supramolecular antigenic constructs that control the conformation of the presented antigen to generate conformation-specific antibodies against a given target in a specific conformational state (WO 2012 / 055933 and WO 2012 / 020124). Conformation-specific antibodies offer numerous advantages because they can distinguish between disease-associated conformations of these proteins and the functional endogenous conformation. This approach offers numerous advantages in therapeutic applications, as such antibodies target misfolded isoforms of disease-related proteins while being less likely to adsorb to proteins in their normal conformation. Similarly, for diagnostic applications, such antibodies recognize only the structural states of proteins associated with disease, which is important for the development of sensitive and specific diagnostics.

[0016] The use of TDP-43-based biomarkers in TDP-43 proteinopathies remains to be established, and such evaluation has been hampered in part by the lack of high-affinity antibodies that can be employed in suitable immunoassays for quantification of pathogenic TDP-43 in biological fluids (Feneberg et al., Molecular Neurobiology, 2018).

[0017] Thus, there is a clear need for biomarkers that can detect misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43, particularly in human samples, to diagnose various types of TDP-43 proteinopathies and / or monitor the effectiveness of therapeutic agents used to treat TDP-43-related, particularly TDP-43 aggregate-related, diseases, disorders, and disorders, or TDP-43 proteinopathies.

[0018] TDP-43 proteinopathies are defined as a set of neurodegenerative disorders characterized by pathogenic TDP-43.

[0019] IX. Prior art Patent application WO2008 / 151055 discloses methods and materials for determining whether a mammal has a neurodegenerative disease using levels of TDP-43 polypeptide and / or TDP-43 polypeptide cleavage products (e.g., 25 kD and 35 kD TDP-43 polypeptide cleavage products) in biological fluids.

[0020] Patent application WO2013 / 061163 discloses TDP-43-specific binding molecules including polypeptides such as human antibodies and fragments, derivatives, and variants thereof.

[0021] Patent application WO2020 / 234473 discloses specific binding molecules comprising polypeptides such as murine antibodies or antigen-binding fragments thereof.

[0022] The dosing regimen is an important element of a drug's target product profile. Because brain penetration of biologics is suboptimal, drug exposure is currently achieved only by repeated antibody administration. To ensure efficient drug distribution necessary for the antibody's desired pharmacological effect, a favorable pharmacokinetic profile is essential for its in vivo therapeutic application. For some antibodies, optimizing pharmacokinetic parameters such as clearance and / or volume of distribution can maximize drug exposure in vivo.

[0023] Antibodies are recycled through the interaction of their Fc domain with the fetal Fc receptor (FcRn). Increasing the affinity for FcRn can be achieved by protein engineering, resulting in improved recycling profiles and prolonged antibody half-lives. Several sets of mutations have been described, such as the YTE (Dall' Acqua et al. Journal of Immunology, 2002, 69:5171-5180) or LS (Zalevsky, J et al., Nat Biotechnol 28(2):157-9, 2010) mutations, which resulted in improved in vivo pharmacokinetic profiles. In some cases, high pI and positively charged patches have been found to adversely affect antibody clearance (Igawa et al., PEDS. 2010; vol. 23 no. 5 pp. 385-392, Bumbaca et al., JBC, VOL. 290, NO. 50, pp. 29732-29741, 2015). Summary of the Invention [Problem to be solved by the invention]

[0024] Currently, there are no TDP-43 antibody therapies on the market, and therefore there is a need for anti-TDP-43 antibodies with suitable pharmacokinetic parameters for in vivo therapeutic use. Without wishing to be bound by any particular theory, it is believed that the binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, contain a humanized acceptor framework that provides optimal half-life for in vivo therapeutic use. The binding molecules of the present invention (particularly hACI-7069-633B12-Ab1_H33L27 and hACI-7069-633B12-Ab1_H32L27) are shown herein to offer beneficial pharmacokinetic and pharmacodynamic properties. See Examples 5-14 and Tables 9, 10, and 11 herein. The binding molecules of the present invention exhibit suitable half-life and clearance values ​​for therapeutic use.

[0025] There is a need for humanized anti-TDP-43 binding molecules that bind to misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43 with suitable pharmacokinetic parameters for in vivo therapeutic use. Such humanized binding molecules, particularly antibodies and antigen-binding fragments thereof, can bind to specific epitopes of human TDP-43 (SEQ ID NO: 1). Furthermore, the development of sensitive and specific biomarkers that enable differentiation between pathogenic forms within the FTD spectrum is an urgent task. [Means for solving the problem]

[0026] The technical problem is solved by the embodiments provided herein.

[0027] The binding molecules of the present invention are humanized forms of mouse antibodies, particularly humanized forms of mouse monoclonal antibodies that bind to human TDP-43 (SEQ ID NO: 1). The antibody designated ACI-7069-633B12-Ab1 herein was selected for development of the humanized binding molecule. As described herein, this antibody is derived from hybridoma clone 633B12C8. It binds to an epitope within amino acids 397-411 of human TDP-43 (SEQ ID NO: 1). More specifically, it binds to an epitope derived from amino acids 400-405 of human TDP-43 (SEQ ID NO: 1). This antibody has (is encoded by) the VH nucleotide sequence set forth as SEQ ID NO: 28 and the VL nucleotide sequence set forth as SEQ ID NO: 29. See Table 10 of WO2020 / 234473. This antibody has the VH amino acid sequence set forth as SEQ ID NO: 20 and the VL amino acid sequence set forth as SEQ ID NO: 24. See Table 11 of WO2020 / 234473. This antibody has a VH CDR1 amino acid sequence set forth as SEQ ID NO:21, a VH CDR2 amino acid sequence set forth as SEQ ID NO:22, and a VH CDR3 amino acid sequence of ES. See Table 11 of WO2020 / 234473. This antibody has a VL CDR1 amino acid sequence set forth as SEQ ID NO:25, a VL CDR2 amino acid sequence set forth as SEQ ID NO:16, and a VL CDR3 amino acid sequence set forth as SEQ ID NO:27. See Table 11 of WO2020 / 234473.

[0028] The selected CDR sequences may be mutated at specific positions. In some embodiments, such mutations are made to avoid potential post-translational modification sites. In certain embodiments, one or more, up to all, of the following residues in the VH region (CDRH2) are mutated: N53, N54, and G55. Particular mutations include N53G, N53S, N53Q, N54Q, N54G, and G55A. Residues are numbered according to Kabat. In certain embodiments, one or more, up to all, of the following residues in the VL region (CDRL1 and / or CDRL2 and / or CDRL3) are mutated: K24, D28, G29, D55, S56, and W89. Residues are numbered according to Kabat. Particular mutations include K24R, D28E, D28G, G29A, D55E, S56A, W89Y, W89F, and W89L.

[0029] In certain embodiments, the humanized binding molecule, particularly the humanized antibody or antigen-binding fragment thereof of the invention, comprises framework sequences of human heavy chain variable domain subfamily 1. More specifically, the humanized binding molecule, particularly the humanized antibody or antigen-binding fragment thereof of the invention, comprises framework sequences of IGHV1-69-2 (IMGT accession numbers KF698734, Z29977, Z12305; UniProtKB A0A0G2JMI3), IGHV1-3 (IMGT accession numbers X62109, X62107, MK540645, MH779622, and MN337616; UniProtKB-A0A0C4DH29), IGHV1-2 (IMGT accession numbers X07448, X62106, X92208, KF698733, HM855674, MH267285, and MN337615; UniProtKB-P23083), and IGH The VH framework sequence may comprise a V1-46 (IMGT accession numbers X92343, J00240, L06612, and MK540650; UniProtKB-P01743), IGHV1-24 (IMGT accession number M99642; UniProtKB-A0A0C4DH33), IGHV5-51 (IMGT accession numbers M99686, M18806, X56368, X56367, Z27449, MK321694, IMGT000055; UniProtKB A0A0C4DH38), or IGHV3-43 (IMGT accession numbers M99672, HM855392; UniProtKB A0A0B4J1X8) VH framework sequence, preferably an IGHV1-69-2 VH framework sequence.

[0030] In certain embodiments, the humanized binding molecules, particularly the humanized antibodies or antigen-binding fragments thereof of the invention, comprise human light chain variable domain kappa subfamily 2 framework sequences. More specifically, the humanized binding molecules, particularly the humanized antibodies or antigen-binding fragments thereof of the invention, comprise human light chain variable domain kappa subfamily 2 framework sequences. IGKV2-30 (IMGT Accession Nos. X63403 and FM164408; UniProtKB-P06310), IGKV2-29 (IMGT Accession Nos. X63396, U41645, and AJ783437; UniProtKB-A2NJV5), IGKV2D-29 (IMGT Accession Nos. M31952 and U41644; UniProtKB-A0A075B6S2), IGKV2-28 (IMGT Accession No. X63397; UniProtKB-A0A0 75B6P5), IGKV2-24 (IMGT accession number X12684; UniProtKB-A0A0C4DH68), IGKV2D-28 (IMGT accession number X12691; UniProtKB-P01615), IGKV2-40 (IMGT accession numbers X59314 and X59317; UniProtKB-A0A087WW87), IGKV2D-40 (IMGT accession number X59311; UniProtKB-P01614), or IGKV4-1 (IMGT accession numbers Z00023 and MW316673; UniProtKB-P06312) The VL framework sequence may comprise, preferably, the IGKV2-40, IGKV2D-40, IGKV2D-28, IGKV4-1, or IGKV2-28 VL framework sequence, most preferably the IGKV2-28 (IMGT accession number X63397; UniProtKB-A0A075B6P5) VL framework sequence.

[0031] In one embodiment, a humanized binding molecule of the invention, particularly a humanized antibody or antigen-binding fragment thereof of the invention, comprises IGHV1-69-2 and IGKV2-28 framework sequences.

[0032] Selected framework sequences may be mutated at specific positions. In some embodiments, such mutations are made to favorably affect the CDR loop conformation and / or variable domain packing between the VH and VL domains. In certain embodiments, one or more, up to all, of the residues listed in Table 2 below according to Kabat numbering are mutated. In certain embodiments, one or more, up to all, of the following VH mutations are made according to Kabat numbering: V24T, Y27F, M48I, Q64K, A71V, T73K, T94R. In certain embodiments, one or more, up to all, of the following VL mutations are made (according to Kabat numbering): I2V, Y36L, Q45K, L46R, G57R. In certain embodiments, one or more, up to all of the following VH mutations are made: V24T, Y27F, M48I, Q64K, A71V, T73K, T94R, and one or more, up to all of the following VL mutations are made: I2V, Y36L, Q45K, L46R, G57R, all according to Kabat numbering.

[0033] Thus, the present invention relates to humanized binding molecules, particularly humanized antibodies and antigen-binding fragments thereof, that specifically recognize misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43. In the present invention, misfolded TDP-43 includes misfolded monomeric, misfolded oligomeric, misfolded aggregated, and / or post-translationally modified and / or misfolded truncated TDP-43. Post-translationally modified TDP-43 includes phosphorylated, ubiquitinated, acetylated, sumoylated, and / or methylated TDP-43. Physiological TDP-43 includes soluble nuclear TDP-43. It is demonstrated herein that the humanized 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 humanized binding molecules, particularly humanized antibodies or antigen-binding fragments thereof, that specifically recognize misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43. Such binding molecules are referred to herein as humanized "pan-TDP-43" binding molecules, particularly humanized pan-TDP-43 antibodies. As described herein, the humanized 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 bind to one over the other. The present invention also provides humanized binding molecules, particularly humanized antibodies or antigen-binding fragments thereof, for preventing, ameliorating, treating, and / or diagnosing diseases, disorders, and disorders associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies. The present invention also provides humanized binding molecules, particularly humanized antibodies or antigen-binding fragments thereof, for detecting and / or understanding (i.e., identifying) specific types of pathogenesis that cause neurodegeneration. The use of these molecules as diagnostic biomarkers is also envisioned, enabling more efficient and accurate subject selection for long-term monitoring in clinical studies and supporting the development of novel therapies for TDP-43 proteinopathies.

[0034] The present invention also provides a humanized TDP-43-binding molecule, particularly a humanized antibody or an antigen-binding fragment thereof, as a pharmaceutical (therapeutic agent).

[0035] Without wishing to be bound by theory, the present invention was developed based on the assumption that modified conformation-specific antigenic peptides and peptide fragments derived from the TDP-43 protein or the entire TDP-43 protein, and humanized antibodies obtained by using said peptides or fragments or the entire TDP-43 protein as antigens, block the cell-to-cell spread of TDP-43, disaggregate TDP-43 aggregates, block the seeding of TDP-43, neutralize TDP-43 capable of seeding, inhibit the aggregation of the TDP-43 protein or fragments thereof, and / or promote the clearance of TDP-43. The humanized binding molecules of the present invention, particularly humanized polypeptides, more particularly humanized antibodies or antigen-binding fragments thereof, bind to misfolded aggregated TDP-43, particularly cytoplasmic and extracellular misfolded TDP-43. The humanized binding molecules of the present invention, particularly humanized polypeptides, more particularly humanized antibodies or antigen-binding fragments thereof, bind to full-length TDP-43 and / or truncated TDP-43. In one embodiment, the humanized binding molecules of the present invention, particularly humanized polypeptides, more particularly humanized antibodies or antigen-binding fragments thereof, specifically bind to cytoplasmic misfolded TDP-43. In one embodiment, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, bind to and neutralize TDP-43 with dissemination capacity. In one embodiment, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, bind to extracellular and / or aggregated TDP-43 and promote clearance of TDP-43 by immune cells, such as microglia, via antibody-dependent cellular phagocytosis (ADCP).

[0036] Misfolded, aggregated TDP-43, or pathogenesis-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-inclusion bodies, as well as in TDP-43-immunoreactive neuronal and glial cytoplasmic inclusions (NCIs and GCIs, respectively), neuronal intranuclear inclusions (NIIs), and dystrophic neurites (DNs).

[0037] Non-aggregated physiological TDP-43 is a physiologically functional TDP-43 protein that is located primarily in the nucleus and shuttles to the cytoplasm, ready to exhibit its desired functions in the cellular environment in vivo.

[0038] The humanized TDP-43 binding molecules of the present invention, in particular humanized anti-TDP-43 antibodies or antigen-binding fragments thereof, surprisingly have at least one, preferably two, more preferably three, even more preferably four, even more preferably five, even more preferably six, and most preferably all seven of the following properties: - Blocking cell-to-cell transmission of TDP-43; - disaggregating TDP-43 aggregates; - inhibiting the aggregation of TDP-43 protein or fragments thereof; - Blocking TDP-43 seeding; - Neutralizing the seeding-capable TDP-43; - Blocking the spread of TDP-43; and - Facilitate clearance of TDP-43.

[0039] Regardless of the combination of one, two, three, four, five, six, or seven of the properties listed above, the humanized anti-TDP-43 binding molecule, preferably the humanized anti-TDP-43 antibody or antigen-binding fragment thereof of the invention, may ameliorate / inhibit / reduce the production of TDP-43 pathology in an in vivo model of TDP-43 proteinopathy, and more importantly in patients with TDP-43 pathology.

[0040] The humanized anti-TDP-43 binding molecule binds to a region within amino acids 397-411 of human TDP-43 (SEQ ID NO: 1), and more specifically, the humanized TDP-43 binding molecule binds to an epitope within amino acid residues 400-405 of human TDP-43 (SEQ ID NO: 1).

[0041] According to the present invention, a. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; b. A VH-CDR2 selected from the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 102, SEQ ID NO: 112, or SEQ ID NO: 122; c. VH-CDR3 containing the amino acid sequence ES (Glu-Ser); d. A VL-CDR1 selected from the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; e. A VL-CDR2 selected from the amino acid sequence of SEQ ID NO: 16; f. A VL-CDR3 selected from the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87: and g. an acceptor framework for a heavy chain variable domain (VH) selected from the group consisting of IGHV1-69-2, IGHV5-51, or IGHV3-43, preferably IGHV1-69-2; and / or h. An acceptor framework for the light chain variable domain (VL) selected from the group consisting of IGKV2-28, IGKV2-24, IGKV2D-28, IGKV2-40, IGKV2D-40, or IGKV4-1, preferably IGKV2-28. The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0042] In a preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.M48I; c.A71V; d.T73K; and e.T94R and / or the following VL mutations: f.I2V; g.Y36L; h.Q45K; i.L46R; and j.G57R and an acceptor framework for VL comprising one or more of: (numbering according to Kabat).

[0043] In a preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.Y27F; c.M48I; d.A71V; e.T73K; and f.T94R and / or the following VL mutations: g.I2V; h.Y36L; i.Q45K; j.L46R; and k.G57R and an acceptor framework for VL comprising one or more of: (numbering according to Kabat).

