Anti-TDP-43 Binding Molecules and Uses Thereof
20220315648 · 2022-10-06
Inventors
Cpc classification
G01N2800/52
PHYSICS
G01N2800/2835
PHYSICS
A61P25/28
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention is in the field of transactive response DNA binding protein with a molecular weight of 43 kDa (TARDB or also TDP-43). The invention relates to TDP-43 specific binding molecules, in particular to anti-TDP-43 antibodies or an antigen-binding fragment or a derivative thereof and uses thereof. The present invention provides means and methods to diagnose, prevent, alleviate and/or treat a disease, disorder and/or abnormality 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 Encelopathy (CTE), and limbic-predominant age-related TDP-43 encephalopathy (LATE).
Claims
1. A TDP-43 binding molecule, which binds misfolded aggregated TDP-43 and non-aggregated physiological TDP-43.
2. The TDP-43 binding molecule of claim 1, which binds misfolded aggregated human TDP-43 and non-aggregated physiological human TDP-43.
3. The TDP-43 binding molecule of claim 1 or 2, which exhibits one or more, up to all of the following characteristics: a) inhibits the aggregation of TDP-43 protein or fragments thereof, b) blocks TDP-43 cell-to-cell propagation, c) disaggregates TDP-43 aggregates; and d) blocks TDP-43 seeding.
4. The TDP-43 binding molecule of any one of the preceeding claims, which reduces TDP-43 pathology in vivo.
5. The TDP-43 binding molecule of any one of the preceeding claims, which reduces levels of aggregated TDP-43 and/or phosphorylated TDP-43 in vivo.
6. The TDP-43 binding molecule of any one of the preceding claims, which binds to an epitope within amino acids residues 181-195, 199-213, 307-321, 352-366, 389-411, 397-411 or 140-200, of human TDP-43 (SEQ ID NO: 1) or to an equivalent epitope in non-human TDP-43.
7. The TDP-43 binding molecule of any one of the preceding claims, which binds to an epitope within amino acids residues 183-188, 203-213, 204-208, 204-211, 205-210, 316-323, 358-361, 400-405, 400-406 or 400-412 of human TDP-43 (SEQ ID NO: 1) or to an equivalent epitope in non-human TDP-43.
8. The TDP-43 binding molecule of any one of the preceding claims, which binds to an epitope within amino acids residues 400-405, 400-406 or 400-412 of human TDP-43 (SEQ ID NO: 1) or to an equivalent epitope in non-human TDP-43.
9. The TDP-43 binding molecule of any one of the preceding claims, which is an antibody or an antigen-binding fragment thereof.
10. The TDP-43 binding molecule of any one of the preceding claims, which comprises: a) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 11; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and VH-CDR3 comprising the amino acid sequence ES (Glu-Ser); VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 15; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or b) 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); 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 c) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 31; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 32; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 33; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 35; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 36; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 37; or d) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 45; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 46; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 47; or e) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 61; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 62; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 63; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 65; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 66; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 67; or f) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 71; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 72; and VH-CDR3 comprising the amino acid sequence 73; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 75; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 77; or g) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 81; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 83; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 86; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or h) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 101; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 102; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 103; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 105; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 106; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 107; or i) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 121; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 122; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 123; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 125; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 16; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 127; or j) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 141; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 142; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 143; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 145; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 146; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 147; or k) VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 151; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 152; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 153; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 155; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 156; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 157.
11. The TDP-43 binding molecule of any one of the preceding claims, which comprises: a. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 10 or a Heavy Chain Variable Region (VH) having at least 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 10; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 14 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: 14; or b. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 20 or a Heavy Chain Variable Region (VH) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 24 or a Light Chain Variable Region (VL) having at least 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 24; or c. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 30 or a Heavy Chain Variable Region (VH) having at least 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 30; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 34 or a Light Chain Variable Region (VL) having at least 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34; or d. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 40 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: 40; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 44; or e. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 60 or a Heavy Chain Variable Region (VH) having at least 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: 64 or a Light Chain Variable Region (VL) having at least 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: 70 or a Heavy Chain Variable Region (VH) having at least 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: 74 or a Light Chain Variable Region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 74; or g. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 80 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: 80; 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 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 84; or h. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 100 or a Heavy Chain Variable Region (VH) having at least 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 104 or a Light Chain Variable Region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 104; or i. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 120 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: 120; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 124 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: 124; or j. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 140 or a Heavy Chain Variable Region (VH) having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 140; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 144; or k. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 150 or a Heavy Chain Variable Region (VH) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 150; and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 154 or a Light Chain Variable Region (VL) having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 154.
12. The TDP-43 binding molecule of any one of the preceding claims, which comprises: a. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 10 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 14; or b. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 20 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 24; or c. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 30 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 34; or d. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 40 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 44; or e. 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: 64; or f. 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: 74; or g. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 80 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 84; or h. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 100 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 104; or i. 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: 124; or j. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 140 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 144; or k. a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 150 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 154.
13. The TDP-43 binding molecule of any one of the preceding claims, which 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); 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.
14. The TDP-43 binding molecule of any one of the preceding claims, which comprises a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 20 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 24.
15. The TDP-43 binding molecule of any one claims 1 to 12, which comprises VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 81; VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 82; and VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 83; VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 85; VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 86; and VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 87.
16. The TDP-43 binding molecule of any one claims 1 to 12 or 15, which comprises a Heavy Chain Variable Region (VH) comprising the sequence of SEQ ID NO: 80 and a Light Chain Variable Region (VL) comprising the sequence of SEQ ID NO: 84
17. The TDP-43 binding molecule of any one of the preceding claims, which is a monoclonal antibody or an antigen-binding fragment thereof.
18. The TDP-43 binding molecule of any one of the preceding claims, which is a murine, a chimeric, a humanized or a human antibody or an antigen-binding fragment thereof.
19. The TDP-43 binding molecule of any one of the preceding claims, which is an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4 antibody or antigen-binding fragment thereof.
20. The TDP-43 binding molecule of any one of the preceding claims for use in human or veterinary therapy and/or diagnosis.
21. The TDP-43 binding molecule of any one of the preceding claims for use in human or veterinary therapy and/or diagnosis, wherein the TDP-43 binding molecule is a diagnostic or a therapeutic tool.
22. The TDP-43 binding molecule of any one of claims 1 to 19 for research use, in particular as an analytical tool or reference molecule.
23. The TDP-43 binding molecule of any one of the preceding claims for use in the prevention, alleviation, treatment and/or diagnosis of diseases, disorders and/or abnormalities associated with TDP-43.
24. The TDP-43 binding molecule of any one of the preceding claims for use in the prevention, alleviation, treatment and/or diagnosis of a TDP-43 proteinopathy.
25. The TDP-43 binding molecule for use of claim 21 for use as a diagnostic tool to monitor a TDP-43 proteinopathy.
26. The TDP-43 binding molecule for use according to any one of claims 23 to 25, wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Frontotemporal dementia (FTD, such as Sporadic or familial with or without motor-neuron disease (MND), with progranulin (GRN) mutation, with C9orf72 mutations, with TARDBP mutation, with valosine-containing protein (VCP) mutation, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), Argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), Nonfluent Variant Primary Progressive Aphasia (nfvPPA) and the like), Amyotrophic lateral sclerosis (ALS, such as Sporadic ALS, with TARDBP mutation, with angiogenin (ANG) mutation), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), Chronic Traumatic Encelopathy, 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 under Machado Joseph Disease)), Hippocampal sclerosis dementia and Myopathies (Sporadic inclusion body myositis, Inclusion body myopathy with a mutation in the valosin-containing protein (VCP); also Paget disease of bone and frontotemporal dementia), Oculo-pharyngeal muscular dystrophy with rimmed vacuoles, Myofibrillar myopathies with mutations in the myotilin (MYOT) gene or mutations in the gene coding for desmin (DES), Traumatic Brain Injury (TBI), Dementia with Lewy Bodies (DLB) or Parkinson's disease (PD).