[0044] In a more preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.M48I; c.A71V; d.T73K; e.T94R and / or the following VL mutations: f.Y36L; g.L46R; and h.G57R The acceptor framework of the VL comprises one or more, preferably all, of the following (numbering according to Kabat):

[0045] In a more preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.Y27F; c.M48I; d.A71V; e.T73K; and f.T94R and / or the following VL mutations: i.Y36L; j.L46R; and k.G57R and VL acceptor frameworks comprising one or more, preferably all, of the following (numbering according to Kabat):

[0046] In a preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.M48I; c.A71V; d.T73K; and e.T94R and the following VL mutations: f.Y36L; g.L46R; and h.G57R and VL acceptor frameworks comprising one or more, preferably all, of the following (numbering according to Kabat):

[0047] In a preferred embodiment, the humanized TDP-43 binding molecule comprises the following VH mutations: a.V24T; b.Y27F; c.M48I; d.A71V; e.T73K; and f.T94R and the following VL mutations: i.Y36L; j.L46R; and k.G57R and VL acceptor frameworks comprising one or more, preferably all, of the following (numbering according to Kabat):

[0048] The humanized TDP-43 binding molecule, particularly the humanized TDP-43 antibody or antigen-binding fragment thereof, a. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; b. A VH-CDR2 selected from the amino acid sequence of SEQ ID NO: 112, SEQ ID NO: 122, or SEQ ID NO: 102; c. VH-CDR3 containing the amino acid sequence ES (Glu-Ser); d. A VL-CDR1 selected from the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; e. A VL-CDR2 selected from the amino acid sequence of SEQ ID NO: 16; and f. A VL-CDR3 selected from the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87 may include:

[0049] In a preferred embodiment, the humanized TDP-43 binding molecule, particularly the humanized TDP-43 antibody or antigen-binding fragment thereof, a. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; b. A VH-CDR2 selected from the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 122; c. VH-CDR3 containing the amino acid sequence ES (Glu-Ser); d. A VL-CDR1 selected from the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; e. A VL-CDR2 selected from the amino acid sequence of SEQ ID NO: 16; and f. A VL-CDR3 selected from the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87 Includes.

[0050] In some embodiments, the humanized TDP-43 binding molecule of the invention comprises: - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; - VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 Includes.

[0051] The present invention relates, inter alia, to (i) immunoconjugates comprising humanized TDP-43-binding molecules, (ii) labeled antibodies comprising humanized TDP-43-binding molecules, (iii) pharmaceutical compositions comprising humanized TDP-43-binding molecules and a pharmaceutically acceptable carrier and / or excipient and / or diluent, (iv) humanized TDP-43-binding molecules for human or veterinary use, and (v) humanized TDP-43-binding molecules for use in preventing, ameliorating, or treating TDP-43-associated diseases, disorders, and / or disorders, or TDP-43 proteinopathies. TDP-43 binding molecules, (vi) humanized TDP-43 binding molecules for diagnostic uses (particularly for in vivo diagnosis, but also for in vitro testing), (vii) humanized TDP-43 binding molecules for research uses, particularly as analytical tools or reference molecules, (viii) humanized TDP-43 binding molecules for use as diagnostic tools for monitoring TDP-43-related diseases, disorders, and / or abnormalities, or TDP-43 proteinopathies, (ix) treating individuals with humanized TDP-43 binding molecules (xii) recombinant expression vectors comprising the nucleic acid molecules of the invention; (xiii) host cells comprising the nucleic acids and / or vectors of the invention; (xiv) cell-free expression systems containing the recombinant expression vectors of the invention; (xv) methods of producing the humanized TDP-43 binding molecules; (xvi) methods of quantifying TDP-43 in a sample obtained from a subject using the humanized TDP-43 binding molecules; and (xvii) kits comprising the humanized TDP-43 binding molecules of the invention and / or nucleic acids, expression vectors, host cells, and / or cell-free expression systems for producing the same.

[0052] The humanized TDP-43-binding molecules of the present invention, particularly humanized anti-TDP-43 antibodies or antigen-binding fragments thereof, can recruit and / or activate microglia. More specifically, the humanized TDP-43-binding molecules of the present invention can affect the morphology of microglia in terms of cell size and activation state. This may contribute to the reduced pathogenicity of TDP-43 demonstrated by the TDP-43-binding molecules of the present invention.

[0053] In the present invention, humanized binding molecules, particularly humanized antibodies or antigen-binding fragments thereof, specifically recognize TDP-43. Humanized binding molecules of the present invention include humanized polypeptides and / or humanized antibodies and / or antigen-binding fragments thereof specific for the TDP-43 protein. "Specifically recognizing TDP-43" means that the humanized binding molecules of the present invention specifically, generally, and collectively bind to TDP-43, particularly to some epitopes in TDP-43, particularly to exposed / accessible epitopes in one or more pathogenic conformations of the TDP-43 protein, with greater affinity than other epitopes. Humanized binding molecules of the present invention, particularly humanized polypeptides, more particularly humanized antibodies or antigen-binding fragments thereof, that specifically bind to TDP-43 specifically recognize misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43.

[0054] The humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, bind to both unaggregated physiological TDP-43 and aggregated TDP-43. That is, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, can bind approximately equally well to soluble and aggregated TDP-43. The humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, can bind approximately equally to aggregated TDP-43 compared to unaggregated TDP-43. More specifically, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, can bind approximately equally to aggregated TDP-43 in the cytoplasm compared to unaggregated TDP-43 in the nucleus. In other embodiments, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, can preferentially bind to aggregated TDP-43 compared to unaggregated TDP-43, but bind to both species. More specifically, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, may preferentially bind to aggregated TDP-43 in the cytoplasm compared to unaggregated TDP-43 in the nucleus, but bind to both species. Alternatively, in other embodiments, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, may preferentially bind to unaggregated TDP-43 compared to aggregated TDP-43, but bind to both species. More specifically, the humanized TDP-43-binding molecules of the present invention, particularly humanized antibodies or antigen-binding fragments thereof, may preferentially bind to unaggregated TDP-43 in the nucleus compared to aggregated TDP-43 in the cytoplasm, but bind to both species. These binding properties can be demonstrated, for example, using immunohistochemistry.

[0055] In some embodiments, the present invention encompasses humanized binding molecules, particularly the humanized antibodies and antigen-binding fragments thereof of the present invention described herein that specifically bind to TDP-43, and the use of these humanized binding molecules for diagnosing, preventing, ameliorating, and / or treating TDP-43-related, particularly TDP-43 aggregate-related, diseases, disorders, and / or conditions, or TDP-43 proteinopathies, including, but not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE). The methods and compositions disclosed herein have applications in the diagnosis, prevention, amelioration, and / or treatment of TDP-43-related, particularly TDP-43 aggregate-related, diseases, disorders, and / or abnormalities, or TDP-43 proteinopathies, including, but not limited to, frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Preferably, the use of these humanized binding molecules for diagnosing, preventing, ameliorating, and / or treating TDP-43-related, particularly TDP-43 aggregate-related, diseases, disorders, and / or abnormalities, or TDP-43 proteinopathies, is directed toward amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), or frontotemporal dementia (FTD). More preferably, the use is directed toward amyotrophic lateral sclerosis (ALS). More preferably, the use is directed toward Alzheimer's disease (AD). More preferably, the use is directed toward frontotemporal dementia (FTD).

[0056] In another embodiment, the humanized TDP-43 binding molecule, particularly the humanized anti-TDP-43 antibody or antigen-binding fragment thereof described herein specific for TDP-43, is used to treat frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), cerebrocortical fibrosis, cerebrospinal fluid syndrome (CF), cerebrospinal fluid syndrome (SFS), cerebrospinal fluid syndrome (CF ... Substantia basalis degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of 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-predominant age-related TDP-43 LATE encephalopathy, chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (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 valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), limbic micromyelopathy, and cerebrospinal fluid disorders. The sample is contacted with a sample to detect, diagnose, and / or monitor a disease, disorder, and / or abnormality associated with TDP-43, particularly TDP-43 aggregate-associated, or TDP-43 proteinopathy, selected from oculopharyngeal muscular dystrophy with cytoplasmic vesicles, myofibrillar myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD).

[0057] In one embodiment, the present invention encompasses the use of humanized binding molecules, particularly the humanized antibodies or antigen-binding fragments thereof described herein that specifically bind to TDP-43, and these humanized binding molecules, particularly these humanized antibodies, to detect the presence of TDP-43 in a sample. Thus, the humanized TDP-43-binding molecules of the present invention, such as the humanized anti-TDP43 antibodies described herein, can be used to screen clinical samples, particularly human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF), and / or urine, for the presence of TDP-43 in a sample, for example, by using an ELISA-based assay or a surface-compatible assay. In some situations, tissue samples, such as brain tissue samples, may also be used. The methods and compositions of the present invention also have applications in presymptomatic disease diagnosis and / or monitoring disease progression and / or treatment effectiveness.According to some embodiments, a humanized antibody specific for TDP-43 (e.g., a full-length humanized antibody or a fragment or derivative of a humanized antibody that binds to TDP-43) is used to treat frontotemporal dementia (e.g., sporadic or familial dementia with or without motor neuron disease (MND) linked to chromosome 9p, with mutations in progranulin (GRN), with mutations in C9orf72, with mutations in TARDBP, with mutations in valosin-containing protein (VCP)). FTD, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of 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-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine 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 valsin-containing protein (VCP); The humanized TDP-43-binding molecules of the present invention are contacted with a sample (e.g., blood, urine, cerebrospinal fluid (CSF), interstitial fluid (ISF), or brain tissue) to detect, diagnose, and / or monitor diseases such as 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 in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD). The humanized TDP-43-binding molecules of the present invention can be used to quantify TDP-43 in suitable samples, particularly clinical samples such as blood, brain tissue, CSF, ISF, or urine, 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), 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. Increasing levels over time may indicate disease progression. Decreasing levels over time may indicate disease regression. Methods may also be performed to monitor therapy, particularly the effectiveness of a particular treatment. Successful treatment can be measured in relation to stabilization or reduction in TDP-43 levels following treatment. It has been demonstrated in Example 12 of WO2020 / 234473 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 or separately from the test samples. In some embodiments, the control level is determined from a series of control samples taken from healthy subjects under similar or identical experimental conditions and used as a comparison control for the levels determined in the test sample. The method of quantifying TDP-43 in a suitable sample using the humanized binding molecules of the present invention may also be used to select a therapy (for further treatment of the subject). That is, a personalized treatment method is envisioned. Samples are taken before and after treatment. If treatment with the therapy results in a stable or preferably decreasing TDP-43 level after treatment, the therapy may be selected for the subject. If the therapy does not result in a stable or preferably decreasing TDP-43 level after treatment, the therapy is not selected for the subject. The therapy may be any suitable candidate therapeutic agent for the treatment of a TDP-43 proteinopathy. In a preferred embodiment, the therapy comprises a humanized TDP-43-binding molecule of the present invention, typically in the form of a pharmaceutical composition as described herein.

[0058] The humanized TDP-43-binding molecules of the present invention may also be used to classify diseases into specific types or subtypes. a. carrying out a method of the invention in which the level of TDP-43 is quantified relative to a suitable control; b. Identifying mutations in a sample from the subject, including, but not limited to, mutations in progranulin (GRN), mutations in C9orf72, mutations in TARDBP, mutations in angiogenin (ANG), mutations in valosin-containing protein (VCP), mutations in the myotilin (MYOT) gene, and mutations in the gene encoding desmin (DES), as appropriate; and c. Classifying diseases, disorders, and / or disorders associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies A method is provided for classifying diseases, disorders, and / or abnormalities associated with TDP-43, particularly associated with TDP-43 aggregates, or for classifying TDP-43 proteinopathies, comprising:

[0059] Similarly, there is provided a method for classifying a TDP-43-associated, particularly TDP-43 aggregate-associated, disease, disorder, and / or disorder, or a TDP-43 proteinopathy, comprising performing a method of the invention to quantify the level of TDP-43 in a sample obtained from a subject having the TDP-43-associated disease, disorder, and / or disorder, or TDP-43 proteinopathy, where the level is compared with control samples taken from subjects having a different type or subtype of the TDP-43-associated, particularly TDP-43 aggregate-associated, disease, disorder, and / or disorder, or TDP-43 proteinopathy (i.e., a representative set of control levels is determined for the type or subtype of interest), and classifying the TDP-43-associated, particularly TDP-43 aggregate-associated, disease, disorder, and / or disorder, or TDP-43 proteinopathy based on the comparison. That is, the classification is based on determining the closest match between the test sample and one or more of the control samples. These methods may further include identifying mutations in the sample, including but not limited to mutations in progranulin (GRN), mutations in C9orf72, mutations in TARDBP, mutations in angiogenin (ANG), mutations in valosin-containing protein (VCP), mutations in the myotilin (MYOT) gene, and mutations in the gene encoding desmin (DES). The identified mutations may also be used to classify diseases, disorders, and / or disorders associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies. For the avoidance of doubt, identification of mutations in the sample may be performed by any suitable method, for example, based on nucleic acid sequencing of nucleic acid molecules in the sample. The sample may be separated and distinct from the sample in which the TDP-43 level was determined, but is a sample from the same subject.

[0060] In other embodiments, the present invention provides methods for preventing, ameliorating, and / or treating diseases, disorders, and / or conditions associated with TDP-43, particularly those associated with TDP-43 aggregates, or TDP-43 proteinopathies. According to one embodiment, the methods of the present invention comprise administering to a subject an effective concentration of a humanized binding molecule, particularly a humanized antibody of the present invention specific for TDP-43 described herein (e.g., a full-length antibody or a fragment or derivative of an antibody that binds to TDP-43). In another embodiment, the present invention provides methods for preventing, ameliorating, and / or treating TDP-43 proteinopathies. According to some embodiments, a humanized binding molecule, particularly a humanized antibody of the present invention or an antigen-binding fragment thereof specific for TDP-43 described herein, is administered to treat, ameliorate, and / or prevent frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). In another embodiment, the humanized binding molecule, particularly the humanized antibody or antigen-binding fragment thereof described herein specific for TDP-43, is administered to prevent, ameliorate, and / or treat a neurodegenerative disease selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD, including sporadic and familial forms of AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0061] In another embodiment, the humanized binding molecule, particularly the humanized antibody or antigen-binding fragment thereof described herein specific for TDP-43, is used to treat frontotemporal dementia (e.g., sporadic or familial dementia with or without motor neuron disease (MND), including those linked to chromosome 9p, with mutations in progranulin (GRN), with mutations in C9orf72, with mutations in TARDBP, with mutations in valosin-containing protein (VCP), FTD, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (e.g., sporadic ALS, ALS with TARDBP mutations, ALS with angiogenin (ANG) mutations), alkene-like neuropathy (ALS), and cerebrospinal fluid syndrome (ALS). Sander's disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine 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, valosin-containing protein (( The compound is administered to prevent, ameliorate, and / or treat a disease selected from inclusion body myopathy with mutations in the gene encoding myotilin (VCP); also Paget's disease of bone and frontotemporal dementia (also Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofiber myopathy with mutations in the gene encoding myotilin (MYOT) or desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD). DETAILED DESCRIPTION OF THE INVENTION

[0062] X.Definition As used herein, an "antigen-binding molecule" refers to any molecule capable of specifically or selectively binding to an antigen, particularly TDP-43. The binding molecule includes or is an antibody or fragment thereof. An anti-TDP-43 binding molecule is a molecule that binds to the TDP-43 protein at a specific recognition site, i.e., epitope, such as an anti-TDP-43 antibody or fragment thereof. That is, the antigen-binding molecule of the present invention binds to an epitope within the amino acid sequence of SEQ ID NO: 1. The antigen-binding molecules provided herein, particularly antibodies or antigen-binding fragments thereof, recognize full-length TDP-43. Other anti-TDP-43 binding molecules may also include multivalent molecules, multispecific molecules (e.g., diabodies), fusion molecules, aptamers, avimers, or other naturally occurring or recombinantly produced molecules. Illustrative antigen-binding molecules useful in the present invention include antibody-like molecules. Antibody-like molecules are molecules that can exhibit a function by binding to a target molecule (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), and include, for example, DARPins (WO2002 / 020565), Affibodies (WO1995 / 001937), Avimers (WO2004 / 044011; WO2005 / 040229), Adnectins (WO2002 / 032925), and fynomers (WO2013 / 135588).

[0063] As used herein, the terms "anti-TDP-43 antibody" and "antibody that binds to TDP-43," or simply "antibody," refer to an antibody that can bind to TDP-43 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent targeting TDP-43. In general, the term "antibody" is used in the broadest sense herein and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific or biparatopic antibodies), fully human antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. The antibodies of the present invention may be chimeric antibodies, recombinant antibodies, antigen-binding fragments of recombinant antibodies, humanized antibodies, or antibodies displayed on the surface of phage or chimeric antigen receptor (CAR) T cells.

[0064] An "antigen-binding fragment" of an antibody or a "functional fragment thereof" refers to a molecule other than an intact or full-length antibody that comprises 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-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Antigen-binding fragments are also referred to as "functional fragments" because they retain the binding function of the original antibody from which they are derived.

[0065] An "antibody that binds to an epitope" within a defined region of a protein is an antibody that requires the presence of one or more amino acids within that region in order to bind to that protein.