27. The TDP-43 binding molecule for use according to claim 26, wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Chronic Traumatic Encelopathy (CTE), or limbic-predominant age-related TDP-43 encephalopathy (LATE).
28. The TDP-43 binding molecule for use according to claim 26 or 27, wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is amyotrophic lateral sclerosis (ALS).
29. The TDP-43 binding molecule for use according to claim 26 or 27, wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Alzheimer's disease (AD).
30. The TDP-43 binding molecule for use according to claim 26 or 27, wherein the disease, disorder, and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Frontotemporal dementia (FTD).
31. A pharmaceutical composition comprising the TDP-43 binding molecule of any one of the preceding claims and a pharmaceutically acceptable carrier and/or excipient.
32. A nucleic acid molecule encoding the TDP-43 binding molecule of any one of the preceding claims.
33. A nucleic acid molecule comprising a nucleotide sequence set forth as: a. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 18 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 19; or b. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 28 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 29; or c. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 38 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 39; or d. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 48 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 49; or e. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 68 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 69; or f. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 78 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 79; or g. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 88 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 89; or h. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 108 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 109 or i. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 128 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 129; or j. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 148 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 149; or k. a Heavy Chain Variable Region (VH) encoding sequence of SEQ ID NO: 158 and a Light Chain Variable Region (VL) encoding sequence of SEQ ID NO: 159.
34. A recombinant vector comprising the nucleic acid of claim 32 or 33.
35. A host cell comprising the nucleic acid of claim 32 or 33 and/or the vector of claim 34.
36. A host cell that expresses a TDP-43 binding molecule according to any one of claims 1 to 30.
37. An expression vector comprising the nucleic acid molecule of claim 32 or 33.
38. A cell-free expression system containing the expression vector of claim 37.
39. A method for producing a TDP-43 binding molecule, in particular an antibody or antigen-binding fragment thereof, comprising the steps of: a) culturing the host cell of claim 35 or 36 or cell-free expression system of claim 38 under conditions suitable for producing the binding molecule, in particular the antibody or antigen-binding fragment thereof, and b) isolating the binding molecule, in particular the antibody or antigen-binding fragment thereof.
40. A method of quantifying TDP-43 in a sample obtained from a subject, the method comprising contacting the sample with a TDP-43 binding molecule according to any one of claims 1 to 30 and comparing the TDP-43 levels in the sample to those in a control sample or samples.
41. A method for diagnosing a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy comprising performing the method of claim 40 wherein higher levels of TDP-43 in the sample compared with a control level based on healthy subjects are indicative of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy.
42. A method for diagnosing a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy comprising performing the method of claim 40 or 41 wherein similar or higher levels of TDP-43 in the sample compared with a diseased control are indicative of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy.
43. A method for classifying a disease, disorder and/or abnormality associated with TDP-43 or for classifying a TDP-43 proteinopathy comprising: a. performing the method of claim 41 and/or 42, b. optionally identifying mutations in the sample including but not limited to progranulin (GRN) mutation, C9orf72 mutations, TARDBP mutation, with valosine-containing protein (VCP) mutation, TARDBP mutation, angiogenin (ANG) mutation), mutation in the valosin-containing protein (VCP), mutation in the myotilin (MYOT) gene or mutations in the gene coding for desmin (DES), and c. classifying the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy.
44. A method for classifying a disease, disorder and/or abnormality associated with TDP-43 or for classifying a TDP-43 proteinopathy comprising: a. performing the method of claim 42 in a sample obtained from a subject with a disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, wherein the comparison with a diseased control is based on a plurality of control samples from subjects with different types or subtypes of disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy; and b. classifying the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy based on the comparison.
45. A method for monitoring a disease, disorder and/or abnormality associated with TDP-43 or for monitoring a TDP-43 proteinopathy at two or more time points using samples from a subject, the method comprising contacting the samples with a TDP-43 binding molecule according to any one of claims 1 to 30 wherein higher levels of TDP-43 in the later sample compared with one or more earlier samples are indicative of progression of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy.
46. A method for monitoring a disease, disorder and/or abnormality associated with TDP-43 or for monitoring a TDP-43 proteinopathy at two or more time points using samples from a subject, the method comprising contacting the samples with a TDP-43 binding molecule according to any one of claims 1 to 30 wherein lower levels of TDP-43 in the later sample compared with one or more earlier samples are indicative of regression of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy.
47. A method for monitoring treatment of a disease, disorder and/or abnormality associated with TDP-43 or for monitoring treatment of a TDP-43 proteinopathy at two or more time points using samples from a subject treated with a particular therapy, the method comprising contacting the samples with a TDP-43 binding molecule according to any one of claims 1 to 30 wherein lower levels of TDP-43 in the later sample compared with one or more earlier samples are indicative of successful treatment of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy.
48. The method of any one of claims 45 to 47 wherein a first time point is prior to treatment with the therapy and a second time point is following treatment with the therapy.
49. A method for selecting a therapy for treatment of a disease, disorder and/or abnormality associated with TDP-43 or selecting a therapy for treatment of a TDP-43 proteinopathy, the method comprising contacting samples taken before and after treatment with the therapy with a TDP-43 binding molecule according to any one of claims 1 to 30 wherein lower levels of TDP-43 in the sample taken after treatment compared with the sample taken before treatment are indicative of successful treatment of a disease, disorder and/or abnormality associated with TDP-43 or a TDP-43 proteinopathy and thus the therapy is selected for treatment.
50. The method of claim 49 wherein the therapy comprises a TDP-43 binding molecule according to any one of claims 1 to 30 or a pharmaceutical composition as claimed in claim 31.
51. The method according to any one of claims 40 to 50 wherein the sample comprises a blood, CSF, ISF or urine sample.
52. The method according to any one of claims 40 to 51 wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Frontotemporal dementia (FTD, such as Sporadic or familial with or without motor-neuron disease (MND), with progranulin (GRN) mutation, with C9orf72 mutations, with TARDBP mutation, with valosine-containing protein (VCP) mutation, linked to chromosome 9p, corticobasal degeneration, frontotemporal lobar degeneration (FTLD) with ubiquitin-positive TDP-43 inclusions (FTLD-TDP), Argyrophilic grain disease, Pick's disease, semantic variant primary progressive aphasia (svPPA), behavioural variant FTD (bvFTD), Nonfluent Variant Primary Progressive Aphasia (nfvPPA) and the like), Amyotrophic lateral sclerosis (ALS, such as Sporadic ALS, with TARDBP mutation, with angiogenin (ANG) mutation), Alexander disease (AxD), limbic-predominant age-related TDP-43 encephalopathy (LATE), Chronic Traumatic Encelopathy, 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 under Machado Joseph Disease)), Hippocampal sclerosis dementia and Myopathies (Sporadic inclusion body myositis, Inclusion body myopathy with a mutation in the valosin-containing protein (VCP); also Paget disease of bone and frontotemporal dementia), Oculo-pharyngeal muscular dystrophy with rimmed vacuoles, Myofibrillar myopathies with mutations in the myotilin (MYOT) gene or mutations in the gene coding for desmin (DES), Traumatic Brain Injury (TBI), Dementia with Lewy Bodies (DLB) or Parkinson's disease (PD).