[0066] In certain embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutational analysis in which amino acids in the protein are mutated and it is determined that antibody binding to the resulting altered protein (e.g., an altered protein containing the epitope) is at least 20% of binding to the unaltered protein. In some embodiments, an "antibody that binds to an epitope" within a defined region of a protein is identified by mutational analysis in which amino acids in the protein are mutated and it is determined that antibody binding to the resulting altered protein (e.g., an altered 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 binding to the unaltered protein. In certain embodiments, antibody binding is determined by FACS, WB, or a suitable binding assay, such as ELISA.

[0067] The term "binding to" as used in the context of the present invention defines the mutual binding (interaction) of at least two "antigen-interaction sites." The term "antigen-interaction site," according to the present invention, defines a polypeptide motif, i.e., a portion of the antibody or antigen-binding fragment of the present invention, which exhibits the ability of specific interaction with a specific antigen or a specific group of antigens of TDP-43. Said binding / interaction is also understood to define "specific recognition." The term "specifically recognizing," according to the present invention, means that the antibody is capable of specifically interacting / binding with at least two amino acids of TDP-43 as defined herein, in particular with at least two amino acids within amino acid residues 397-411 of human TDP-43 (SEQ ID NO: 1), and even more particularly with at least two amino acids within amino acid residues 400-405, 400-406, or 400-412 of human TDP-43 (SEQ ID NO: 1).

[0068] The term "pan-TDP-43 antibody" refers to an antibody that binds to misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43, including monomeric TDP-43, oligomeric TDP-43, post-translationally modified TDP-43 (e.g., phosphorylated, ubiquitinated, acetylated, sumoylated, and / or methylated), aggregated TDP-43, and cleaved TDP-43.

[0069] The term "specific interaction" as used in accordance with the present invention means that the antibody or antigen-binding fragment thereof of the present invention does not cross-react or essentially does not cross-react with (poly)peptides of similar structure. Thus, the antibody or antigen-binding fragment thereof of the present invention specifically binds / interacts with the structure of TDP-43 formed by a specific amino acid sequence within amino acid residues 397-411 of human TDP-43 (SEQ ID NO: 1), and more specifically, binds / interacts with the structure of TDP-43 formed by a specific amino acid sequence within amino acid residues 400-405, 400-406, or 400-412 of human TDP-43 (SEQ ID NO: 1).

[0070] The cross-reactivity of a panel of antigen-binding molecules, particularly antibodies or antigen-binding fragments thereof, under consideration can be tested, for example, by assessing the binding of said panel of antibodies or antigen-binding fragments thereof to the (poly)peptide of interest and several more or less closely (structurally and / or functionally) related (poly)peptides under conventional conditions (see, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988) and Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1999)). Only constructs (i.e., antibodies, antigen-binding fragments thereof, etc.) that bind to a certain structure of TDP-43 defined herein, e.g., a specific epitope or (poly)peptide / protein of TDP-43 defined herein, but do not or essentially do not bind to any other epitope or (poly)peptide of the same TDP-43, are considered specific for the epitope or (poly)peptide / protein of interest and are selected for further study by the methods provided herein. These methods may include, inter alia, binding studies, blocking and competition studies with structurally and / or functionally closely related molecules, including FACS analysis, surface plasmon resonance (SPR, e.g., by BIACORE™), analytical centrifugation, isothermal titration calorimetry, fluorescence anisotropy, fluorescence spectroscopy, or radiolabeled ligand binding assays.

[0071] Thus, specificity can be determined experimentally by methods known in the art and described herein, including but not limited to Western blot, ELISA-, RIA-, ECL-, IRMA-tests, and peptide scans.

[0072] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture and are essentially uncontaminated by other immunoglobulins. The modified "monoclonal" indicates the nature of the antibody as being within a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. As noted above, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method described by Kohler, Nature 256 (1975), 495.

[0073] As used herein, the term "polyclonal antibody" refers to an antibody produced among or in the presence of one or more other non-identical antibodies. Generally, polyclonal antibodies are produced from a B lymphocyte in the presence of several other B lymphocytes that produce non-identical antibodies. Usually, polyclonal antibodies are obtained directly from immunized animals.

[0074] As used herein, the term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. A fully human antibody may contain mouse carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, a "mouse antibody" or "murin antibody" refers to an antibody that contains only mouse / murin immunoglobulin protein sequences. Alternatively, a "fully human antibody" may contain rat carbohydrate chains if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell. Similarly, the term "rat antibody" refers to an antibody that contains only rat immunoglobulin protein sequences. Fully human antibodies may also be produced, for example, by phage display, a widely used screening technology that allows for 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. Patent Nos. 5,403,484, 5,969,108, and 5,885,793. Another technology that allows for the development of fully human antibodies involves modifications of mouse hybridoma technology. Mice are transgenic to contain human immunoglobulin loci in place of their own genes (see, eg, US Pat. No. 5,877,397).

[0075] The term "chimeric antibody" refers to an antibody comprising a variable region of the present invention fused or chimerized with an antibody region (e.g., constant region) from another human or non-human species (e.g., mouse, horse, rabbit, dog, cow, chicken).

[0076] The term "antibody" also relates to recombinant human antibodies, xenoantibodies, and heterohybrid antibodies. The term "recombinant (human) antibody" includes all human sequence antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes; antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant combinatorial human antibody libraries, or antibodies prepared, expressed, created, or isolated by any other means, including splicing of human immunoglobulin gene sequences into 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 when animals transgenic for human Ig sequences are used) and therefore the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that do not naturally occur within the human antibody germline repertoire in vivo.

[0077] A "heterologous antibody" is defined in relation to the transgenic non-human organism producing such an antibody. This term refers to an antibody that has an amino acid sequence corresponding to, or encoding a nucleic acid sequence for, an antibody found in an organism not comprised of a transgenic non-human animal, and that is generally derived from a species other than that of the transgenic non-human animal.

[0078] The term "heterohybrid antibody" refers to an antibody having light and heavy chains of different organismal origin. For example, an antibody having a human heavy chain in combination with a murine light chain is a heterohybrid antibody. Examples of heterohybrid antibodies include chimeric antibodies and humanized antibodies.

[0079] The present invention specifically relates to humanized antibodies. "Humanized" forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, i.e., immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are often human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. Thus, when a particular human framework sequence, such as an IGHV1-3 VH framework sequence, is referred to herein, it is intended to encompass not only the germline sequence but also mutated versions. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are human immunoglobulin consensus sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a 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. For a description of antibody humanization methods, including specific mutations, that can be employed in accordance with the present invention, see Example 1.

[0080] A well-known method for antibody humanization involves CDR grafting, in which a functional antigen-binding site from a non-human "donor" antibody is grafted onto a human "acceptor" antibody. CDR grafting methods are known in the art and are described, for example, in U.S. Pat. Nos. 5,225,539, 5,693,761, and 6,407,213. Another related method is the production of humanized antibodies from transgenic animals that have been genetically engineered to contain one or more humanized immunoglobulin loci that are capable of undergoing gene rearrangement and gene conversion (see, e.g., U.S. Pat. No. 7,129,084).

[0081] Thus, in the context of the present invention, the term "antibody" relates to complete immunoglobulin molecules as well as to portions of such immunoglobulin molecules (i.e., "antigen-binding fragments thereof"). Furthermore, the term relates to engineered and / or modified antibody molecules, as discussed above. The term also relates to recombinantly or synthetically produced / synthesized antibodies. The term also relates to intact antibodies as well as antibody fragments thereof, such as separated light and heavy chains, Fab, Fv, Fab', Fab'-SH, F(ab')2. The term "antibody" also includes, but is not limited to, fully human antibodies, chimeric antibodies, humanized antibodies, CDR-grafted antibodies, and antibody constructs, such as single-chain Fv (scFv) or antibody fusion proteins.

[0082] "Single-chain Fv" or "scFv" antibody fragments, in the context of the present invention, comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. Techniques described for the production of single-chain antibodies are described, for example, in Plueckthun in The Pharmacology of Monoclonal Antibodies, Rosenburg and Moore eds. Springer-Verlag, NY (1994), 269-315.

[0083] As used herein, a "Fab fragment" consists of one light chain and the CH1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0084] The "Fc" region comprises two heavy chain fragments containing the CH2 and CH3 domains of an antibody, held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domains.

[0085] A "Fab'" fragment contains one light chain and a portion of one heavy chain containing the VH domain, the CH1 domain, and also the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0086] "F(ab')2 fragment" refers to a fragment of two light chains and a C H 1 Domain and C H It contains two heavy chains with a portion of the constant region between the two domains, thereby forming an interchain disulfide bond between the two heavy chains, i.e., an F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0087] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0088] The humanized antibodies, humanized antibody constructs, humanized antibody fragments, humanized antibody derivatives (all Ig-derived) employed in accordance with the present invention, or their corresponding immunoglobulin chains, can be further modified using conventional techniques known in the art, for example, by amino acid deletion, insertion, substitution, addition, and / or recombination, and / or any other modification known in the art, alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an 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" particularly relates to a (poly)peptide construct comprising at least one CDR. Fragments or derivatives of the recited Ig-derived domains define (poly)peptides that are portions of the above-mentioned antibody molecules and / or that have been modified by chemical / biochemical or molecular biological methods. Corresponding methods are known in the art and are described, inter alia, in laboratory manuals (see Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, 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)).

[0089] The term "CDR" as used herein refers to "complementarity-determining region," which is well known in the art. CDRs are the portions of immunoglobulins that determine the specificity of the molecule and contact specific ligands. CDRs are the most variable portions of the molecule and contribute to the diversity of these molecules. There are three CDR regions in each V domain: CDR1, CDR2, and CDR3. CDR-H represents the CDR region of the variable heavy chain, and CDR-L relates to the CDR region of the variable light chain. VH refers to the variable heavy chain, and VL refers to the variable light chain. The CDR regions of Ig-derived regions can be determined as described in Kabat "Sequences of Proteins of Immunological Interest," 5th edition, NIH Publication no. 91-3242, US Department of Health and Human Services (1991). The CDR sequences provided herein are defined according to Kabat. However, one of skill in the art will appreciate that the present invention is intended to encompass binding molecules in which the CDR sequences are 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; 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) numbering scheme may be adopted.

[0090] Thus, in the context of the present invention, the humanized antibody molecule described herein above is selected from the group consisting of a complete antibody (such as an immunoglobulin, IgG1, IgG2, IgA1, IgGA2, IgG3, IgG4, IgA, IgM, IgD, or IgE), a F(ab)-, Fab'-SH-, Fv-, Fab'-, F(ab')2-fragment, a chimeric antibody, a CDR-grafted antibody, a fully human antibody, a bivalent antibody construct, an antibody fusion protein, a synthetic antibody, a bivalent single-chain antibody, a trivalent single-chain antibody, and a multivalent single-chain antibody.

[0091] "Humanization approaches" are well known in the art and have been particularly described with respect to antibody molecules, e.g., Ig-derived molecules. The term "humanized" refers to humanized forms of non-human (e.g., murine) antibodies or fragments thereof (Fv, Fab, Fab', F(ab'), scFv, or other antigen-binding subsequences of antibodies) that contain some portion of sequence derived from the non-human antibody. Humanized antibodies include human immunoglobulins in which residues from the complementarity-determining regions (CDRs) of a human immunoglobulin are replaced by residues from the CDRs of a non-human species, such as mouse, rat, or rabbit, possessing the desired binding specificity, affinity, and capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically all of two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Optimally, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, inter alia, Jones et al., Nature 321 (1986), 522-525; Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acids introduced from a source that is non-human and retains the binding activity of the original antibody. Methods for humanizing antibodies / antibody molecules are described in further 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.Examples of humanized antibodies, such as antibodies directed against EpCAM, are known in the art (see, for example, LoBuglio, Proceedings of the American Society of Clinical Oncology Abstracts (1997), 1562 and Khor, Proceedings of the American Society of Clinical Oncology Abstracts (1997), 847).

[0092] Thus, in the context of the present invention, there is provided an antibody molecule or antigen-binding fragment thereof that has been humanized and can be successfully employed in a pharmaceutical composition.

[0093] The specificity of the humanized antibody or antigen-binding fragment thereof of the present invention is expressed not only by the amino acid sequence properties of the humanized antibody or antigen-binding fragment as defined above, but also by the epitope to which the antibody can bind. That is, in one embodiment, the present invention relates to an anti-misfolded humanized TDP-43 antibody or antigen-binding fragment thereof that recognizes the same epitope as the antibody of the present invention.

[0094] 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 chemically synthesized peptides. The amino acid positions are indicated solely to indicate the location of the corresponding amino acid sequence in the sequence of the TDP-43 protein. The present invention encompasses all peptides containing epitopes. Peptides may be part of a polypeptide longer than 100 amino acids, or may be small peptides less than 100, preferably less than 50, more preferably less than 25, and even more preferably less than 16 amino acids. The amino acids in such peptides may be natural or unnatural amino acids (e.g., beta-, gamma-, or D-amino acids), or combinations thereof. Furthermore, the present invention may encompass retro-inverso peptides of each epitope. Peptides may be unconjugated or conjugated. They may be conjugated, 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.), to proteins, fatty acids, sugar moieties, or may be inserted into membranes.

[0095] To test whether the antibody of interest and the antibody of the present invention recognize the same epitope, the following competition study is performed: Vero cells infected at 3 MOI (multiplicity of infection) are incubated for 1 hour with various concentrations of the antibody of interest as a competitor after 20 hours. In the 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 of interest indicates that both antibodies recognize the same epitope. However, many other assays that can be used are known in the art.

[0096] The present invention also relates to the production of specific antibodies against natural and recombinant TDP-43 polypeptides. This production is based on the immunization of animals, such as mice. However, other animals for antibody / antiserum production are also contemplated within the present invention. For example, monoclonal and polyclonal antibodies can be produced in rabbits, mice, goats, donkeys, and other animals. A polynucleotide encoding a correspondingly selected TDP-43 polypeptide can be subcloned into an appropriate vector, and the recombinant polypeptide can be expressed in an organism capable of expression, such as bacteria. That is, the expressed recombinant protein can be injected intraperitoneally into mice, and the resulting specific antibodies can be obtained from mouse serum provided, for example, by intracardiac blood puncture. The present invention also contemplates the production of specific antibodies against natural and recombinant polypeptides using DNA / RNA vaccine strategies, as exemplified in the accompanying examples. DNA vaccine strategies are well known in the art and include gene gun or jet injection, and liposome-mediated delivery via intramuscular or intradermal injection. That is, antibodies against a TDP-43 polypeptide or protein or epitope, particularly an epitope of the antibody provided herein, can be obtained by directly immunizing an animal via intramuscular injection of a vector expressing a desired TDP-43 polypeptide or protein or epitope, particularly an epitope of the antibody of the present invention within amino acid residues 397-411 of SEQ ID NO: 1, more specifically within amino acid residues 400-405, 400-406, or 400-412 of SEQ ID NO: 1. The amount of specific antibody obtained can be quantified using ELISA, as described herein and below. Further methods for producing antibodies are well known in the art. See, for example, Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press, Cold Spring Harbor, 1988.

[0097] That is, under specified assay conditions, the identified antibody and the corresponding epitope of TDP-43 bind to each other and do not bind significantly to other components present in the sample. Specific binding to a target analyte under such conditions may require a binding moiety selected for its specificity for the particular target analyte. Various immunoassay formats can be used to select antibodies specifically reactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically reactive with an analyte. For descriptions 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, a specific or selective reaction will have a signal-to-noise ratio of at least 2-fold, more typically 10-100-fold greater than 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 adapted to avoid unwanted cross-reactivity; for example, polyclonal antibodies can be readily purified and selected by known methods (see Shepherd and Dean, supra).

[0098] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0099] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding.

[0100] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Target sites for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in Table 1 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading of "Exemplary Substitutions" and are further described below with reference to classes of amino acid side chains. Amino acid substitutions are introduced into the antibody of interest, and the products are screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0101] [Table 1]

[0102] Amino acids can be grouped according to common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe

[0103] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0104] One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have an alteration (e.g., improvement) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, and the mutated antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0105] For example, to improve the affinity of an antibody, alterations (e.g., substitutions) may be made in CDRs. Such alterations are made in CDR "hot spots," i.e., residues encoded by codons frequently mutated during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDRs (a-CDRs), and the resulting VH or VL variants are tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries 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 affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified using, for example, alanine scanning mutagenesis or modeling. CDR-H3 and especially CDR-L3 are often targeted.

[0106] In certain embodiments, substitutions, insertions, or deletions may be made in one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be outside the "hot spots" of the CDRs, i.e., the SDRs. In certain embodiments of the VH and VL sequences of the variants provided above, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0107] 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 residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody and antigen is affected. Further substitutions are introduced at the amino acid positions, and functional sensitivity to the initial substitution is demonstrated. Alternatively or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and nearby residues can be targeted or excluded as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0108] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0109] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0110] If the antibody contains an Fc region, the carbohydrate attached thereto can vary. Natural antibodies produced by mammalian cells typically contain branched and biantennary oligosaccharides, generally attached by an N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to generate antibody variants with improved properties.