53. The method according to claim 52 wherein the disease, disorder and/or abnormality associated with TDP-43 or TDP-43 proteinopathy comprises Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Chronic Traumatic Encelopathy (CTE), or limbic-predominant age-related TDP-43 encephalopathy (LATE).
54. The method according to claim 52 wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is amyotrophic lateral sclerosis (ALS).
55. The method according to claim 52, wherein the disease or disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Alzheimer's disease (AD).
56. The method according to claim 52, wherein the disease, disorder and/or abnormality associated with TDP-43, or TDP-43 proteinopathy, is Frontotemporal dementia (FTD).
57. A kit for diagnosis of a disease, disorder and/or abnormality associated with TDP-43, or a TDP-43 proteinopathy, or for use in a method of any one of claims 40 to 56 comprising a TDP-43 binding molecule according to any one of claims 1 to 30.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0359]
[0360]
[0361]
[0362]
[0363]
[0364]
[0365]
EXAMPLES
Example 1: Preparation of a TDP-43 Vaccine Composition
[0366] The liposome-based vaccines were prepared according to the protocols published in WO2012/055933. Vaccines containing full length TDP-43 (FL TDP-43) protein as antigen (Table 2, SEQ ID NO: 1) were used for antibody generation.
TABLE-US-00002 TABLE 2 TDP-43 protein and peptide antigen description SEQ ID NO Definition Amino acid sequence (1-letter code) SEQ ID NO: 1 Q13148 (UniProt) MSEYIRVTEDENDEPIEIPSEDDGTVLLSTVTAQFPGAC TADBP_HUMAN GLRYRNPVSQCMRGVRLVEGILHAPDAGWGNLVYVV TAR DNA-binding NYPKDNKRKMDETDASSAVKVKRAVQKTSDLIVLGL protein 43 PWKTTEQDLKEYFSTFGEVLMVQVKKDLKTGHSKGF aa 1-414 GFVRFTEYETQVKVMSQRHMIDGRWCDCKLPNSKQS QDEPLRSRKVFVGRCTEDMTEDELREFFSQYGDVMDV FIPKPFRAFAFVTFADDQIAQSLCGEDLIIKGISVHISNA EPKHNSNRQLERSGRFGGNPGGFGNQGGFGNSRGGG AGLGNNQGSNMGGGMNFGAFSINPAMMAAAQAALQ SSWGMMGMLASQQNQSGPSGNNQNQGNMQREPNQA FGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGS SMDSKSSGWGM
Example 2: Generation of Anti-TDP-43 Antibodies
[0367] A. Mouse Immunization
[0368] Female C57BL/6JOlaHsd (C57BL/6) and BALB/c OlaHsd (BALB/c) wild-type mice (Harlan, USA) were received at 9 weeks of age. Vaccinations started at 10 weeks. Mice were vaccinated with full-length TDP-43 protein presented on the surface of liposomes in the presence of Monophosphoryl Hexa-acyl Lipid A, 3-Deacyl (Synthetic) (3D-(6-acyl) PHAD®) as adjuvant.
[0369] Mice were vaccinated by subcutaneous injection (s.c.) on days 0, 4, 8, 21, 35, and 60. Mice were bled and heparinized plasma prepared 7 days before immunization (pre-immune plasma) and on days 14, 28, 42, 81 and 121 after first immunization. Mice used for myeloma fusion were additionally vaccinated with three daily booster injections of TDP-43 protein per i.p. injection without adjuvant.
[0370] Vaccine response was measured in mouse plasma. Binding of plasma derived antibodies from immunized mice to immobilized recombinant full-length (FL) TDP-43 indicated high titers for antibodies against TDP-43.
[0371] B. Generation of Hybridomas and Selection for Subcloning
[0372] Mice were euthanized and fusion with myeloma cells was performed using splenocytes from four individual mice. Screening for antibodies from the successfully fused hybridoma cell lines were performed as follows. Diluted (1:32) cell culture supernatants were analyzed using Luminex bead-based multiplex assay (Luminex, The Netherlands). Luminex beads were conjugated to FL TDP-43 and with capturing IgGs with anti-mouse IgG-Fc antibodies specific for the IgG1, IgG2a, IgG2b, IgG2c, and IgG3 subclasses (Jackson Immunoresearch, USA). Binding to beads conjugated to FL TDP-43 identified 386 hits derived from mice immunized with the FL TDP-43 liposomal vaccine.
[0373] Viable hybridomas were grown using serum-containing selection media. Clones with preferential binding to TDP-43 inclusions in human FTD brain and clones binding to C-terminus of TDP-43 were selected for further subcloning. Following limiting dilution, the clonal hybridomas were grown in low immunoglobulin containing medium and stable colonies were selected for antibody screening and selection. Antibodies shown in Table 3 were identified from this screen.
Example 3: Determination of Binding Efficacy (EC50)
[0374] Luminex assays with serial dilution of antibodies were performed as described before to determine half maximal effective concentration (EC50) of binding of antibodies to FL TDP-43. All EC50 values are summarized in Table 3. In summary, all tested antibodies bind to full length TDP-43 with high affinity.
TABLE-US-00003 TABLE 3 EC50 values determined by Luminex Assay EC50 (μM) Hybridoma κ, λ, TDP-43 Clone Antibody Name Isotype chain protein 631B2A2 ACI-7069-631B2-Ab1 IgG1 κ <10 633B12C8 ACI-7069-633B12-Ab1 IgG2b κ <10 634H10H7 ACI-7069-634H10-Ab2 IgG2b κ <10 636E5B8 ACI-7069-636E5-Ab1 IgG2b λ <10 641H1E7 ACI-7069-641H1-Ab2 IgG2b κ <10 642A10B11 ACI-7069-642A10-Ab1 IgG1 κ 20 642D12B4 ACI-7069-642D12-Ab1 IgG2b κ <10 646B7F7 ACI-7069-646B7-Ab1 IgG2b κ <10 712A6B10 ACI-7071-712A6-Ab1 IgG2c κ, λ 180 809D9C2 ACI-7071-809D9-Ab2 IgG2b κ <10 809F12D8 ACI-7071-809F12-Ab1 IgG2b κ <10
Example 4: Antibody Binding to Human FL TDP-43
[0375] Antibody binding to human FL TDP-43 was determined using an indirect ELISA. ELISA plate coating with 1 μg/ml human FL TDP-43 was performed overnight in carbonate buffer at 4° C. Plates were washed with 0.05% Tween-20/PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20/PBS for 1 hour at 37° C. Antibodies purified from hybridoma supernatants were then added in a 3-fold serial dilution starting at 1 μg/ml, and incubated for 2 hours at 37° C. after which the plates were washed. An AP-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, United Kingdom) was added at 1/1000 dilution in 0.05% Tween-20/PBS for 1 hour at 37° C. After the final wash, plates were incubated with pNPP (Sigma-Aldrich, Switzerland) phosphatase substrate solution, and read at 405 nm using an ELISA plate reader (Tecan, Switzerland). All tested clones bind to full length TDP-43 with different EC50 values ranging from 10-1567 ng/ml (Table 4).