[0111] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, e.g., as described in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately 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, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, depending on minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 013214 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); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Publication No. 2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Bioteeh. Bioeng. 87: 614 (2004); Kanda, Y. et al., Bioteehnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0112] Antibody variants with truncated oligosaccharides, for example, antibody variants in which biantennary oligosaccharides attached to the Fc region of the antibody are cleaved by GlcNAc, are also provided. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0113] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0114] In certain embodiments, the antibodies provided herein bind to pathogenic TDP-43 and form immune complexes that are removed by antibody-dependent cellular phagocytosis (ADCP), thereby promoting the clearance of TDP-43. ADCP is mediated by the interaction of the Fc fragment of the antibody with an Fc receptor, such as an Fc gamma receptor, expressed on the surface of fetal immune cells, such as microglia or dendritic cells. By modifying the Fc portion of the antibody, Fc-mediated functions can be tuned to achieve desired effects.

[0115] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for uses in which in vivo antibody half-life is important but certain effector functions (e.g., complement activation and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes and microglia express FcγRI, FcγRII, and FcγRIII. FcR expression on 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499- 1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).

[0116] Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA; and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.

[0117] Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in a animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. sci. USA 95:652-656 (1998).

[0118] To confirm that an antibody cannot bind to C1q and therefore lacks CDC activity, C1q binding assays can be performed.See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402.To assess complement activation, CDC assays can be performed (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)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0119] 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. Pat. No. 6,737,056). Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581), or the so-called "DANG" Fc mutant with substitutions of residue 265 to alanine and 297 to glycine. Alternatively, antibodies with reduced effector function include antibodies with one or more substitutions of Fc region residues 234, 235, and 329, the so-called "PG-LALA" Fc mutant with substitutions of residues 234 and 235 to alanine and 329 to glycine (Lo, M. et al., Journal of Biochemistry, 292, 3900-3908). Other known mutations at positions 234, 235, and 321, including the CH2 domain mutations L234F / L235E / P331S, can be used, such as so-called TM mutants (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies derived from the human IgG4 isotype include the mutations S228P / L235E, which stabilize the hinge and reduce FgR binding (Schlothauer et al., PEDS, 29 (10):457-466). The numbering of the constant domain is according to the EU numbering system.

[0120] Other Fc variants include variants having a substitution at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821.

[0121] In certain embodiments, the Fc region is mutated to increase its affinity for FcRn at pH 6.0, resulting in an extended half-life of the antibody. Antibodies with enhanced affinity for FcRn include antibodies with one or more substitutions of Fc region residues 252, 253, 254, 256, 428, 434, including the so-called YTE mutation with substitutions 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)).

[0122] In certain embodiments, it is desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby located at accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0123] In certain embodiments, the antibodies provided herein may be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Moieties suitable for derivatization of antibodies 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 homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has manufacturing advantages due to its stability in water. Polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, etc.

[0124] In another embodiment, a conjugate of an antibody and a nonproteinaceous moiety is provided that is selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, a wavelength that heats the nonproteinaceous moiety to a temperature that is not damaging to normal cells but that kills cells in proximity to the antibody and nonproteinaceous moiety.

[0125] 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 described herein is provided. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., YO, NSO, Sp20). In one embodiment, a method of making an antibody against misfolded TDP-43 is provided, the method comprising culturing a host cell comprising nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or culture medium of the host cell).

[0126] For recombinant production of an antibody against misfolded TDP-43, for example, nucleic acids encoding the antibody are isolated 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 capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0127] 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, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0128] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0129] Suitable host cells for the expression of glycosylated antibodies are also derived 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.

[0130] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTBODIES™ technology for producing antibodies in transgenic plants).

[0131] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40-transformed macaque kidney CV1 lines (COS-7); human embryonic kidney lines (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 carcinoma cells (HeLa); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (WI38); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g., as described in Mather et al., Annals N. Y. Aead. Sei. 383:44-68 (1982)); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. cii. USA 77:4216 (1980)); and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Val. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0132] There are several approaches known in the art to deliver molecules across the blood-brain barrier (BBB), such as altering the route of administration, disrupting the BBB and altering its permeability, nanoparticle delivery, Trojan horse approaches, receptor-mediated transport, and cell and gene therapy.

[0133] Altered routes 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 et al., Nature Med. 9: 589-595 (2003); and Gliadel Wafers™, Guildford Pharmaceutical), or intranasal administration to bypass the BBB (Mittal et al., Drug Deliv. 21(2):75-86 (2014)).

[0134] 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 the permeability enhancer A-7 (see, e.g., U.S. Patent Nos. 5,112,596, 5,268,164, 5,506,206, and 5,686,416).

[0135] 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 Publication Nos. 2002 / 0065259, 2003 / 0162695, and 2005 / 0124533); activating potassium channels (see, e.g., U.S. Patent Publication No. 2005 / 0089473), and inhibiting ABC drug transporters (see, e.g., U.S. Patent Publication No. 2003 / 0073713).

[0136] Trojan horse delivery methods for delivering humanized antibodies or humanized antibody fragments thereof across the blood-brain barrier include, but are not limited to, cationization of the antibody (see, e.g., U.S. Pat. No. 5,004,697) and the use of cell-penetrating peptides such as Tat peptide to gain entry into the CNS (e.g., Dietz et al., J. Neurochem. 104:757-765 (2008)).

[0137] Nanoparticle delivery methods for delivering a humanized antibody or antigen-binding fragment thereof across the blood-brain barrier include, but are not limited to, encapsulating the antibody or antigen-binding fragment thereof in an extracellular vesicle, such as a liposome or exosome, linked without limitation to the antibody or antigen-binding fragment or alternatively to a peptide that binds to a receptor on the vascular endothelium of the blood-brain barrier (see, e.g., U.S. Patent Publication 20020025313), and coating the antibody or antigen-binding fragment thereof onto low-density lipoprotein particles (see, e.g., U.S. Patent Publication 20040204354) or apolipoprotein E (see, e.g., U.S. Patent Publication 20040131692).

[0138] The humanized antibodies of the present invention can be further modified to enhance penetration of the blood-brain barrier.

[0139] The humanized antibody or antigen-binding fragment thereof of the present invention can be fused to a polypeptide that binds to a blood-brain barrier receptor. BBB receptors include, but are not limited to, transferrin receptor, insulin receptor, or low-density lipoprotein receptor. The polypeptide can be a peptide, receptor ligand, single-domain antibody (VHH), scFv, or Fab fragment.

[0140] The humanized antibodies of the present invention can also be delivered as corresponding nucleic acids encoding the humanized antibodies. Such nucleic acid molecules can be part of a viral vector for targeted delivery to the blood-brain barrier or any other cell type in the CNS. The viral vector can be a recombinant adeno-associated viral vector (rAAV) selected from any AAV serotype known in the art, including, without limitation, AAV1 to AAV12, that allows the humanized antibody, humanized antibody fragment, or humanized antibody derivative to be expressed intracellularly or in the brain parenchyma.

[0141] Cell therapy methods for delivering the humanized antibodies, or humanized antibody fragments, or humanized antibody derivatives of the invention across the blood-brain barrier include, but are not limited to, using the homing ability of endothelial progenitor cells (EPCs) transfected ex vivo with our proprietary vectors to overcome the strong filtering activity of the BBB (see, e.g., Heller and al., J Cell Mol Med. 00:1-7 (2020)) and the secretion and delivery of antibodies or antibody fragments by these cells to the brain, or using polymeric cell implantation devices loaded with genetically engineered cells to secrete antibodies or antibody fragments (see, e.g., Marroquin Belaunzaran et al. PLoS ONE 6(4): e18268 (2011)).

[0142] Pharmaceutically acceptable carriers, diluents, adjuvants, and excipients are well known in the pharmaceutical art and can be found, 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 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, 2003; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992), the disclosures of which are hereby incorporated by reference.

[0143] Carriers, diluents, adjuvants, and pharmaceutical excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice. The compounds must be acceptable in the sense of not being harmful to the recipients thereof. 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, 2003; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992), the disclosures of which are hereby incorporated by reference.

[0144] An "effective amount" of a compound to be administered to a subject is a dosage appropriate for treating, preventing, or ameliorating a disease, disorder, or condition according to sound medical judgment. Specific dose levels and dosage frequency can depend on various factors, including, for example, the activity of the specific compound employed, the metabolic stability and duration of action of the compound, the mode and time of administration. An "effective amount" of a compound to be administered to a subject is a dosage appropriate for treating, preventing, or ameliorating a condition, disease, disorder, or condition according to sound medical judgment. Specific dose levels and dosage frequency can depend on various factors, including, for example, the activity of the specific compound employed, the metabolic stability and duration of action of the compound, the mode and time of administration, the rate of excretion, and the drug combination. Patient-specific factors, such as age, weight, general health, sex, diet, and the severity of a particular condition, can also affect the amount to be administered.

[0145] The term "clearance" (also referred to as "clearance value" or "CL" or "systemic clearance") relates to the efficiency of elimination of a substance from the body. The clearance of a substance (in this case, the binding molecule of the present invention) is the sum of urinary and extrarenal clearance. For substances eliminated by both 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 of mAbs is cellular uptake and subsequent proteolysis. Because mAbs have low clearance from the systemic circulation, they can be administered less frequently than peptides or small molecules, which is often more convenient for patients (Betts et al., MABs. 2018).

[0146] XI. Inventive Embodiments of TDP-43-Specific Binding Molecules In some embodiments, humanized TDP-43 binding molecules, particularly a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or b) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or c) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); d) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or e) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or f) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser). The present invention provides a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0147] In some embodiments, humanized TDP-43 binding molecules, particularly a) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or b) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or c) VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 The present invention provides a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0148] In some embodiments, humanized TDP-43 binding molecules, particularly a) i. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or ii. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or iii. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 92; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or iv. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or v. A VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or vi. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a heavy chain variable region (VH) comprising: b) i. VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or ii. VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or iii. VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 A light chain variable region (VL) containing The present invention provides a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0149] In some embodiments, a) i. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 22; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or ii. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 82; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or iii. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 92 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 92; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or iv. a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 102 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 102; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or v. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 112; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or vi. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 21; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser). a heavy chain variable region (VH) comprising: b) i. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or a VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or a VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or a VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 27; or ii. a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85, or a VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 85; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, or a VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87, or a VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 87; or iii. VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or a VL-CDR1 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or a VL-CDR2 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or a VL-CDR3 comprising an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87. A light chain variable region (VL) containing The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0150] In some embodiments, a) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 22; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); or b) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 112; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); c) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 122; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and d) a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 27; or e) a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 85; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87; or f) a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87. The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0151] More specifically, in some embodiments, a) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 122; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and b) a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 85; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87. The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0152] More specifically, in some embodiments, a) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 112; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and b) a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87. The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0153] More specifically, in some embodiments, a) a heavy chain variable region (VH) comprising: a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122, or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 122; and a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a light chain variable region (VL) comprising: a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 80%, 90%, 95%, or 100% sequence identity with SEQ ID NO: 87. The present invention provides a humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, comprising:

[0154] In some embodiments, the humanized TDP-43 antibody comprises CDRs selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 112, or SEQ ID NO: 122; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87.

[0155] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27.

[0156] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27.

[0157] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0158] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27.

[0159] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0160] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0161] In some embodiments, the humanized TDP-43 antibody comprises a CDR selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; (b) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; (c) a VH-CDR3 comprising the amino acid sequence ES(Glu-Ser); (d) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; (e) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and (f) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.

[0162] In another embodiment, the humanized TDP-43 antibody comprises a heavy chain variable domain (VH) selected from the group consisting of SEQ ID NOs: 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120, including post-translational modifications of said sequence. In a specific embodiment, the heavy chain variable domain (VH) comprises at least one, two, or three CDRs selected from: (a) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, (b) a VH-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 82, 92, 102, 112, and 122, and (c) a VH-CDR3 comprising the amino acids ES (Glu-Ser).

[0163] In another embodiment, the humanized TDP-43 antibody comprises a light chain variable domain (VL) selected from the group consisting of SEQ ID NOs: 34, 44, 54, 64, 74, 84, and 94, including post-translational modifications of that sequence. In a specific embodiment, the light chain variable domain (VL) comprises at least one, two, or three CDRs selected from (a) VL-CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 85, (b) VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and (c) VL-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 87.

[0164] In some embodiments, the humanized TDP-43 antibody comprises: a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 30 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 60; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; or f. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 80 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 80; or g. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 90 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 90; or h. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 100 or having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100; or i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 or having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; or j. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120. Includes.

[0165] In some embodiments, the humanized TDP-43 antibody comprises: a. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 34 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or b. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or c. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or d. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64 or having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or e. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 74 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or f. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84 or having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or g. A light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94. Includes.

[0166] In some embodiments, the humanized TDP-43 binding molecule, particularly a humanized TDP-43 antibody or antigen-binding fragment thereof, a) i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 30 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; or ii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40; or iii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; or iv. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 60; or v. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; or vi. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 80 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 80; or vii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 90 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 90; or viii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 100 or having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100; or ix. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 or having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; or x. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120. a heavy chain variable region (VH) selected from b) i. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 34 or having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or ii. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or iii. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or iv. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or v. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 74 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or vi. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84 or having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or vii. A light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94. The variable region comprises a light chain (VL) selected from:

[0167] In some embodiments, the humanized TDP-43 binding molecule, particularly a humanized TDP43 antibody or antigen-binding fragment thereof, a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60 or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 60, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54 or a light chain variable region (VL) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or f. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or g. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or h. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84 or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94; or j. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 or having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94. Includes.

[0168] In some embodiments, the humanized TDP-43 antibody comprises: a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or f. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or g. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or h. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94; or j. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 Includes.

[0169] In some embodiments, the humanized TDP-43 antibody comprises: a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94; or c. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 Includes.

[0170] In some embodiments, the humanized TDP-43 antibody comprises: a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94; or c. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120 and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94 Includes.

[0171] In some embodiments, the present invention provides hACI-7069-633B12-Ab1_H27L23, hACI-7069-633B12-Ab1_H28L22, hACI-7069-633B12-Ab1_H28L23, hACI-7069-633B12-Ab1_H28L24, hACI-7069-633B12-Ab1_H32L24, hACI-7 The present invention relates to a humanized TDP-43 binding molecule selected from hACI-7069-633B12-Ab1_H32L26, hACI-7069-633B12-Ab1_H33L24, hACI-7069-633B12-Ab1_H33L26, hACI-7069-633B12-Ab1_H32L27, or hACI-7069-633B12-Ab1_H33L27.

[0172] Preferably, the humanized TDP-43 binding molecule is selected from the group consisting of hACI-7069-633B12-Ab1_H33L26, hACI-7069-633B12-Ab1_H32L27, or hACI-7069-633B12-Ab1_H33L27.

[0173] Even more preferably, the humanized TDP-43 binding molecule is selected from the group consisting of hACI-7069-633B12-Ab1_H32L26, hACI-7069-633B12-Ab1_H32L27, or hACI-7069-633B12-Ab1_H33L27.

[0174] In one preferred embodiment, the humanized TDP-43 binding molecule is hACI-7069-633B12-Ab1_H33L27.

[0175] In some embodiments, the humanized TDP-43 binding molecules of the invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, comprise an Fv with a pI of less than 7.8, preferably less than 7.5, and more preferably less than 7.0.

[0176] In some embodiments, the humanized TDP-43 binding molecules of the invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, comprise an Fv with a net charge at pH 7.4 of less than 0.2, preferably less than −0.8, and more preferably less than −1.8.

[0177] In some embodiments, the humanized TDP-43 binding molecules of the invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, comprise an Fv with a pI of less than 7.0 and a net charge of less than -1.8.

[0178] In some embodiments of the present invention, the humanized anti-TDP-43 binding molecules of the present invention have a clearance of 0.50 mL / h / kg or less, particularly 0.25 mL / h / kg or less, and more particularly 0.18 mL / h / kg or less in mice. In some embodiments, the humanized TDP-43 binding molecules of the present invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, exhibit a half-life of at least 8 days, preferably at least 10 days, and more preferably at least 12 days in Tg32 mice. In one embodiment, the humanized TDP-43 binding molecule may be hACI-7069-633B12-Ab1_H33L27 or hACI-7069-633B12-Ab1_H32L27. Clearance (e.g., systemic, CL) and half-life (T 1 / 2β For the measurement of ), reference may be made to Example 5.

[0179] In some embodiments of the invention, the humanized anti-TDP-43 binding molecules of the invention have a clearance of 0.27 mL / h / kg or less in NHPs, particularly 0.12 mL / h / kg or less, and more particularly 0.08 mL / h / kg or less. In some embodiments, the humanized TDP-43 binding molecules of the invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, exhibit a half-life of at least 6 days, preferably at least 8 days, e.g., at least 10 or 12 days, in NHPs. In one embodiment, the humanized TDP-43 binding molecule may be hACI-7069-633B12-Ab1_H33L27 or hACI-7069-633B12-Ab1_H32L27. Clearance (e.g., systemic, CL) and half-life (T1 / 2β For the measurement of ), reference may be made to Example 6.

[0180] In some embodiments, the humanized TDP-43-binding molecules of the present invention, particularly humanized TDP-43 antibodies or antigen-binding fragments thereof, exhibit a predicted half-life in humans of at least 20 days, preferably at least 24 days, e.g., at least 26 days. The predicted half-life in humans may be 27-30 days or 17-22 days. The humanized TDP-43-binding molecule may be hACI-7069-633B12-Ab1_H33L27 or hACI-7069-633B12-Ab1_H32L27. See Example 9 for a method for calculating the predicted half-life in humans from the half-lives in Tg32 mice and / or cynomolgus monkeys.

[0181] In some embodiments, (isolated) nucleic acids encoding humanized TDP-43 binding molecules, particularly the humanized TDP-43 antibodies and fragments thereof described herein, are provided.

[0182] In some embodiments, an (isolated) nucleic acid encoding a humanized anti-TPD-43 antibody is provided, comprising SEQ ID NO: 38.

[0183] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 48 encoding a humanized anti-TPD-43 antibody is provided.

[0184] In some embodiments, an (isolated) nucleic acid encoding a humanized anti-TPD-43 antibody is provided, comprising SEQ ID NO: 58.

[0185] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 68 encoding a humanized anti-TPD-43 antibody is provided.

[0186] In some embodiments, an (isolated) nucleic acid encoding a humanized anti-TPD-43 antibody is provided, comprising SEQ ID NO: 78.

[0187] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 88 encoding a humanized anti-TPD-43 antibody is provided.

[0188] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 98 encoding a humanized anti-TPD-43 antibody is provided.

[0189] In some embodiments, an (isolated) nucleic acid encoding a humanized anti-TPD-43 antibody is provided, comprising SEQ ID NO: 108.

[0190] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 118 encoding a humanized anti-TPD-43 antibody is provided.

[0191] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 128 encoding a humanized anti-TPD-43 antibody is provided.

[0192] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 39 encoding a humanized anti-TPD-43 antibody is provided.

[0193] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 49 encoding a humanized anti-TPD-43 antibody is provided.

[0194] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 59 encoding a humanized anti-TPD-43 antibody is provided.

[0195] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 69 encoding a humanized anti-TPD-43 antibody is provided.

[0196] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 79 encoding a humanized anti-TPD-43 antibody is provided.

[0197] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 89 encoding a humanized anti-TPD-43 antibody is provided.

[0198] In some embodiments, an (isolated) nucleic acid comprising SEQ ID NO: 99 encoding a humanized anti-TPD-43 antibody is provided.

[0199] XII. Compositions and Methods The present invention also relates to a pharmaceutical composition comprising a humanized TDP-43-binding molecule, in particular a humanized antibody or antigen-binding fragment thereof of the present invention as described herein, and a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0200] In some embodiments, a pharmaceutical composition is provided comprising an (isolated) humanized antibody described herein and a pharmaceutically acceptable carrier.

[0201] In some embodiments, conjugated binding molecules, particularly antibodies or antigen-binding fragments thereof, are provided, comprising the binding molecules described herein, particularly antibodies or antigen-binding fragments thereof, and conjugated molecules. The conjugates of the present invention are referred to as immunoconjugates. Any suitable conjugate 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., radioactive conjugates), nucleic acid molecules, detectable labels, therapeutic agents, toxins, and blood-brain barrier-penetrating moieties. Conjugation methods are well known in the art, and several techniques for conjugating antibodies to labels or other molecules are commercially available. Conjugation is typically via an amino acid residue (e.g., lysine, histidine, or cysteine) contained in the binding molecules of the present invention. These may rely on methods such as the NHS (succinimidyl) ester method, the isothiocyanate method, the carbodiimide method, and the periodate method. Conjugation can be achieved, for example, through the creation of a fusion protein. This is appropriate when the binding molecule is conjugated to another protein molecule. That is, suitable genetic constructs can be formed that allow 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 between the antibody and the conjugated molecule, such as a detectable label. However, a linker is not required in all cases. In some embodiments, the humanized TDP-43-specific binding molecule of the present invention is linked to a detectable label.

[0202] The present invention also relates to immunoconjugates comprising the humanized TDP-43-binding molecules provided herein conjugated to one or more therapeutic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioisotopes (i.e., radioconjugates), blood-brain barrier-penetrating moieties, or detectable labels. As discussed herein, various approaches exist for improving drug delivery across the blood-brain barrier (BBB), and these considerations apply mutatis mutandis. Non-invasive approaches include the so-called "Trojan horse approach," in which a conjugated molecule delivers the binding molecule of the present invention by binding to and mediating transport through a BBB receptor. Suitable molecules include endogenous ligands or antibodies, particularly monoclonal antibodies, that bind to specific epitopes on BBB receptors.

[0203] In some embodiments, an immunoconjugate is provided comprising an (isolated) humanized antibody described herein and a therapeutic agent. In some embodiments, a labeled humanized antibody is provided comprising a humanized antibody described herein and a detectable label.

[0204] In some embodiments, the humanized TDP-43-specific binding molecule is part of an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent.

[0205] In some embodiments, the humanized TDP-43-specific binding molecule or an immunoconjugate comprising the same is present as a composition comprising the humanized TDP-43-specific binding molecule.

[0206] In some embodiments, the humanized TDP-43-specific binding molecule is part of a pharmaceutical composition comprising the humanized TDP-43-specific binding molecule or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule in combination with a pharmaceutically acceptable carrier and / or excipient and / or diluent.

[0207] In some embodiments, the humanized TDP-43-specific binding molecule is part of a detection and / or diagnostic kit that includes the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition that includes the humanized TDP-43-specific binding molecule.

[0208] Kits containing the humanized binding molecules of the present invention are also provided. In particular, such kits can be useful for practicing the diagnostic methods of the present invention, including classification, monitoring, and treatment selection methods. That is, kits containing the humanized TDP-43-specific binding molecules of the present invention are provided for diagnosing diseases, disorders, and / or disorders associated with TDP-43, particularly TDP-43 aggregate-related diseases, disorders, and / or disorders, or TDP-43 proteinopathies, or for use in the methods of the present invention. Such kits can include all components necessary to practice the methods provided herein. Typically, each component is stored separately in a single overall package. Suitable additional components to be included in the kit include, for example, buffers, detectable dyes, laboratory equipment, reaction vessels, instructions for use, etc. The instructions can be tailored to the particular method in which the kit is employed. Suitably labeled humanized TDP-43-binding molecules of the present invention included in such kits are also provided.

[0209] In some embodiments, the humanized TDP-43-specific binding molecules are used in immunodiagnostic methods for use in the prevention, diagnosis, or treatment of TDP-43 proteinopathies.

[0210] In some embodiments, the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule, administered to a subject in need thereof, is used to diagnose, prevent, ameliorate, or treat a disease, disorder, and / or condition associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy, including, but not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0211] In some embodiments, the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule, is administered to a subject in need thereof, or to a subject with frontotemporal dementia (e.g., chromosome 9p-linked, with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations, with motor deficits, Sporadic or familial FTD with or without neuronal disease (MND), corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of 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-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathies (sporadic inclusion body myositis, inclusion body myopathy with mutations in valsin-containing protein (VCP); Paget's disease of bone and in methods for diagnosing or monitoring a disease, disorder, and / or abnormality associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy, selected from: myotilin (MYOT) gene mutations or myofibrillar myopathy associated with mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD).

[0212] In other embodiments, the present invention relates to any method for detecting, diagnosing, or monitoring a disease, disorder, and / or abnormality associated with TDP-43, particularly associated with TDP43 aggregates, or a TDP-43 proteinopathy, selected from frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0213] Preferably, the TDP-43-related, particularly TDP-43 aggregate-related, disease, disorder, and / or abnormality, or TDP-43 proteinopathy is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and frontotemporal dementia (FTD). More preferably, the TDP-43-related, particularly TDP-43 aggregate-related, disease, disorder, and / or abnormality, or TDP-43 proteinopathy is amyotrophic lateral sclerosis (ALS). More preferably, the TDP-43-related, particularly TDP-43 aggregate-related, disease, disorder, and / or abnormality, or TDP-43 proteinopathy is Alzheimer's disease (AD). More preferably, the TDP-43-related, particularly TDP-43 aggregate-related, disease, disorder, and / or abnormality, or TDP-43 proteinopathy is frontotemporal dementia (FTD).

[0214] In some embodiments, the humanized TDP-43-specific binding molecule is used in a method for diagnosing presymptomatic disease, monitoring disease progression and therapeutic efficacy, predicting response, or selecting subjects likely to respond to treatment with the humanized TDP-43-specific binding molecule. The method is preferably performed using human blood or urine samples. Most preferably, the method comprises an ELISA-based assay or a surface-compatible assay.

[0215] In some embodiments, the humanized TDP-43-specific binding molecules are used in methods of contacting the humanized TDP-43-specific binding molecules of the 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-predominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0216] In some embodiments, the humanized TDP-43-specific binding molecule is used to treat frontotemporal dementia (e.g., sporadic or familial FTD with or without motor neuron disease (MND), corticobasal degeneration, frontotemporal lobar degeneration with ubiquitin-positive TDP-43 inclusions, linked to chromosome 9p, with mutations in progranulin (GRN), with mutations in C9orf72, with mutations in TARDBP, with mutations in valosin-containing protein (VCP), (FTLD) (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of 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-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE) , Perry syndrome, Alzheimer's disease (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 valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), oculopharyngeal myopathy with marginal vesicles The humanized TDP-43-specific binding molecules of the invention are used in methods 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 encephalopathy, myofibrillar myopathy associated with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD).

[0217] In some embodiments, the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule, is administered to a subject in need thereof or used to prevent, ameliorate, or treat a disease, disorder, and / or condition associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy, 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-predominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0218] In some embodiments, the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule, is administered to a subject in need thereof, or to a subject with frontotemporal dementia (e.g., chromosome 9p-linked, with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, valosin-containing protein Sporadic or familial FTD with or without motor neuron disease (MND) with mutations in VCP, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioral variant FTD (bvFTD), non-fluent variant primary progressive aphasia (nfvPPA), amyotrophic lateral sclerosis (e.g., sporadic ALS, TARD) ALS with mutations in angiogenin (ANG), with mutations in BP, Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis and myelopathy. The present invention is used to treat a disease selected from the group consisting of myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with marginal vesicles, myofiber myopathy with mutations in the myotilin (MYOT) gene or the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD). Preferably, the treatment of the disease helps preserve or increase intellectual cognition and / or reduces brain levels of TDP-43 aggregates.

[0219] In some embodiments, the humanized TDP-43-specific binding molecule, or an immunoconjugate in which the humanized TDP-43-specific binding molecule is covalently linked to another suitable therapeutic agent, or a composition comprising the humanized TDP-43-specific binding molecule, administered to a subject in need thereof, is used to manufacture a medicament for treating a disease, disorder, and / or condition associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy, 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-predominant age-related TDP-43 encephalopathy (LATE), and / or Parkinson's disease (PD).

[0220] Pharmaceutical formulations of the humanized anti-TDP-43 antibodies (a preferred type of TDP-43-specific binding molecule) or immunoconjugates described herein are prepared by mixing such humanized antibodies or immunoconjugates having the 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 antibodies or fragments thereof are prepared as lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 residues) benzoates, benzoyl ... polypeptides (less than 1000 bases); 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.).Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases. Pharmaceutically acceptable excipients that can be used to formulate the compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic materials (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 a salt selected from the group consisting of phosphate, acetate, citrate, succinate, and tartrate, and / or the buffer includes histidine, glycine, TRIS glycine, Tris, or a mixture thereof. In the context of the present invention, it is further envisioned that the diluent is a buffer selected from the group consisting of potassium phosphate, acetic acid / sodium acetate, citric acid / sodium citrate, succinic acid / sodium succinate, tartaric acid / sodium tartrate, and histidine / histidine HCl, or a mixture thereof.

[0221] 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 comprising a histidine-acetate buffer.

[0222] The formulations herein may contain more than one active ingredient as necessary for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other.

[0223] The active ingredient may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., 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 techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0224] Sustained-release preparations can also be prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies or immunoconjugates, which matrices are in the form of shaped articles, such as films or microcapsules.Preparations used for in vivo administration are generally sterile.Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0225] Any of the humanized antigen-binding molecules, humanized anti-TDP-43 antibodies, or immunoconjugates provided herein find use in methods, e.g., therapeutic methods.

[0226] In another aspect, a humanized anti-TDP-43 antibody (a preferred form of the humanized TDP-43-specific binding molecule) or immunoconjugate is provided for use as a pharmaceutical. In a further aspect, an anti-misfolded humanized TDP-43 antibody (a preferred form of the humanized TDP-43-specific binding molecule) or immunoconjugate is provided for use in a method of treatment. In certain embodiments, a humanized anti-TDP-43 antibody (a preferred form of the humanized TDP-43-specific binding molecule) or immunoconjugate is provided for use in the prevention, diagnosis, and / or treatment of TDP-43 proteinopathy. In a preferred embodiment of the present invention, there is provided a humanized anti-TDP-43 antibody (a preferred type of humanized TDP-43-specific binding molecule) or immunoconjugate for use in the prevention, diagnosis, and / or treatment of diseases, disorders, and / or conditions associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathies, including, but not limited to, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), and / or limbic-predominant age-related TDP-43 encephalopathy (LATE).

[0227] In a further aspect, the present invention provides the use of a humanized anti-TDP-43 antibody (a preferred type of humanized TDP-43-specific binding molecule) or immunoconjugate in the manufacture or preparation of a medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0228] A "subject" or "individual" according to any of the embodiments may be an animal, a mammal, preferably a human.

[0229] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the humanized anti-TDP-43 antibodies (preferred types of humanized TDP-43-specific binding molecules) or immunoconjugates provided herein, e.g., for use in any of the methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the humanized anti-TDP-43 antibodies (preferred types of humanized TDP-43-specific binding molecules) or immunoconjugates provided herein and a pharmaceutically acceptable carrier and / or excipient and / or diluent (discussed elsewhere herein). In another embodiment, the pharmaceutical formulation comprises any of the humanized anti-TDP-43 antibodies (preferred types of humanized TDP-43-specific binding molecules) or immunoconjugates provided herein and at least one additional therapeutic agent, e.g., as described below.

[0230] The humanized antibodies or immunoconjugates of the present invention can be used therapeutically alone or in combination with other agents. For example, the humanized antibodies (preferred types of humanized TDP-43-specific binding molecules) or immunoconjugates of the present invention can be co-administered with at least one additional therapeutic agent targeting alpha-synuclein, BACE1, tau, beta-amyloid, TDP-43, or a neuroinflammatory protein.

[0231] For example, a humanized antibody (a preferred type of humanized TDP-43-specific binding molecule) or immunoconjugate of the invention may be co-administered with at least one additional therapeutic agent selected from, but not limited to, a neuropharmaceutical drug, an anti-amyloid beta antibody, an anti-tau antibody, a tau aggregation inhibitor (including small molecules), a beta-amyloid aggregation inhibitor (including small molecules), an anti-BACE1 antibody, a BACE1 inhibitor, an anti-alpha synuclein inhibitor, an anti-alpha synuclein antibody, and a neuroinflammation inhibitor.

[0232] Such combination therapy, as noted above, encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration (wherein administration of a humanized antibody (a preferred form of humanized TDP-43-specific binding molecule) or immunoconjugate of the invention occurs before, simultaneously with, and / or after administration of an additional therapeutic agent and / or adjuvant). The humanized antibody (a preferred form of humanized TDP-43-specific binding molecule) or immunoconjugate of the invention can also be used in combination with radiation therapy.

[0233] The humanized antibody (a preferred form of humanized TDP-43-specific binding molecule) or immunoconjugate (and any additional therapeutic agent) of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional, intrauterine, or intravesical administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0234] The humanized antibodies (a preferred form of humanized TDP-43-specific binding molecule) or immunoconjugates of the present invention are formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the particular TDP-43-associated, particularly TDP-43 aggregate-associated disease, disorder, and / or disorder being treated, or TDP-43 proteinopathy, the mammal being treated, the clinical condition of the individual subject, the cause of the TDP-43-associated, particularly TDP-43 aggregate-associated disease, disorder, and / or disorder, or TDP-43 proteinopathy, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to clinicians. The humanized antibodies or immunoconjugates need not, but may, be formulated with one or more drugs currently used to prevent or treat the TDP-43-associated, particularly TDP-43 aggregate-associated disease, disorder, and / or disorder, or the TDP-43 proteinopathy in question. The effective amounts of such other agents depend on the amount of humanized antibody or immunoconjugate present in the formulation, the type or treatment of the disease, disorder, and / or disorder associated with TDP-43, particularly TDP-43 aggregates, or TDP-43 proteinopathy, and other factors discussed above. They are generally used in the same dosages and by any route of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined experimentally / clinically to be appropriate.