TABLE-US-00004 TABLE 4 EC50 values by ELISA Hybridoma EC50 clone name ng/ml 631B2A2 53 633B12C8 32 634H10H7 53 636E5B8 15 641H1E7 16 642A10B11 1567 642D12B4 14 646B7F7 11 712A6B10 102 809D9C2 17 809F12D8 14 2E2D3 Control 20.7
Example 5: Epitope Mapping by ELISA and Peptide Array
[0376] Antibodies purified from serum-free hybridoma supernatants were screened by an indirect ELISA assay to determine binding regions using 40-66 aa linear peptides or a library of 15-mer peptides biotinylated on N-terminus and covering the entire sequence of TDP-43 with 9 aa offset and 6 aa overlap. Peptide sequences are provided in Table 5.
[0377] 96-well plates were coated with 5 μg/ml non-biotinylated peptides overnight in carbonate buffer at 4° C.
[0378] Plates were washed with 0.05% Tween-20/PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20/PBS for 1 hour at 37° C. The antibody purified from hybridoma supernatant was then added at 1 μg/ml, and incubated for 2 hours at 37° C. after which the plates were washed. An AP-conjugated anti-mouse IgG secondary antibody (Jackson Immunoresearch Laboratories, United Kingdom) was added at 1/1000 dilution in 0.05% Tween-20/PBS for 1 hour at 37° C. After the final wash, plates were incubated with pNPP (Sigma-Aldrich, Switzerland) phosphatase substrate solution, and read at 405 nm using an ELISA plate reader (Tecan, Switzerland).
[0379] For biotinylated peptides, 96-well streptavidin-coated ELISA plates were incubated with 5 μg/mL of biotinylated, 15-mer peptides. Plates were washed 3 times with 0.05% Tween-20/PBS and then blocked with 1% bovine serum albumin (BSA) in 0.05% Tween-20/PBS for 1 hour at 37° C. The antibody purified from hybridoma supernatant was then added at 1 μg/ml and incubated for 2 hours at 37° C. after which the plates were washed. An AP-conjugated anti-mouse IgG secondary antibody (Jackson ImmunoResearch Laboratories, United Kingdom) was added at 1/1000 dilution in 0.05% Tween-20/PBS for 1 hour at 37° C. After the final wash, plates were incubated with pNPP (Sigma-Aldrich, Switzerland), an AP substrate solution, and read at 405 nm using an ELISA plate reader (Tecan).
[0380] Determined binding regions are provided in Table 6. Tested antibodies were found to bind to the following peptides: TP-21, TP-23, TP-35, TP-40, TP-48, TDP-6 corresponding respectively to regions 181-195, 199-213, 307-321, 352-366, 389-411, 140-200 of SEQ ID NO: 1.
[0381] More precise linear epitopes were mapped using a library of 15-mer peptides directly synthesized on a solid support and covering the entire sequence of TDP-43 according to SEQ ID NO:1 with 1 aa offset and 14 aa overlap (Pepscan, Netherlands). The peptide arrays were blocked with horse serum and ovalbumin and incubated with purified antibody solution at concentrations between 0.75 and 5 μg/ml overnight at 4° C. After washing, the peptide arrays were incubated with a 1/1000 dilution of rabbit anti-mouse IgG(H+L) HRP conjugate (Southern Biotech, USA) for one hour at 25° C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml of 3% H.sub.2O.sub.2 were added. After one hour, the color development was quantified with a charge coupled device (CCD)—camera and an image processing system. These binding regions were confirmed by epitope mapping and the following epitopes (provided in Table 6) were identified: aa 183-188, 203-213, 204-208, 204-211, 205-210, 316-323, 358-361, 400-405, 400-406, 400-412 of SEQ ID NO:1.
TABLE-US-00005 TABLE 5 Peptides used for determination of binding regions by ELISA Position Peptide according to number SEQ ID NO: 1 TP-21 181-195 TP-23 199-213 TP-35 307-321 TP-40 352-366 TP-48 389-411 TDP-6 140-200
TABLE-US-00006 TABLE 6 Binding regions and epitopes for tested antibodies Hybridoma clone name Binding regions, aa Epitopes, aa 631B2A2 397-411 400-406 633B12C8 397-411 400-405 634H10H7 140-200 183-188 636E5B8 352-366 358-361 641H1E7 199-213 204-211 642A10B11 389-411 400-412 642D12B4 181-195 183-188 646B7F7 199-213 205-210 712A6B10 307-321 316-323 809D9C2 199-213 203-213 809F12D8 199-213 204-208
Example 6: Detection of TDP-43 in Brain Tissues from FTD/ALS Subjects by Immunohistochemistry
[0382] Target engagement was evaluated in immunohistochemistry experiments on tissues from FTD subject brains. Human FTD brain tissues were obtained from The Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam (open access: www.brainbank.nl) and Queen Square Brain Bank for Neurological Disorders, UCL. All material has been collected from donors from whom a written informed consent for brain autopsy and the use of the material and clinical information for research purposes has been obtained by the brain bank. Immunohistochemistry was performed on 10 μm thick frozen sections using fluorescently labeled secondary antibody for detection. The following antibodies were used as controls: rabbit polyclonal pan TDP-43 antibody (Proteintech, 10782-2-AP) to detect pathological inclusions and physiological nuclear TDP-43; rabbit monoclonal phospho TDP-43 p409/410 antibody (Cosmobio, TIP-PTD-P02) to detect pathological aggregated and phosphorylated TDP-43 and secondary antibody without primary antibody (No 1º Ab) to detect non-specific background.
[0383] All antibodies of the present invention bind to nuclear, non-aggregated as well as aggregated TDP-43. Some antibodies of the present invention preferentially bind to aggregated TDP-43 in the cytoplasm in Type A pathology (
TABLE-US-00007 TABLE 7 Detection of TDP-43 in brain tissues from FTD subjects IHC detection IHC detection of aggregated of nuclear non- Antibody Name TDP-43 aggregated TDP-43 ACI-7069-631B2-Ab1 +++ +++ ACI-7069-633B12-Ab1 +++ +++ ACI-7069-634H10-Ab2 ++ ++ ACI-7069-636E5-Ab1 +/− +++ ACI-7069-641H1-Ab2 +++ + ACI-7069-642A10-ab 1 +++ + ACI-7069-642D12-Ab1 ++ + ACI-7069-646B7-Ab1 +++ +++ ACI-7071-712A6-Ab1 ++ + ACI-7071-809D9-Ab2 +++ +++ ACI-7071-809F12-Ab1 +++ +++ NA data not available; − absent; +/− not clear; +weak; ++medium; +++abundant
Example 7: Detection of TDP-43 in Brain Tissues from FTD/ALS Subjects by Western Blot
[0384] A region of brain tissue (frontal cortex) was homogenized at 1:4 (w/v) ratio in the homogenization-solubilization buffer (HS buffer) at 4° C. with precellys using CK mix homogenization tubes (Labgene, BER0092). The following sequence was used for homogenization: 3 cycles of 30 s at 5000 rpm (with 15 s pause between each cycle). Homogenized samples were aliquoted and stored at −80° C. in 1.5 ml low protein binding tubes (Axygen MCT-175-L-C). [0385] HS buffer—10 mM Tris.HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, complete EDTA-free protease inhibitors (Roche, 32524300) and PhosSTOP phosphatase inhibitors (Roche, 4906837001).