[0235] The appropriate dosage of a humanized antibody (a preferred form of a humanized TDP-43-specific binding molecule) or immunoconjugate of the present invention for disease prevention or treatment (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody or immunoconjugate, the severity and course of the disease, whether the antibody or immunoconjugate is being administered for preventive or therapeutic purposes, previous treatments, the subject's medical history, and response to the antibody or immunoconjugate, as well as the discretion of the treating physician. The humanized antibody (a preferred form of a humanized TDP-43-specific binding molecule) or immunoconjugate is suitably administered to the subject once or over a series of treatments. Depending on the type and severity of the disease, a suggested initial dosage of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the humanized antibody (a preferred form of a humanized TDP-43-specific binding molecule) or immunoconjugate can be administered to the subject, for example, in one or more individual administrations or via continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or more, treatment is generally sustained until a desired suppression of disease symptoms occurs, depending on the condition. One exemplary dosage of a humanized antibody or immunoconjugate would be in the range of about 0.05 mg / kg to about 10 mg / kg. That is, one or more doses of about 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, every week or every three weeks (e.g., so that the subject receives about two to about 20 doses, or, for example, about six doses of the antibody). An initial high loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may also be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0236] It is understood that any of the above formulations or methods of treatment may be carried out using both the immunoconjugates of the invention and humanized anti-TDP-43 antibodies (a preferred type of humanized TDP-43-specific binding molecule).

[0237] Another aspect of the present invention provides an article of manufacture containing the above-described materials useful for the treatment, prevention, and / or diagnosis of a disease, disorder, or disorder associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective, by itself or in combination with another composition, for treating, preventing, and / or diagnosing a disease, disorder, or disorder associated with TDP-43, particularly associated with TDP-43 aggregates, or a TDP-43 proteinopathy, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a human antibody or immunoconjugate of the present invention. The label or package insert indicates that the composition is used for treating the selected condition.

[0238] Additionally, the article of manufacture may include (a) a first container containing therein a composition comprising a humanized antibody (a preferred type of humanized TDP-43-specific binding molecule) or immunoconjugate of the present invention; and (b) a second container containing therein a composition comprising an additional therapeutic agent. The article of manufacture in this embodiment of the present invention may further include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture 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 article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0239] In a further embodiment, the present invention relates to a method for preserving or increasing, or preventing and / or delaying the decline of, cognitive memory, motor and language functions in a subject, comprising administering a humanized binding molecule of the invention, an immunoconjugate of the invention, a composition of the invention, or a pharmaceutical composition of the invention.

[0240] In a further embodiment, the present invention relates to a method for reducing the level of TDP-43 comprising administering a humanized binding molecule of the invention, an immunoconjugate of the invention, a composition of the invention, or a pharmaceutical composition of the invention.

[0241] The methods of the present invention may comprise the administration of at least one additional therapy, preferably selected from, but not limited to, antibodies or small molecules targeting alpha synuclein, BACE1, tau, beta amyloid, TDP-43, or neuroinflammatory proteins, in particular selected from neuropharmaceuticals, anti-beta amyloid antibodies, anti-tau antibodies, tau aggregation inhibitors, beta amyloid aggregation inhibitors, anti-BACE1 antibodies, BACE1 inhibitors, anti-alpha synuclein antibodies, and neuroinflammatory inhibitors.

[0242] The present invention further relates to a method for detecting TDP-43, comprising contacting a sample with a humanized binding molecule of the invention, preferably a humanized antibody of the invention, wherein the sample is a brain sample, a cerebrospinal fluid sample, an interstitial fluid (ISF) sample, a urine sample, or a blood sample.

[0243] In a further embodiment, the present invention relates to a method for detecting and / or measuring the level of TDP-43 using single molecule array (SIMOA®) technology, comprising contacting a sample with a humanized binding molecule of the invention, preferably a humanized antibody of the invention, wherein the sample is a human blood sample, a cerebrospinal fluid sample (CSF), an interstitial fluid (ISF) sample, or a urine sample, preferably a CSF sample.

[0244] In certain embodiments, the humanized TDP-43 binding molecules, particularly the humanized TDP-43 antibodies and fragments thereof provided herein, have an activity 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 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 The humanized TDP-43-binding molecules of the invention have a dissociation constant (KD) of 2 nM or less for soluble full-length TDP-43, in certain embodiments, 1 nM or less, and in more certain embodiments, 700 pM or less, preferably 250 pM or less for soluble full-length TDP-43. This is demonstrated with reference to Table 8 in Example 3 for the humanized TDP-43-binding molecules of the invention. In one embodiment, binding affinity for full-length (FL) TDP-43 may be assessed 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 suitable SPR methods that may be employed.

[0245] The humanized TDP-43 binding molecules of the invention may have a KD of 15 nM or less for the TP-51 peptide, and in certain embodiments, 10 nM or less, e.g., 4 nM or less, preferably 2 nM or less. This is also demonstrated in Example 3 for the humanized TDP-43 binding molecules of the invention with reference to Table 8.

[0246] That is, the humanized TDP-43 binding molecule of the present invention has a KD of 575 pM or less for soluble full-length TDP-43 and a KD of 2.2 nM or less for TP-51 peptide, a KD of 575 pM or less for soluble full-length TDP-43 and a KD of 1.6 nM or less for TP-51 peptide, a KD of 550 pM or less for soluble full-length TDP-43 and a KD of 2.2 nM or less for TP-51 peptide, preferably a KD of 550 pM or less for soluble full-length TDP-43 and a KD of 2.2 nM or less for TP-51 peptide. and TP-51 peptide of 2 nM or less, more preferably, a KD of 250 pM or less for soluble full-length TDP-43 and a KD of 1.6 nM or less for TP-51 peptide, even more preferably, a KD of 150 pM or less for soluble full-length TDP-43 and a KD of 1.0 nM or less for TP-51 peptide, and even more preferably, a KD of 130 pM or less for soluble full-length TDP-43 and a KD of 0.8 nM or less for TP-51 peptide.

[0247] The humanized TDP-43 binding molecules of the present invention can neutralize seeding-competent TDP-43. Neutralization of seeding-competent TDP-43 can be assessed by a Real-Time Quaking-induced Conversion (RT-QuIC) assay. See Example 11 for a detailed description of a suitable RT-QuIC assay that can be employed. "Seeding-competent TDP-43" refers to a pathogenic TDP-43 that can induce aggregation of physiological (i.e., non-pathogenic) TDP-43.

[0248] The humanized TDP-43-binding molecules of the present invention can promote the clearance of TDP-43. The promotion of TDP-43 clearance can be evaluated by an in vitro assay using THP-1 cells. See Example 12 for a detailed description of a suitable assay using THP-1 cells that can be employed. Generally, TDP-43 clearance is promoted by the antibodies of the present invention by binding to pathogenic TDP-43 and forming an immune complex that is removed by antibody-dependent cellular phagocytosis (ADCP). ADCP is mediated by the interaction of the Fc fragment of the antibody with an Fc receptor, such as an Fc gamma receptor, expressed on the surface of fetal immune cells, such as microglia or dendritic cells. By modifying the Fc portion of the antibody, Fc-mediated functions can be adjusted to achieve desired effects.

[0249] In some embodiments, the humanized TDP-43 binding molecule of the present invention has an EC50 of less than 250 pM, preferably 237 pM or less, for TDP-43 in human CSF. The humanized TDP-43 binding molecule may be hACI-7069-633B12-Ab1_H33L27. For a method for measuring EC50, see Example 14. [Brief explanation of the drawings]

[0250] [Figure 1]Inhibition of TDP-43 aggregation in vitro by hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H32L27, and hACI-7069-633B12-Ab1_H32L26. Briefly, de novo aggregation of recombinant TDP-43 upon cleavage from the MBP tag was analyzed in the presence of either hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H32L27, or hACI-7069-633B12-Ab1_H32L26 and compared to chimeric antibody cACI-7069-633B12-Ab1 or isotype control over time. The percentage of aggregated TDP-43 at 1.5 hours was normalized to the isotype control condition and demonstrated strong inhibition of aggregation by cACI-7069-633B12-Ab1, hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H32L27, and hACI-7069-633B12-Ab1_H32L26. One-way ANOVA followed by Dunnett's post hoc test for multiple comparisons was used. ****p<0.0001. [Figure 2] Pharmacokinetic profiles in serum of homozygous Tg32 mice after a single IV bolus dose of hACI-7069-633B12-Ab1_H33L27 (circles) or hACI-7069-633B12-Ab1_H32L27 (squares) at 40 mg / kg. Twenty-one male mice were used per compound, with three animals per time point. The total duration of the study was 6 weeks (1008 hours). [Figure 3] Pharmacokinetic profiles in serum of cynomolgus monkeys after a single IV bolus dose of hACI-7069-633B12-Ab1_H33L27 (A) or hACI-7069-633B12-Ab1_H32L27 (B) at 40 mg / kg. Four male cynomolgus monkeys were used per compound for an 8-week (1344 h) study period. [Figure 4]Pharmacokinetic profile of TDP-43 in serum of cynomolgus monkeys after a single IV bolus dose of hACI-7069-633B12-Ab1_H33L27 (A) or hACI-7069-633B12-Ab1_H32L27 (B) at 40 mg / kg. Target engagement of hACI-7069-633B12-Ab1_H33L27 (A) and hACI-7069-633B12-Ab1_H32L27 (B) in serum was assessed. [Figure 5] Real-time shaking-induced conversion (RT-QuIC) aggregation kinetics of synthetic TDP-43 peptide (TP-51) in the presence of cerebrospinal fluid (CSF) from donors with sporadic ALS (Figure 5A) or healthy controls (Figure 5B). Aggregation and fibril formation were quantified using thioflavin T (ThT) fluorescence. [Figure 6A] Uptake of pHrodo™-TDP-43 aggregates by THP-1 cells is significantly accelerated in the presence of hACI-7069-633B12-Ab1_H33L27 or chimeric antibody cACI-7069-633B12-Ab1, but not in the presence of an isotype antibody control. (A) shows pHrodo™ fluorescence intensity (Y-axis) measured hourly (X-axis) over 24 hours for cells incubated with aggregated TDP-43, aggregated TDP-43 and an isotype control, aggregated TDP-43 and cACI-7069-633B12-Ab1, and aggregated TDP-43 and hACI-7069-633B12-Ab1_H33L27. (B) shows the difference in fluorescence intensity normalized to cell confluency at 10 h for cells incubated with aggregated TDP-43, aggregated TDP-43 and isotype control, aggregated TDP-43 and cACI-7069-633B12-Ab1, and aggregated TDP-43 and hACI-7069-633B12-Ab1_H33L27. [Figure 6B] Same as above. [Figure 7A](A) Representative image of immunoblot of TDP-43 in FTLD-TDP brain extracts after immunodepletion with hACI-7069-633B12-Ab1_H33L27 (lane 1), cACI-7069-633B12-Ab1 (lane 2), or an isotype antibody control (lane 3). (B) Quantitative analysis of the signal obtained for total TDP-43 (upper panel) or the C-terminal fragment of TDP-43 (i.e., <25 kDa, CTF) (lower panel) normalized to the corresponding signal in the input. [Figure 7B] Same as above. [Figure 8] Graphical representation of TDP-43 target engagement of hACI-7069-633B12-Ab1_H33L27 in human CSF samples. [Figure 9] Serum exposure of hACI-7069-633B12-Ab1_H33L27 in cynomolgus monkeys after IV infusion once weekly for 4 weeks at doses of 40, 120, and 360 mg / kg. [Figure 10A] (A) Pharmacokinetic profiles in homozygous Tg32 mouse serum after a single IV bolus dose of hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H19L18, or cACI-7069-633B12-Ab1 at 40 mg / kg. (B) Pharmacokinetic profiles in cynomolgus monkey serum after a single IV bolus dose of hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H19L18, or cACI-7069-633B12-Ab1 at 40 mg / kg. [Figure 10B] Same as above. [Example]

[0251] [Example 1]

[0252] Humanization of anti-human TDP-43 mouse monoclonal antibody Design of humanized variable regions The variable domain of ACI-7069-633B12-Ab1 has a theoretical pI of 8.5 and a net charge of +3.1 at physiological pH. In some cases, the high positive net charge of an antibody has been shown to be responsible for the rapid clearance of the antibody in vivo (Igawa et al., PEDS. 2010; vol. 23 no. 5 pp. 385-392, Bumbaca et al., JBC, VOL. 290, NO. 50, pp. 29732-29741, 2015). To optimize the charge profile of the molecule, a pair of acceptor frameworks with low pI was selected for grafting onto the ACI-7069-633B12-Ab1 CDR (the CDR described in WO2020 / 234473). First, the heavy and light chain frameworks were ranked based on sequence identity with the mouse heavy and light chain V regions (SEQ ID NOs: 20 and 24, respectively). To identify such germline variable genes, databases of human and mouse germline variable genes such as the IMGT database (Ehrenmann, F et al., (2010) Nucl. Acids Res., 38(S1):D301-D307) or IgBlast (Ye J. et al., (2013), Nucleic Acids Res. 2013 Jul; 41(Web Server issue):W34-W40) can be used. Next, the theoretical pI of each individual chain was calculated using the web server Isoelectric Point Calculator 2.0 (Kozlowski LP et al., Nucleic Acids Res. 49(W1):W285-W292.). The average pI values ​​from all available models were taken into account along with the net charge calculated at pH 7.4.

[0253] For the heavy chain variable domain, the frameworks IGHV1-69-2, IGHV1-24, IGHV5-51, or IGHV3-43 can be used as acceptor frameworks, while for the light chain variable domain, IGKV2-24, IGKV2-40, IGKV2D-40, IGKV2D-28, IGKV4-1, or IGKV2-28 can be used as acceptor frameworks. All of the listed frameworks have the same or lower theoretical pI and net charge as the variable domain of ACI-7069-633B12-Ab1.

[0254] While a slight reduction in pI was observed with human frameworks such as IGHV1-24 and IGKV2-40, the pair formed by IGHV1-69-2 and IGKV2-28 substantially reduced the net Fv charge and provided a balanced charge distribution. Using IGHV1-69-2 as the human acceptor framework disrupted a large patch of positive charge by replacing mouse positively charged residues with substitutions K64Q, R82aS, and K73T. To a lesser extent, using germline IGKV2-28 as the VL contributes to the charge reduction with substitution K45Q in framework 2, eliminating the positive charge while maintaining the hydrophilic nature of the molecule. From this analysis, novel humanized variants based on the human frameworks of IGHV1-69-2 and IGKV2-28 were generated (Table 3).

[0255] IGHV1-69-2 and IGKV2-28 were selected as the acceptor frameworks for the heavy and light chain variable domains, respectively. IGHV1-69-2 shares 64.9% sequence identity with ACI-7069-633B12-Ab1 VH, while possessing a pI of 4.3 and a net charge of -6.9 at physiological pH. IGKV2-28 shares 72% sequence identity with ACI-7069-633B12-Ab1 VH, while possessing a pI of 4.7 and a net charge of -2.9 at physiological pH. IGHJ2*01 and IGKJ2*02 were used as J-junction genes for the heavy and light chain variable domains, respectively.

[0256] As a starting point for the humanization process, the murine CDRs were grafted onto the human acceptor framework for both the VH and VL regions. To adapt the CDRs to the human acceptor framework, key positions were modified by substituting murine residues for human residues.

[0257] To identify residues that may have the greatest impact on CDR conformation and / or VH / VL orientation, 3D models of human-mouse hybrid VH-VL pairs were generated by homology modeling using the AbodyBuilder server (8). Model analysis allowed the selection of a subset of amino acid positions, including those listed in Table 2. Numbering is according to the Kabat numbering system.

[0258] [Table 2]

[0259] Potential post-translational modification sites were identified within the ACI-7069-633B12-Ab1 CDR sequences. In the variable heavy chain, N53, N54, and G55 were identified as two deamidation sites. In the variable light chain, isomerization sites were recognized at positions D28 and G29, while an oxidation site was identified at position W89 (according to the Kabat numbering system). In some constructs, point mutations including N53Q and / or G55A were introduced into the VH region, while G29A and / or W89F were introduced into the VL region to remove post-translational modification sites in CDRs L1 and L3.

[0260] The pI and charge reductions are illustrated in Table 3.

[0261] [Table 3]

[0262] The backmutations in Table 2 were combined to generate the sequences listed in Tables 4 and 6, respectively. The corresponding nucleic acid sequences encoding the humanized TDP-43 binding molecules are shown in Tables 5 and 7.

[0263] [Table 4-1] [Table 4-2]

[0264] [Table 5-1] [Table 5-2] [Table 5-3]

[0265] [Table 6-1] [Table 6-2]

[0266] [Table 7-1] [Table 7-2] [Table 7-3] [Example 2]

[0267] Production of humanized antibody variants The DNA coding sequences for both the heavy and light chain variable domains were synthesized and cloned into plasmids using standard molecular biology techniques to enable expression in mammalian cells. The heavy chain variable domain was fused to a human IgG1 constant domain, and the light chain variable domain was cloned into a plasmid containing a constant kappa light chain domain. The chimeric antibody and humanized variants were transiently expressed in ExpiCHO-S (ThermoFischer Scientific, A29127) cells by co-transfecting the heavy and light chain plasmids using the ExpiCHO™ Expression System Kit (ThermoFischer Scientific, A29133). After transfection, the cells were maintained at 37°C with 150 rpm agitation and 8% CO2. Six days after transfection, the supernatant was harvested and purified on Protein A (Cytiva, 17127903). The antibody was captured by incubating with Protein A at room temperature for 2 hours while agitating on a roller. The mixture was poured into a gravity-flow chromatography column (BioRad, 7321010), the resin was washed with 10 CV of 2x PBS, and eluted with 0.1 M glycine, pH 3.2. The eluate was then neutralized by adding 0.1 M Tris, pH 7.4. The sample was then dialyzed against PBS buffer. [Example 3]

[0268] Characterization of ACI-7069-633B12-Ab1 humanized variants by surface plasmon resonance (SPR) Measurements were performed on a Biacore 8K instrument (GE Healthcare Life Sciences) by immobilizing soluble TDP-43 on a CM5 Series S sensor chip (GE Healthcare, BR-1005-30).

[0269] Determination of the KD of soluble TDP-43 by SPR The device was filled with operating buffer PBS-P+, and flow cells (Fc) 1 and 2 of channels 1–8 were activated with a fresh solution of EDC / NHS (Amine Coupling Kit, 1:1 ratio of both reagents, GE Healthcare Life Sciences, BR-1006-33) at 10 μL / min for 420 s. Soluble TDP-43 (Selvita) was diluted with sodium acetate, pH 4.5, to a final concentration of 5 μg / mL and injected into Fc2 at a flow rate of 5 μL / min for 900 s. All flow cells were quenched with 1 M ethanolamine (GE Healthcare Life Sciences, BR-1006-33) at 10 μL / min for 420 s. The immobilization level after the ethanolamine quench was 680 RU for all eight channels. Two start-up cycles were performed prior to analysis. Increasing concentrations of mAb, prepared in 3-fold serial dilutions ranging from 1.2 nM to 100 nM in the working buffer, were injected in single-cycle kinetics with a contact time of 300 s, a dissociation time of 3600 s, and a flow rate of 30 μL / min. Each cycle was followed by a single regeneration using 10 mM glycine-HCl, pH 1.7, with a contact time of 30 s and a 30 μL / min regeneration cycle, followed by a stabilization time of 300 s. Results from single-cycle kinetics were double-referenced using blank Fc1 and buffer cycles and evaluated using Biacore 8K evaluation software using a 1:1 kinetic fit model with RI and global Rmax. The following kinetic parameters were obtained: association rate constant (ka), dissociation rate constant (kd), affinity constant (KD), and saturation response (Rmax).

[0270] Determination of the KD of TP-51 peptide by SPR The device was filled with operating buffer PBS-P+. Fc1-2 in channels 1-8 (8K) were activated with a fresh solution of EDC / NHS (Amine Coupling Kit, 1:1 ratio of both reagents, GE Healthcare Life Sciences, BR-1006-33) at 10 μL / min for 420 s, and goat anti-human antibody (GE Healthcare Life Sciences, 29234600) was immobilized at 25 μg / mL in 10 mM sodium acetate, pH 5 for 420 s. All Fc was then quenched with 1 M ethanolamine (GE Healthcare Life Sciences, BR-1006-33) for 420 s. Any noncovalently bound antibody was removed by regeneration twice with 10 mM glycine-HCl, pH 1.7, for 30 s. Following the ethanolamine quench, the immobilization level was assessed (10,000 RU for all channels).

[0271] Each cycle began with noncovalent capture of the humanized variant, diluted to a final concentration of 2 μg / mL in working buffer and injected for 60 seconds at a flow rate of 10 μL / min. TDP-43 mAb was captured in channels 1–8, with Fc1 remaining as the blank Fc. Capture levels were assessed after a 120-second stabilization period following each mAb injection and ranged from 300–500 RU.

[0272] TP-51 consists of amino acids 352-414 of TDP-43 (SEQ ID NO: 1). Injections of the TP-51 peptide (Pepscan) were performed in single-cycle kinetics at increasing concentrations prepared in serial 3-fold dilutions ranging from 1.2 nM to 100 nM. Injections were performed with a contact time of 300 s per injection at a flow rate of 25 μL / min. After the final injection, the dissociation phase lasted 3600 s. The sensor surface was regenerated with a single 120 s injection of 10 mM glycine-HCl, pH 1.7, at a flow rate of 10 μL / min, followed by a 300 s stabilization time. Results from single-cycle kinetics were double-referenced using blank Fc1 and buffer cycles and evaluated using Biacore 8K evaluation software using a 1:1 kinetic fit model with RI and global Rmax. The following kinetic parameters were obtained: association rate constant (ka), dissociation rate constant (kd), affinity constant (KD), and saturation response (Rmax).

[0273] All parameters (except Rmax) are reported in Table 8 as the mean ± SD from 2 to 6 independent experiments.

[0274] [Table 8]

[0275] Overall, all humanized variants retained good affinity for the TDP-43 and TP-51 peptides, as observed for the murine antibody ACI-7069-633B12-Ab1 (Table 8). From the variants tested in this set of experiments, the variants with the best affinity were hACI-7069-633B12-Ab1_H28L24, hACI-7069-633B12-Ab1_H33L24, hACI-7069-633B12-Ab1_H32L26, hACI-7069-633B12-Ab1_H33L26, hACI-7069-633B12-Ab1_H32L27, and hACI-7069-633B12-Ab1_H33L27.

[0276] The humanized variants exhibited similar binding affinities for TDP-43 (ranging from 1156 to 126 pM, Table 8) compared to the parental murine antibody ACI-7069-633B12-Ab1 (328 pM, Table 8), with hACI-7069-633B12-Ab1_H33L27 having the best affinity (lowest KD) for TDP-43. Notably, the changes in pI and net charge of the humanized variant hACI-7069-633B12-Ab1_H33L27 did not affect its thermal stability, measured by differential scanning fluorometry (DSF), which remained above the 70 °C expected for standard human IgG1, nor did it affect its affinity for FcRn. [Example 4]

[0277] The ACI-7069-633B12-Ab1 humanized variant inhibits TDP-43 aggregation in vitro. hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H32L27, and hACI-7069-633B12-Ab1_H32L26 were characterized in an in vitro functional assay based on the aggregation properties of TDP-43 (Wang et al. EMBO 2018). To this end, TDP-43 was fused to recombinant maltose-binding protein (TDP-43-MBP) at its C-terminus, and aggregation was induced by removing the MBP protein using tobacco etch virus (TEV) protease. Aggregation was then monitored over time by measuring absorbance at 600 nm. It has been reported that an increase in absorbance at 600 nm correlates with the amount of aggregation. hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H32L27, and hACI-7069-633B12-Ab1_H32L26 maintained similar functional potency to the human IgG chimeric cACI-7069-633B12-Ab1 antibody (a chimera of the VH and VL of the ACI-7069-633B12-Ab1 mouse antibody with the human IgG1 constant region) in inhibiting TDP-43 aggregation at 1.5 hours. In contrast, addition of an isotype control mAb did not inhibit TDP-43 aggregation (Figure 1). "Example 5"

[0278] Pharmacokinetic evaluation of two ACI-7069-633B12-Ab1 humanized variants in Tg32 mice Transgenic Tg32 mice overexpress human fetal Fc receptors, presenting strong interest for PK evaluation and planned human PK modeling (Avery et al., 2016). A single IV bolus (40 mg / kg) of hACI-7069-633B12-Ab1_H33L27 or hACI-7069-633B12-Ab1_H32L27 was administered to 21 male Tg32 mice per compound. Blood samples were collected over a 6-week period (as shown on the y-axis in Figure 2), and serum samples (three mice per time point) were analyzed. Overall, both antibodies displayed favorable and similar PK parameters, making them desirable candidates for applications where in vivo antibody half-life is important, such as therapeutic use in humans (Figure 2, Table 9).

[0279] [Table 9] [Example 6]

[0280] Pharmacokinetic evaluation of two ACI-7069-633B12-Ab1 humanized variants in cynomolgus monkeys To evaluate the pharmacokinetics of hACI-7069-633B12-Ab1_H33L27 and hACI-7069-633B12-Ab1_H32L27 in non-human primates, a single IV bolus (40 mg / kg) was administered to four male cynomolgus monkeys per compound. Blood samples were collected over a 56-day (1344-hour) period (as shown on the y-axis in Figure 3), and serum samples (four cynomolgus monkey samples per time point) were analyzed. Three of eight animals were detected to have anti-drug antibodies (ADAs, confirmed by ELISA), a percentage consistent with previously described rates for human IgG administration in non-human primates (NHPs) (Valente et al., Mabs 2020). PK parameters were calculated after excluding animals with ADAs. Both antibodies exhibited favorable PK parameters (Figure 3, Table 10), making them suitable for further clinical development as therapeutic candidates.

[0281] [Table 10]

[0282] To preliminary evaluate tolerability, toxicity, and toxicokinetic data in non-human primates, hACI-7069-633B12-Ab1_H33L27 was administered intravenously to cynomolgus monkeys at doses of 40, 120, and 360 mg / kg once weekly for 4 weeks. Animals were sacrificed one week after the last dose. Serum samples were collected at various time points and analyzed for hACI-7069-633B12-Ab1_H33L27 concentrations. A dose-proportional increase in serum hACI-7069-633B12-Ab1_H33L27 exposure was observed, with no signs of immunogenicity during the study period (Figure 9). Furthermore, no adverse effects were observed on body weight, clinical observations, clinical pathology, urinalysis, microscopy, or organ weights in cynomolgus monkeys, confirming that the ACI-7069-633B12-Ab1 humanized variant is suitable for further clinical development. [Example 7]

[0283] Comparison of the pharmacokinetic profiles of ACI-7069-633B12-Ab1_H33L27 and the parental antibody in Tg32 mice To further evaluate the pharmacokinetics of the ACI-7069-633B12 humanized variants, a single IV bolus (40 mg / kg) of hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H19L18 (described in WO2022 / 034228), or cACI-7069-633B12-Ab1 was administered to 21 male Tg32 mice per compound. Blood collection and serum analysis were performed as described in Example 5. Data are presented as the mean ± standard deviation for 3-4 animals per group (Figure 10A). The humanized variant ACI-7069-633B12-Ab1_H33L27 exhibited a superior PK profile compared to the parent hACI-7069-633B12-Ab1_H19L18 and cACI-7069-633B12-Ab1 antibodies (Figure 10A), making it suitable for further clinical development. [Example 8]

[0284] Comparison of the pharmacokinetic profiles of ACI-7069-633B12-Ab1_H33L27 and the parent antibody in cynomolgus monkeys The pharmacokinetics of ACI-7069-633B12 humanized variants were further evaluated in NHPs (four male cynomolgus monkeys per antibody) with a single IV bolus dose (40 mg / kg) of hACI-7069-633B12-Ab1_H33L27, hACI-7069-633B12-Ab1_H19L18 (described in WO2022 / 034228), or cACI-7069-633B12-Ab1. Blood collection and serum analysis were performed as described in Example 6. Data are presented as mean ± standard deviation for 3-4 animals per group (Figure 10B). As seen in Tg32 mice, the humanized variant ACI-7069-633B12-Ab1_H33L27 exhibited a superior PK profile compared to the parent hACI-7069-633B12-Ab1_H19L18 and cACI-7069-633B12-Ab1 antibodies (Figure 10B). This result supports the suitability of the PK profile of hACI-7069-633B12-Ab1_H33L27 for further clinical development. [Example 9]

[0285] Prediction of human PK from Tg32 mice and cynomolgus monkeys Tg32 mice and cynomolgus monkeys were used as in vivo tools for predicting human clearance of mABs. The predicted PK parameters from Tg32 and cynomolgus monkeys were scaled up to 70 kg humans by allometric scaling using fixed exponents reported in the literature (Betts et al., 2018) (Table 11). The predicted human clearance in Tg32 mice and cynomolgus monkeys was 0.195 mL / h / kg and 0.256 mL / h / kg for hACI-7069-633B12-Ab1_H19L18 (described in WO2022 / 034228), compared with 0.082 mL / h / kg and 0.043 mL / h / kg for hACI-7069-633B12-Ab1_H33L27 (Table 11). This resulted in predicted human half-lives of 27 to 30 days for hACI-7069-633B12-Ab1_H33L27 and 17 to 22 days for hACI-7069-633B12-Ab1_H19L18, respectively, confirming that the predicted human PK profile of hACI-7069-633B12-Ab1_H33L27 was favorable.

[0286] [Table 11] [Example 10]

[0287] NHP serum pharmacokinetics of two ACI-7069-633B12-Ab1 humanized variants To assess serum pharmacokinetics, unbound TDP-43 levels were measured in NHPs dosed with the compounds using single molecule array (SIMOA®) technology. Baseline TDP-43 levels averaged 500 pg / ml at the pre-dose time point (Figures 4A-B). A rapid decline (>80%) in free / unbound TDP-43 was measured 3 minutes after IV injection of hACI-7069-633B12-Ab1_H33L27 or hACI-7069-633B12-Ab1_H32L27 (0.05 hours), indicating complete and rapid target saturation by these two mAbs with the native TDP-43 protein present in the serum of these NHPs. With the exception of animals that developed ADA (described in Example 6), TDP-43 remained bound to the antibodies until the end of the study, demonstrating target engagement of the administered antibodies (Figures 4A-B). [Example 11]

[0288] Neutralization of disseminating TDP-43 in sporadic ALS cerebrospinal fluid by one ACI-7069-633B12-Ab1 humanized variant To confirm the potency and mode of action of the ACI-7069-633B12-Ab1 humanized variant, we performed a real-time shaking-induced conversion (RT-QuIC) assay using a synthetic TDP-43 peptide (TP-51) as a substrate (used at 10 μM) as previously described (Scialo, C. et al. Brain Commun, fcaa142- (2020)). In the presence of cerebrospinal fluid (CSF) from a sporadic ALS donor (Figure 5A) compared to a healthy control (Figure 5B), accelerated aggregation of the substrate was observed, confirming the presence of seeding-competent TDP-43 species in ALS CSF. Each CSF used as a seed in this assay was then preincubated with hACI-7069-633B12-Ab1_H33L27 or an isotype control (used at 0.006 μM). A significant delay in aggregation of the TDP-43 peptide (TP-51) substrate was observed in the ACI-7069-633B12-Ab1_H33L27 humanized variant in CSF from ALS donors (Figure 5A-B), demonstrating that the antibody can bind to and neutralize disseminating-competent TDP-43 in the CSF of ALS patients. [Example 12]

[0289] ACI-7069-633B12-Ab1 humanized variant enhances TDP-43 uptake by THP-1 cells To evaluate and confirm the direct involvement of microglia in the clearance of TDP-43 aggregates via an Fc-dependent mechanism, an in vitro assay using THP-1 cells (a human leukemia-monocytic cell line) was set up as previously described (Lindner et al., 2020). To this end, recombinant TDP-43 aggregates were labeled with pHrodo™ dye (ThermoFisher Scientific, P36013) according to the manufacturer's instructions. pHrodo™ dye becomes fluorescent upon internalization into acidic cellular compartments, allowing for real-time monitoring of TDP-43 aggregate internalization by THP-1 cells. pHrodo™ fluorescence intensity was then automatically quantified every hour over 24 hours (Figure 6A). Data represent the mean ± SEM of 4–6 replicates per condition. Differences in fluorescence intensity normalized to cell confluency for the 10-hour time point were compared between conditions by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons (Figure 6B). Limited internalization of TDP-43 aggregates was observed in the presence of 30 nM TDP-43 aggregates alone or 30 nM isotype control antibody. However, in the presence of 30 nM hACI-7069-633B12-Ab1_H33L27 or the chimeric antibody cACI-7069-633B12-Ab1, a significant increase in uptake of pHrodo™-labeled TDP-43 aggregates was observed compared to negative control pHrodo™-labeled TDP-43 alone and in combination with the isotype control antibody (Figure 6A-B). These results confirmed that binding of the ACI-7069-633B12 humanized mutant to TDP-43 aggregates can promote the uptake of aggregates by immune cells such as microglia, and therefore their clearance. [Example 13]

[0290] Immunodepletion of TDP-43 from human brain extracts by ACI-7069-633B12-Ab1 humanized variant To evaluate the effect of the ACI-7069-633B12-Ab1 humanized variant on templated TDP-43 aggregates in vitro, immunodepletion experiments were performed using aggregated and phosphorylated TDP-43 enriched from FTLD-TDP brain extracts as previously described (WO2022 / 034228). Immunodepleted fractions demonstrated significant depletion of TDP-43 compared to the isotype control using hACI-7069-633B12-Ab1_H33L27 or the chimeric antibody cACI-7069-633B12-Ab1 (Figure 7A). For quantitative analysis, the signals obtained for total TDP-43 or C-terminal fragments of TDP-43 (i.e., <25 kDa, CTF) were normalized to the corresponding signals in the input (Figure 7B). These results confirmed the ability of the ACI-7069-633B12-Ab1 humanized variant to bind to aggregated and phosphorylated TDP-43 from the brains of FTLD-TDP patients, confirming its therapeutic potential. [Example 14]

[0291] ACI-7069-633B12-Ab1 humanized variant binds with high affinity to human TDP-43 in CSF To characterize the binding affinity of the ACI-7069-633B12-Ab1 humanized variant to TDP-43 in human CSF, CSF samples from healthy donors were preincubated with increasing concentrations of hACI-7069-633B12-Ab1_H33L27, and the amount of unbound TDP-43 was measured using a SIMOA-based target engagement assay as described in Example 10. An EC50 of 35.58 ng / ml (237 pM) was obtained for the binding of hACI-7069-633B12-Ab1_H33L27 to human TDP-43 in CSF samples (Figure 8). This result confirmed the ability of the ACI-7069-633B12-Ab1 humanized variant to bind TDP-43 in CSF with high affinity.