[0386] Brain homogenates were thawed on ice and resuspended in HS buffer to obtain final concentration of 2% Sarkosyl, 1 unit/μL Benzonase and 1 mM MgCl.sub.2. The samples were then incubated at 37° C. under constant shaking at 600 rpm on a thermomixer for 45 min. The supernatants were collected in a new tube. The pellet was resuspended in 1000 μl of myelin floatation buffer and centrifuged at 20,000 g for 60 min at 4° C. on the benchtop centrifuge. The supernatant was carefully removed to remove all the floating lipids. This step was repeated if all the lipids could not be removed in a single step. The pellet was subsequently washed with PBS and centrifuged for 30 min at 4° C. on the benchtop centrifuge. The final pellet was resuspended on 200 μl PBS and stored at −80° C. The samples were analyzed by immunoblotting in denaturing conditions. [0387] HS buffer with Sarkosyl, Benzonase and MgCl.sub.2— 10 mM Tris.HCl pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 4% Sarkosyl, 1 Unit/μL Benzonase (Novagen 70746-4), 4 mM MgCl.sub.2 Complete EDTA-free protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche). [0388] Myelin floatation buffer—HS buffer with 1% Triton X-100 and 30% Sucrose
[0389] Western blots were performed on Bolt 12% Bis-Tris Plus gel 1.0 mm (Thermofisher) using MES SDS running buffer (Thermofisher). Samples (30 μl/sample) were loaded on the gel once diluted in PBS, loading buffer (lx, Licor, 928-40004) containing 100 mM of DTT. Proteins were resolved under constant voltage for 100 V for 1 hour. After electrophoresis, proteins were transferred on nitrocellulose membrane (Thermofisher, IB23001) using iBLOT (Thermofisher, IB21001) at 20 volts for 7 mins. Following protein transfer, membranes were blocked for 1 hour in Licor blocking buffer (Odyssey blocking buffer 927-40000) diluted 1:3 in PBS. Membranes were incubated overnight with the following primary antibodies: total TDP-43 (Proteintech, 60019-2-Ig or 10782-2-AP), pTDP-43 (Cosmobio, TIP-PTD-M01). For primary antibodies, blocking buffer was diluted 1:1 in PBS-T (PBS with 0.4% Tween-20). After 4 washes with PBS-T (PBS with 0.1% Tween-20), membranes were incubated with secondary antibodies coupled with the LICOR dye. Secondary antibodies—donkey anti-mouse (catalog number 926-68072) or goat anti-rabbit (catalog number 926-32211)—were used at a dilution of 1:10000 in Licor blocking buffer diluted 1:1 with PBS-T (PBS with 0.4% Tween-20) for 1 hour at room temperature. The membranes were washed again 4 times with PBS-T (PBS with 0.1% Tween-20) and scanned using the LICOR system.
Example 8a: Avidity Measurements Using SPR
[0390] Binding avidity to soluble or aggregated FL TDP-43 was evaluated by determining the dissociation constants (KD) using surface plasmon resonance (SPR; Biacore T200, GE Healthcare Life Sciences). Recombinant human soluble or aggregated FL TDP-43 were immobilized on a CM5 Series S sensor chip (GE Healthcare Life Sciences) by amine coupling. Soluble TDP-43 was immobilized at a concentration of 5 μg/ml in 10 mM sodium acetate (pH 4.5) with a flow rate of 5 μl/min for 420 seconds resulting in an immobilization level of 150 RU. Aggregated TDP-43 was immobilized at a concentration of 50 μg/ml in 10 mM sodium acetate (pH 4.5) with a flow rate of 5 μl/min for 840 seconds resulting in an immobilization level of 110 RU. Biotinylated TP-73 peptide (aa 181-190 of SEQ ID NO: 1) was immobilized on a Series S Sensor Chip SA (GE Healthcare Life Sciences) at a concentration of 5 μg/ml in PBS-P.sup.+ with a flow rate of 5 μl/min for 30 seconds resulting in an immobilization level of 400 RU. To evaluate KD values, the purified antibodies and the control antibody (2E2-D3) were injected at 3-fold dilutions in PBS-P.sup.+ starting from 333 nM and dilute down to 0.15 nM. The antibodies were injected at a flow rate of 50 μl/min for 90 seconds contact time and 700 seconds dissociation phase followed by three regenerations with 10 mM glycine-HCl pH 1.7. For the optimized SPR protocol the antibodies were diluted 3-fold starting from 300 nM and dilute down to 1.2 nM and injected for 300 seconds at 30 μl/min followed by 600 seconds dissociation. The surface was regenerated by one injection with 10 mM glycine-HCl pH 1.7. Results obtained from binding kinetics were double-referenced using a blank flow cell and a buffer cycle and were evaluated with a global 1:1 fitting model with RI. Avidities for 11 antibodies and two Fab fragments are depicted in Table 8. The antibodies of the invention bind aggregated TDP-43 with a KD ranging from 0.62 nM to 4.64 nM. In addition, some antibodies show preferential binding to aggregated TDP-43 as compared with soluble TDP-43. Two Fab fragments bind to soluble TDP-43 with a KD ranging from 2.8 nM to 21.8 nM and show similar KD for aggregated TDP-43. Two antibodies (marked with *) were re-analyzed using an optimized SPR protocol with longer association and dissociation phase which allows more accurate KD determination especially for antibodies with slow dissociation rates. The two antibodies bind to soluble TDP-43 with a KD ranging from 0.22 nM to 3.9 nM and to aggregated TDP-43 with a KD ranging from 0.18 nM to 0.69 nM. The antibody ACI-7069-642D12-Ab1 binds to TP-73 peptide with KD of 3.6 nM.
TABLE-US-00008 TABLE 8 Characterization of binding by SPR Soluble TDP-43 Aggregated TDP-43 Hybridoma ka kd KD Rmax ka kd KD Rma × clone name (1/Ms) (1/s) (nM) (RU) (1/Ms) (1/s) (nM) (RU) 631B2A2 1.59E+03 7.74E−04 486 243.4 8.61E+04 7.10E−05 0.82 72 633B12C8 8.73E+02 3.19E−04 365 323.3 8.21E+04 5.01E−05 0.62 74.1 633B12C8 Fab 5.51E+04 1.46E−04 2.8 69.0 3.22E+04 2.15E-04 6.9 102.4 633B12C8* 6.18E+04 1.08E−05 0.22 107.7 8.40E+04 1.32E-05 0.18 134.6 634H10H7 4.43E+04 8.74E−04 19.7 16 1.10E+05 1.66E−04 1.51 117.9 636E5B8 1.56E+04 6.82E−04 43.84 23.3 1.13E+05 2.74E−04 2.43 80.8 641H1E7 1.25E+05 8.41E−04 6.76 50.8 1.21E+05 1.93E−04 1.6 128.7 642A10B11 NA NA NA NA 1.26E+06 5.85E−03 4.64 53.2 642D12B4 1.59E+05 8.38E−04 5.28 43.1 1.48E+05 1.31E−04 0.88 170.2 642D12B4 Fab 1.28E+05 2.67E−03 21.8 43.0 9.40E+04 1.76E-03 21.8 275.9 642D12B4* 1.15E+05 4.50E−04 3.9 74.3 1.49E+05 9.87E-05 0.69 498.7 646B7F7 1.78E+05 6.50E−04 3.66 67.3 1.44E+05 1.84E−04 1.28 148.3 712A6B10 1.34E+03 5.45E−04 406 85.7 3.48E+04 2.36E−05 0.68 29.4 809D9C2 7.71E+04 8.21E−04 10.6 47.7 9.59E+04 2.16E−04 2.25 88.4 809F12D8 9.94E+04 1.67E−04 1.68 70.6 9.47E+04 7.63E−05 0.81 132.3 NA, not applicable since less than three curves available for fit *Characterization of binding using the optimized SPR protocol on recombinantly produced IgG2a isotype antibodies.