[0292] References [Table 12] [Table 13] Table 14 Table 15 Table 16

Claims

1. a. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; b. a VH-CDR2 selected from the amino acid sequence of SEQ ID NO: 22, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 102, SEQ ID NO: 112, or SEQ ID NO: 122; c. VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); d. a VL-CDR1 selected from the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; e. a VL-CDR2 selected from the amino acid sequence of SEQ ID NO: 16; f. a VL-CDR3 selected from the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87; and g. An acceptor framework for the heavy chain variable domain (VH) of IGHV1-69-2; and h. Acceptor framework for the light chain variable domain (VL) of IGKV2-28 A humanized TDP-43 binding molecule that is an antibody or an antigen-binding fragment thereof, comprising:

2. The VH acceptor framework has the following VH mutations: a. V24T; b. Y27F; c. M48I; d. A71V; e. T73K; and f. T94R and / or the acceptor framework of the VL comprises one or more of the following VL mutations: g. I2V; h.Y36L; i. Q45K; j.L46R; and K. G57R 2. The humanized TDP-43 binding molecule of claim 1, comprising one or more of:

3. The VH acceptor framework has the following VH mutations: a. Y27F; and b. T94R and the VL acceptor framework comprises the following VL mutations: c.Y36L; d. L46R; and e. G57R 3. The humanized TDP-43 binding molecule of claim 2, comprising:

4. a. VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; b. a VH-CDR2 selected from the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 122; c. VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); d. a VL-CDR1 selected from the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 85; e. a VL-CDR2 selected from the amino acid sequence of SEQ ID NO: 16; and f. A VL-CDR3 selected from the amino acid sequence of SEQ ID NO: 27 or SEQ ID NO: 87 2. The humanized TDP-43 binding molecule of claim 1, comprising:

5. a. a heavy chain variable domain (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 22; a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or b. a heavy chain variable domain (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or c. a heavy chain variable domain (VH) comprising a VH-CDR1 having the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 having the amino acid sequence of SEQ ID NO: 112; a VH-CDR3 having the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 having the amino acid sequence of SEQ ID NO: 85; a VL-CDR2 having the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 87; or d. a heavy chain variable domain (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 27; or e. a heavy chain variable domain (VH) comprising a VH-CDR1 having the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 having the amino acid sequence of SEQ ID NO: 122; a VH-CDR3 having the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 having the amino acid sequence of SEQ ID NO: 85; a VL-CDR2 having the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 having the amino acid sequence of SEQ ID NO: 87; or f. a heavy chain variable domain (VH) comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 112; a VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or g. A heavy chain variable domain (VH) comprising a VH-CDR1 having the amino acid sequence of SEQ ID NO: 21; a VH-CDR2 having the amino acid sequence of SEQ ID NO: 122; a VH-CDR3 having the amino acid sequence ES (Glu-Ser); and a light chain variable domain (VL) comprising a VL-CDR1 having the amino acid sequence of SEQ ID NO: 25; a VL-CDR2 having the amino acid sequence of SEQ ID NO: 16; and a VL-CDR3 having the amino acid sequence of SEQ ID NO:

87.

2. The humanized TDP-43 binding molecule of claim 1, comprising:

6. a. a heavy chain variable region (VH) chosen from: i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 30, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; or ii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 40, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 40; or iii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 50, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 50; or iv. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 60; or v. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; or vi. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 80, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 80; or vii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 90, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 90; or viii. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 100, or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100; or ix. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; or x. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, or having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120; and b. A light chain variable region (VL) chosen from: i. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 34, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or ii. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or iii. A light chain variable region (VL) comprising the sequence of SEQ ID NO: 54, or a light chain variable region (VL) having at least 98%, or 99%, sequence identity to the amino acid sequence of SEQ ID NO: 54; or iv. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or v. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 74, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or vi. a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or vii. A light chain variable region (VL) comprising the sequence of SEQ ID NO: 94, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94; 2. The humanized TDP-43 binding molecule of claim 1, comprising:

7. a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 60; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54, or a light chain variable region (VL) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54, or a light chain variable region (VL) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 54; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, or a heavy chain variable region (VH) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 70; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or f. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or g. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 64; or h. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84, or a light chain variable region (VL) having at least 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, or a heavy chain variable region (VH) having at least 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 110; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94; or j. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, or a heavy chain variable region (VH) having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 120; and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94, or a light chain variable region (VL) having at least 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

94.

2. The humanized TDP-43 binding molecule of claim 1, comprising:

8. a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 60, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 44; or c. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 54; or d. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 70, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or e. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or f. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or g. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 64; or h. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or i. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94; or j. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

94.

2. The humanized TDP-43 binding molecule of claim 1, comprising:

9. a. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 84; or b. a heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 110, and a light chain variable region (VL) comprising the sequence of SEQ ID NO: 94; or c. A heavy chain variable region (VH) comprising the sequence of SEQ ID NO: 120, and a light chain variable region (VL) comprising the sequence of SEQ ID NO:

94.

2. The humanized TDP-43 binding molecule of claim 1, comprising:

10. a. Binds to misfolded, aggregated TDP-43 and non-aggregated physiological TDP-43; b. binds to monomeric and / or oligomeric and / or aggregated and / or post-translationally modified and / or cleaved TDP-43, preferably human TDP-43; c. Binds to misfolded, aggregated human TDP-43 and non-aggregated physiological human TDP-43; d. Neutralizes seeding-competent TDP-43; e. Promotes the clearance of TDP-43; f. binds to an epitope within amino acid residues 397-411 of human TDP-43 (SEQ ID NO: 1); g. has a dissociation constant (KD) for binding to soluble TDP-43 (SEQ ID NO: 1) of less than 1 nM, preferably less than 250 pM; and / or h. Has a dissociation constant (KD) for binding to soluble TP-51 (amino acids 352-414 of SEQ ID NO: 1) peptide of less than 4 nM, preferably less than 2 nM; The humanized TDP-43 binding molecule of claim 1.

11. a. Reduces TDP-43 pathology in vivo; and / or b. reducing the levels of misfolded, aggregated TDP-43 and / or phosphorylated TDP-43 in vivo; The humanized TDP-43 binding molecule of claim 1.

12. The humanized TDP-43 binding molecule of claim 1, wherein the humanized TDP-43 binding molecule is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody or antigen-binding fragment thereof, preferably an IgG1 or IgG4 antibody, and / or the humanized TDP-43 binding molecule comprises an Fc mutation, preferably an S228P mutation.

13. The humanized TDP-43 binding molecule of claim 1, wherein the humanized TDP-43 binding molecule (a) comprises an Fv having a pI of less than 7.5, preferably less than 7.0, and / or (b) comprises an Fv having a net charge at pH 7.4 of less than 0.2, preferably less than -0.8, more preferably less than -1.

8.

14. 14. An immunoconjugate comprising the humanized TDP-43 binding molecule of any one of claims 1 to 13, optionally crossing the blood-brain barrier using a delivery vehicle or a blood-brain barrier moiety, and optionally further comprising: a. the delivery vehicle comprises a liposome or extracellular vesicle; b. The humanized TDP-43 binding molecule is linked to a blood-brain barrier portion; c. the blood-brain barrier moiety is a polypeptide or small molecule, preferably a peptide, a receptor ligand, a single domain antibody (VHH), an scFv, or a Fab fragment; d. the blood-brain barrier moiety binds to a blood-brain barrier receptor, optionally the blood-brain barrier receptor includes, but is not limited to, a transferrin receptor, an insulin receptor, or a low-density lipoprotein receptor; 10. An immunoconjugate comprising:

15. A labeled binding molecule comprising the humanized TDP-43 binding molecule of any one of claims 1 to 13.

16. A pharmaceutical composition comprising a humanized TDP-43 binding molecule according to any one of claims 1 to 13 or an immunoconjugate comprising the humanized TDP-43 binding molecule according to any one of claims 1 to 13, and a pharmaceutically acceptable carrier and / or excipient and / or diluent.

17. 14. A pharmaceutical composition comprising a humanized TDP-43 binding molecule of any one of claims 1 to 13, or an immunoconjugate comprising the humanized TDP-43 binding molecule of any one of claims 1 to 13, for human or veterinary use.

18. 10. A pharmaceutical composition comprising the humanized TDP-43 binding molecule of claim 1 or an immunoconjugate comprising the humanized TDP-43 binding molecule of claim 1 for use in the prevention, amelioration, or treatment of a disease, disorder, and / or disorder associated with TDP-43, or a TDP-43 proteinopathy.

19. 10. A pharmaceutical composition comprising the humanized TDP-43 binding molecule of claim 1, or an immunoconjugate comprising the humanized TDP-43 binding molecule of claim 1, or a labeled binding molecule comprising the humanized TDP-43 binding molecule of claim 1, for use in the diagnosis of a TDP-43-associated disease, disorder, and / or disorder, or a TDP-43 proteinopathy, optionally for use as a diagnostic tool for monitoring a TDP-43-associated disease, disorder, and / or disorder, or a TDP-43 proteinopathy.

20. 14. A humanized TDP-43 binding molecule according to any one of claims 1 to 13, or an immunoconjugate comprising the humanized TDP-43 binding molecule according to any one of claims 1 to 13, or a labeled binding molecule comprising the humanized TDP-43 binding molecule according to any one of claims 1 to 13, or a pharmaceutical composition comprising the humanized TDP-43 binding molecule according to any one of claims 1 to 13, for research use, in particular as an analytical tool or reference molecule.

21. Diseases, disorders, and / or abnormalities associated with TDP-43 or TDP-43 proteinopathies are known to be associated with frontotemporal dementia (e.g., chromosome 9p-linked, with progranulin (GRN) mutations, with C9orf72 mutations, with TARDBP mutations, with valosin-containing protein (VCP) mutations), sporadic or familial FTD with or without motor neuron disease (MND), large intestine, thyroid cancer ... Corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), argyrophilic grain disease, Pick's disease, semantic variant of primary progressive aphasia (svPPA), behavioral variant of FTD (bvFTD), non-fluent variant of primary progressive aphasia (nfvPPA), etc.), amyotrophic lateral sclerosis (e.g., sporadic ALS, with TARDBP mutations, with angiogenin (ANG) mutations) ALS), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy, Perry syndrome, Alzheimer's disease (AD, including sporadic and familial forms of AD), Down's syndrome, familial British dementia, polyglutamine diseases (Huntington's disease and spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease)), hippocampal sclerosis dementia and myopathy (sporadic inclusion body myositis, inclusion body myopathy with mutations in valosin-containing protein (VCP); also Paget's disease of bone and frontotemporal dementia), oculopharyngeal muscular dystrophy with limbic vesicles, myofiber myopathy with mutations in the myotilin (MYOT) gene or mutations in the gene encoding desmin (DES), traumatic brain injury (TBI), dementia with Lewy bodies (DLB), or Parkinson's disease (PD), preferably The TDP-43 related disease, disorder, and / or abnormality, or TDP-43 proteinopathy, is selected from the group consisting of: a. Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), chronic traumatic encephalopathy (CTE), or limbic-predominant age-related TDP-43 encephalopathy (LATE); b. Amyotrophic lateral sclerosis (ALS); c. Alzheimer's disease (AD); or d) Frontotemporal dementia (FTD), The pharmaceutical composition according to claim 18 or 19.

22. (a) A method for maintaining or increasing recognition memory performance or delaying memory loss in an individual having a TDP-43-related disease, disorder, and / or abnormality, or TDP-43 proteinopathy; or (b) a method for reducing the level of aggregated TDP-43 and / or phosphorylated TDP-43 in an individual; 14. A pharmaceutical composition comprising a humanized TDP-43 binding molecule of any one of claims 1 to 13 or an immunoconjugate comprising the humanized TDP-43 binding molecule of any one of claims 1 to 13 for use in A pharmaceutical composition optionally comprising administering at least one additional therapeutic agent, optionally wherein the additional therapeutic agent targets alpha synuclein, BACE1, tau, beta amyloid, TDP-43, or a neuroinflammatory protein.

23. A gene encoding the humanized TDP-43 binding molecule of any one of claims 1 to 13; b. encoding a humanized TDP-43 binding molecule comprising the nucleotide sequence set forth in SEQ ID NO:38, SEQ ID NO:48, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:78, SEQ ID NO:88, SEQ ID NO:98, SEQ ID NO:108, SEQ ID NO:118, SEQ ID NO:128, SEQ ID NO:39, SEQ ID NO:49, SEQ ID NO:59, SEQ ID NO:69, SEQ ID NO:79, SEQ ID NO:89, or SEQ ID NO:99; and / or c. below: a) a heavy chain variable region (VH) encoded by SEQ ID NO:68 and a light chain variable region (VL) encoded by SEQ ID NO:59; or b) a heavy chain variable region (VH) encoded by SEQ ID NO:78 and a light chain variable region (VL) encoded by SEQ ID NO:49; or c) a heavy chain variable region (VH) encoded by SEQ ID NO:78 and a light chain variable region (VL) encoded by SEQ ID NO:59; or d) a heavy chain variable region (VH) encoded by SEQ ID NO:78 and a light chain variable region (VL) encoded by SEQ ID NO:69; or e) a heavy chain variable region (VH) encoded by SEQ ID NO: 118 and a light chain variable region (VL) encoded by SEQ ID NO: 69; or f) a heavy chain variable region (VH) encoded by SEQ ID NO: 118 and a light chain variable region (VL) encoded by SEQ ID NO: 89; or g) a heavy chain variable region (VH) encoded by SEQ ID NO: 128 and a light chain variable region (VL) encoded by SEQ ID NO: 69; or h) a heavy chain variable region (VH) encoded by SEQ ID NO: 128 and a light chain variable region (VL) encoded by SEQ ID NO: 89; or i) a heavy chain variable region (VH) encoded by SEQ ID NO: 118 and a light chain variable region (VL) encoded by SEQ ID NO: 99; or j) a heavy chain variable region (VH) encoded by SEQ ID NO: 128 and a light chain variable region (VL) encoded by SEQ ID NO: 99 encoding a humanized TDP-43 binding molecule comprising the nucleotide sequence shown as Nucleic acid molecule.

24. The nucleic acid molecule of claim 23, wherein the nucleic acid is part of a viral vector for targeted delivery to the blood-brain barrier or any other cell type in the CNS, and optionally the viral vector is a recombinant adeno-associated viral vector (rAAV), preferably a recombinant adeno-associated viral vector selected from AAV1 to AAV12.

25. 24. A recombinant expression vector comprising the nucleic acid molecule of claim 23. (a) comprising a nucleic acid encoding the humanized TDP-43 binding molecule of claim 1 and / or a vector comprising a nucleic acid encoding the humanized TDP-43 binding molecule of claim 1; or (b) a host cell expressing the humanized TDP-43 binding molecule of claim 1.

27. A cell-free expression system containing a recombinant expression vector comprising a nucleic acid encoding the humanized TDP-43 binding molecule of claim 1.

28. The process of claim 27: a. culturing the host cell of claim 26 or the cell-free expression system of claim 27 under conditions suitable for producing a humanized TDP-43 binding molecule; and b. Isolating the humanized TDP-43 binding molecule A method for producing a humanized TDP-43 binding molecule, comprising:

29. A method for detecting and / or quantifying TDP-43 in a sample obtained from a subject, the method comprising contacting the sample with a humanized TDP-43 binding molecule according to any one of claims 1 to 13, and comparing the level of TDP-43 in the sample with the level of TDP-43 in one or more control samples, optionally wherein the sample is human blood, cerebrospinal fluid (CSF), interstitial fluid (ISF) and / or urine, preferably CSF.

30. A kit for the diagnosis of a TDP-43-related disease, disorder and / or disorder, or a TDP-43 proteinopathy, comprising the humanized TDP-43 binding molecule of any one of claims 1 to 13.

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