Example 8B3: Affinity Measurements Using SPR
[0391] Binding affinity to soluble FL TDP-43 was evaluated by determining the dissociation constants (KD) using surface plasmon resonance (SPR; Biacore T200, GE Healthcare Life Sciences). Goat-anti mouse capture antibody was immobilized on a CM5 Series S sensor chip (GE Healthcare Life Sciences) by amine coupling. Antibodies were captured at a concentration of 2-5 μg/ml in PBS-P+(GE Healthcare Life Sciences) with a flow rate of 10 μl/min for 120 seconds resulting in a capture level of 350-1000 RU. To evaluate KD values, FL TDP-43 or TP-51 peptide (aa 352-414 of SEQ ID NO: 1) was injected at a flow rate of 30 μl/min in single-cycle kinetics for 300 sec contact time in 3-fold dilutions PBS-P.sup.+ starting from 1.2 nM up to 100 nM. Dissociation was recorded for 1 h followed by one regeneration with 10 mM glycine-HCl pH 1.7. Results obtained from binding kinetics were double-referenced using a blank flow cell and a buffer cycle and were evaluated with a global 1:1 fitting model with RI. On-rates (ka), off-rates (kd) and affinities (KD) for 3 antibodies are shown in Table 9 as mean values±SD of 12 (ACI-7069-633B12-Ab1), 2 (ACI-7069-642D12-Ab1) or 3 (ACI-7071-809F12-Ab1) replicates. The antibodies ACI-7069-633B12-Ab1, ACI-7069-642D12-Ab1 and ACI-7071-809F12-Ab1 bind to soluble TDP-43 with affinities ranging from 15 to 135 pM, from 226 to 272 pM and from 389 to 457 pM, respectively. The antibody ACI-7069-633B12-Ab1 binds to TP-51 peptide with affinity ranging from 1184 to 1316 pM.
TABLE-US-00009 TABLE 9 Affinities for soluble FL TDP-43 and TP-51 peptide by SPR ACI-7069-633B12-Ab1 ACI-7069-642D12-Ab1 ACI-7071-809F12-Ab1 Hybridoma ka kd KD ka kd KD ka kd KD clone name (1/Ms) (1/s) (pM) (1/Ms) (1/s) (pM) (1/Ms) (1/s) (pM) Soluble 4.39E+04 3.45E−06 75 1.10E+05 2.76E−05 249 3.20E+04 1.36E−05 423 TDP-43 Mean Soluble 5.35E+03 3.03E−06 60 8.00E+03 4.40E−06 23 2.16E+03 1.88E−06 34 TDP-43 SD TP-51 1.10E+05 1.38E−04 1250 Mean TP-51 9.41E+03 1.72E−05 66 SD
Example 9: Antibody Sequencing
[0392] Clonal hybridoma cell lysates were used for gene sequencing of the variable region. Mouse hybridomas were harvested and lysed using a lysis buffer containing guanidinium salts to deactivate RNases. Genomic DNA was then eliminated by RNase-free DNase, and RNA was purified with a silica-based affinity column using multiple washes and eluted from the column using RNase-free water. Once the RNA was extracted, its purity and concentration was measured spectrophotometrically. The integrity of the RNA was assessed on a denaturing agarose gel and RNA was reverse transcribed into cDNA using reverse transcriptase (RT). Before adding the RT reaction mixture, the RNA was heated to 70° C. for 10 min in order to disrupt RNA secondary structures. The RT products were directly used for PCR amplification. For high-fidelity PCR amplification of the cDNA, each of the variable region primers corresponding to the different gene families encoding for antibodies were individually mixed with the constant primer, for VH and VL separately in a total reaction volume of 50 μl. Initially, a degenerate primer pool was used (12 for VH and 12 for VL) and, depending on the results, a second pool was used to obtain PCR products. After the PCR reaction, the products were analyzed by gel electrophoresis on 2% agarose gels stained with ethidium bromide. The PCR products for VL and VH were individually purified on an agarose gel using tris-acetate-EDTA (TAE). The purified fragments excised from the gel were sequenced using the dye-terminator sequencing method using the same primers as those used for PCR. Sequencing was carried out in both directions to provide overlap at both ends. The sequences were analyzed using multiple sequence alignment (Clustal tool) and annotated using the algorithm of Kabat as described in Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242 (1991). Nucleotide sequences of the Heavy Chain and Light Chain Variable Domains (VH and VL) are shown in Table 10. Translated protein sequences for selected Heavy (VH) and Light (VL) Chain Variable Domains, and their complementarity-determining regions (CDRs) are shown in Table 11.
TABLE-US-00010 TABLE 10 Nucleotide sequences of the Heavy Chain and Light Chain Variable Domains (VH and VL) Antibody Hybridoma Name Code VH VL ACI- 631B2A2 GAGGTCCAGCTGCAACAGTCT GATGTTGTGATGACCCAGAC 7069- GGACCTGAACTGGTGAAGCCT TCCACTCACTTTGTCGGTTAC 631B2- GGGGCTTCAGTGAAGATATCC CATTGGACAACCAGCCTCCA Ab1 TGCAAGACTTCTGGATACACAT TCTCTTGCAAGTCAAGTCAGA TCACTGAATACTCCATACACTG GCCTCTTAAATAGTGATGGA GGTGAAACAGAGCCATGGAGA AAGACATATTTGAATTGGTTG GAGCCTTGAGTGGATTGGAGG TTACAGAGGCCAGGCCAGTC TATTAATCCTGACAATGGTGGT TCCAAAGCGCCTAATCTATCT ACTAGGTACAACCAGAAGTTC GGTGTCTAAACTGGACTCTA AAGGGCAAGGCGACATTGACT GAATCCCTGACAGGTTCACT GTAGACAAGTCCTCCAGCACA GGCAGTGGATCAGGGACAGA GCCTACATGGACCTCCGCAGC TTTCACACTGAAAATCAGCA CTGACATCTGAGGATTCTGCAG GAGTGGAGGCTGAGGATTTG TTTATTATTGTGCAAGAGAGTC GGAGTTTATTATTGCTGGCAA CTGGGGCCAAGGCACCACTCT GGTACACATTTTCCTCACACG CACAGTCTCCTCT TTCGGTTCTGGGACCAAGCTG (SEQ ID NO: 18) GAGCTGAAA (SEQ ID NO: 19) ACI- 633B12C8 GAGGTCCAGCTGCAACAGTCT GATGTTGTGATGACCCAGAC 7069- GGACCTGAACTGGTGAAGCCT TCCACTCACTTTGTCGGTTAC 633B12- GGGGCTTCAGTGAAGATATCC CATTGGACAACCAGCCTCCA Ab1 TGCAAGACTTCTGGATTCACAT TCTCTTGCAAGTCAAGTCAGA TCACTGAATACTCCATGCACTG GCCTCTTACATAGTGATGGA GGTGAAACAGAGCCATGGAAA AAGACATATTTGAATTGGTTG GAGCCTTGAGTGGATTGGAGG TTACAGAGGCCAGGCCAGTC TATTAATCCTAACAATGGTGGT TCCAAAGCGCCTAATCTATCT ACTAGCTACAACCAGAAGTTC GGTGTCTAAACTGGACTCTA AAGGGCAAGGCCACATTGACT GAATCCCTGACAGGTTCACT GTAGACAAGTCCTCCAGCACA GGCAGTGGATCAGGGACAGA GCCTACATGGAGCTCCGCAGC TTTCACACTGAAAATCAGCA CTAACATCTGAGGATTCTGCAG GAGTGGAGGCTGAGGATTTG TCTATTACTGTGCAAGAGAGTC GGAGTTTATTATTGCTGGCAA CTGGGGCCAAGGCACCACTCT GGTACACATTTTCCTCACACG CACAGTCTCCTCA TTCGGTGCTGGGACCAAGCT (SEQ ID NO: 28) GGAGCTGAAA (SEQ ID NO: 29) ACI- 634H10H7 GAGGTTCAGCTGCAGCAGTCT GACATCAAGATGACCCAGTC 7069- GGGGCAGAGCTTGTGAAGCCA TCCATCCTCCATGTATGCATC 634H10- GGGGCCTCAGTCAGGTTGTCCT GTTGGGAGAGAGAGTCACTA Ab2 GCACAGCTTCTGGCTTCAACAT TCACTTGCAAGGCGAGTCAG TAAAGACACCTATATGCACTG GACATTAAAAGCTATTTAAG GGTGAAGCAGAGGCCTGAACA CTGGTACCAGCATAAACCAT GGGCCTGGAATGGATTGGAAG GGAAATCTCCTAAGGCCCTG GATTGATCCTGCGAATAGTAAT ATCTATTATGCTACAAGCTTG ACTAAATTTGACCCGAAGTTCC GCAGATGGGGTCCCATCAAG AGGGCAAGGCCACTATAACAT ATTCAGTGGCAGTGGATCTG CAGACACATCCTCCAACACAG GGCAAGATTATTCTCTAACCA CCTACCTGCAGCTCAGCAGCCT TCAGCAGCCTGGAGTCTGAC GACATCTGAGGACACTGCCGT GATACAGCAACTTACTACTGT CTATTACTGTGCTAGATTCTAC CTACAGCAAGGTGAGAGCCC GGTGGTAGCCACTGGTACTTCG GTACACGTTCGGAGGGGGGA ATGTCTGGGGCGCAGGGACCA CCAAGCTGGAAATAAAA CGGTCACCGTCTCCTCA (SEQ ID NO: 39) (SEQ ID NO: 38) ACI- 636E5B8 GAGGTACATCTGGTGGAGTCT CAACTTGTGCTCACTCAGTCA 7069- GGGGGAGACTTAGTGATGCCT TCTTCAGCCTCTTTCTCCCTG 636E5- GGAGGGTCCCTGAAGCTCTCCT GGAGCCTCAGCAAAACTCAC Ab1 GTGCAGCCTCTGGATTCACTTT GTGCACCTTGAGTAGTCAGC CAGTAACTATGGCATGTCTTGG ACAGTACGTACACCATTGAA GTTCGCCAGACTCCAGACAAG TGGTATCAGCAACAGCCACT AGGCTGGAGTGGGTCGCAACC CAAGCCTCCTAAGTATGTGAT ATTAGTAGTGGTGGTAAATAT GGAGCTTAAGAAAGATGGAA ATCAACTACTTAGACAGTTTGA GCCACAGCACAGGTGATGGG AGGGGCGATTCACCATCTCCA ATTCCTGATCGCTTCTCTGGA GAGACAATGCCAAGAACACCC TCCAGCTCTGGTGCTGATCGC TATACCTGCAAATGAGCAGTCT TACCTTAGCATTTCCAACATC GAAGTCTGAGGATACAGCCAT CAGCCTGAAGATGAAGCAAT GTATTACTGTGCAAAAGACTA ATACATCTGTGGTGTGGGTG CGGTAGTGGCTGGGCCTGGTTT ATACAATTAAGGAACAATTT GCTTACTGGGGCCAAGGGACT GTGTATGTTTTCGGCGGTGGA CTGGTCACTGTCTCTGCA ACCAAGGTCACTGTCCTA (SEQ ID NO: 48) (SEQ ID NO: 49) ACI- 641H1E7 CAGGTGCAGCTGAAGGAGTCA GATGTTTTGATGACCCAAACT 7069- GGACCTGGCCTGGTGGCGCCC CCACTCTCCCTGCCTGTCAGT 641H1- TCACAGAGCCTGTCCATCACTT CTTGGAGATCAAGCCTCCATC Ab2 GTACTGTCTCTGGGTTTTCATT TCTTGCAGATCTAGTCAGAGC AACCAACTATGGTGTACACTG ATTGTACATACTATTGGAAAC GGTTCGCCAGCCTCCAGGAAA ACCTATTTAGAATGGTACCTG GGGTCTGGAGTGGCTGGGACT CAGAAACCAGGCCAGTCTCC AATGTGGGCTGGTGGAAGCAC AAAGCTCCTGATCTACAAAG AAATTATAATTCGGCTCTCATG TTTCCAACCGGTTTTCTGGGG TCCAGACTGAGCATCAGCAAA TCCCAGACAGGTTCAGTGGC GACAACTCCAAGAGTCAAGTT AGTGGATCAGGGACAGATTT TTCTTAAAAATGAACAGTCTGC CACACTCAAGATCAGCAGAG AAACTGATGACACAGCCATGT TGGAGGCTGAGGATCTGGGA ACTACTGTGTCATCTATAGGAC GTTTATTACTGCTTTCAAGGT GGGGTTTGCTTACTGGGGCCA TCACATGTTCCATTCACTTTC AGGGACTCTGGTCACTGTCTCT GGCTCGGGGACAAAGTTGGA GCA AATAAAA (SEQ ID NO: 68) (SEQ ID NO: 69) ACI- 642A10B11 CAGGTCCAACTGCAGCAGCCT GATGTTGTGATGACCCAGAC 7069- GGGGCTGAACTGGTGAAGCCT TCCACTCACTTTGTCGGTTAC 642A10- GGGGCTTCAGTGAAGCTGTCCT CATTGGACAACCAGCCTCCA ab1 GCAAGGCTTCTGGCTACACCTT TCTCTTGCAAGTCAAGTCAGA CACCAAGTACTGGATGCACTG GCCTCTTTGATCGTGATGGAA GGTGAAGCAGAGGCCTGGACA AGACATATTTGAATTGGTTGT AGGCCTTGAGTGGATTGGAGA TACAGAGGCCAGGCCAGTCT GATTAATCCTAGCAACGGTCGT CCAAAGCGCCTAATCTATCTG ACTAACTACAATGAGAAGTTC GTGTCTAAACTGGACTCTGG AAGAGCAAGGCCACACTGACT AGTCCCTGACAGGTTCACTG GTAGACAAATCCTCCAGCACA GCAGTGGATCAGGGACAGAT GCCTACATGCAACTCAGCAGC TTCACACTGAAAATCAGCAG CTGACATCTGAGGACTCTGCG AGTGGAGGCTGAGGATTTGG GTCTATTACTGTGCAAGATATA GAGTTTATTATTGCTGGCAAG TGGACTACTGGGGTCAAGGAA GTACACATTTTCCGTGGACGT CCTCAGTCACCGTCTCCTCA TCGGTGGAGGCACCAAGCTG (SEQ ID NO: 78) GAAATCAAA (SEQ ID NO: 79) ACI- 642D12B4 GAGGTTCAGCTGCAGCAGTCT GACATCAAGATGACCCAGTC 7069- GGGGCAGAGCTTGTGAAGCCG TCCATCCTCCATGTATGCATC 642D12- GGGGCCTCAGTCAGGTTGTCCT GTTGGGAGAGAGAGTCACTA Ab1 GCACAGCTTCTGGCTTCAACAT TCACTTGCAAGGCGAGTCAG TAAAGACCCCTATATGCACTG GACATTAAAAGGTATTTAAG GGTCAGGCAGAGGCCTAAACA CTGGTACCAGCAGAAACCAT GGGCCTGGAGTGGATTGGAAG GGAAATCTCCTAAGATCCTG GATTGATCCTGCGGATGGTAAT ATCTATTATGCAACAAGCTTG ACTAAATATGACCCGAAGTTC GCAGATGGGGTCCCATCAAG CAGGGCAAGGCCACTTTAACA ATTCAGTGGCACTGGATCTG GCAGACACATCCTCCAATGTA GACAAGATTATTCTCTAACCA GCCTACCTGCACCTCAGCAGCC TCAGCAGCCTGGAGTCTGAC TGACATCTGAGGACACTGCCG GATGTAGCAACTTACTACTGT TCTATTACTGTGCTAGATTCTA CTACAGCAAGGTGAGAGCCC CGGTAGTAGCCACTGGTATTTC GTACACGTTCGGAGGGGGGA GATGTGTGGGGCGCAGGGACC CCAAGCTGGAAATAAAA ACGGTCACCGTCTCCTCA (SEQ ID NO: 89) (SEQ ID NO: 88) ACI- 646B7F7 CAGGTGCAACTGAAGGAGTCA GATGTTTTGATGACCCAAACT 7069- GGACCTGGCCTGGTGGCGCCC CCACTCTCCCTGCCTGTCAGT 646B7- TCACAGAGCCTGTCCATCACTT CTTGGAGATCAAGCCTCCATC Ab1 GTACTGTCTCTGGATTTTCATT TCTTGCAGATCTAGTCAGAGC AACCAACTTTGGTGTACACTGG ATTGTACATGCTATTGGAAAC GTTCGCCAGCCTCCAGGAAAG ACCTATTTAGAATGGTACCTG GGTCTGGAGTGGCTGGGAATA CAGAAACCAGGCCAGTCTCC ATGTGGGCTGGTGGAAGCACA AAAGCTCCTGATCTACAAAG AATTATAATTCGGCTCTCATGT TTTCCAACCGGTTTTCTGGGG CCAGACTGAGCATCAGCAAAG TCCCAGACAGGTTCAGTGGC ACAACTCCAAGAGTCAAGTTTT AGTGGATCAGGGACAGATTT CTTAAAAATGAACAGTCTCCA CACACTCAAGATCAGCAGAG AACTGATGACACAGCCATGTA TGGAGGCTGAGGATCTGGGA CTACTGTGTCATCTATAAGACG GTTTATTACTGCTTTCAAGGT GGGTTTGCTTACTGGGGCCAA TCACATGTTCCATTCACGTTC GGGACTCTGGTCACTGTCTCTG GGCTCGGGGACAAAGTTGGA CA AATAAAA (SEQ ID NO: 108) (SEQ ID NO: 109) ACI- 712A6B10 CAGGTCCAGCTGCAGCAGTCT GATGTTGTGATGACCCAGATT 7071- GGAGCTGAGCTGGTGAAACCC CCACTCACTTTGTCGATTACC 712A6- GGGACATCAGTGAAGCTGTCC ATTGGACAACCAGCCTCCAT Ab1 TGTAAGGCTTCTGCCTACACCT CTCTTGCAAGTCAAGTCAGA TCACTGAATATACTATACACTG GCCTCTTACCTAGTGATGGAA GATAAAGCAGAAATCTGGACA AGACATATTTGAATTGGTTGT GGGTCTTGAGTGGATTGGGTG TACAGAGGCCAGGCCAGTCT GTTTCACCCTGAAAATGATAAT CCAAAGCGCCTAATCTATCTG ATAAAGTACAATGAGAATTTC GTGTCTAAACTGGACTCTGG AAGGACAAGGCCACATTGACT AGTCCCTGACAGGTTCACTG GCGGACAGATCCTCCAGCACA GCAGTGGATCAGGGACAGAT GTCTATATGGAACTTAGTAGAT TTCACACTGAAAATCAGCAG TGACATCTGAAGACTCTGCGGT AGTGGAGGCTGACGATTTGG CTATTTCTGTGCAGGGACGTCA GAGTTTATTATTGCTGGCAAG GGCTACGGAGACTACTGGGGC GTACACATTTTCCTCCTACGT CAAGGCACCACTCTCACAGTCT TCGGTGCTGGGACCAAGCTG CTTCA GAACTGAAA (SEQ ID NO: 128) (SEQ ID NO: 129) ACI- 809D9C2 CAGGTCCAGCTGCAGCAGTCT GATATTGTGATGACTCAGGCT 7071- GGGGCTGAGCTGGTGAGGCCT GCACCCTCTATACCTGTCACT 809D9- GGGGTCTCAGTGAAGATTTCCT CCTGGAGAGTCAGTATCCAT Ab2 GCAAGGGTTCTGGCTACAAAT CTCCTGCAGGTCTAGTAAGA TCACTGATTATTCTATGCACTG GTCTCCTGCATAGTAATGGCA GGTGAAACAGAGTCATACAAA ACACTTACTTGTATTGGTTCC GAGTCTAGAGTGGATTGGAGT TGCAGAGGCCAGGCCAGTCT TATTAGTACTTACTATGGTGAT CCTCAGCTCCTGATATATCGG ACTACCTACAACCAGAAATTC ATGTCCAACCTTGCCTCAGGA AAGGGCAAGGCCACAATCACT GTCCCAGACAGGTTCAGTGG GTAGACAAATCCTCCAGCACA CAGTGGGTCAGGAACTGCTT GCCTATATGGAACTTGCCAGA TCACACTGAGAATCAGTAGA CTGACATCTGAGGATTCTGCCA GTGGAGGCTGAGGATGTGGG TCTATTACTGTGCAACGTACGG TGTTTATTACTGTATGCAACA TAACTTCCCGGCCTCATTTTCT TCTAGAATATCCATTCACGTT TACTGGGGCCAAGGGACTCTG CGGCTCGGGGACAAAGTTGG GTCACTGTCTCTGCA AAATAAAA (SEQ ID NO: 148) (SEQ ID NO: 149) ACI- 809F12D8 CAGGTGCAGCTGAAGGAGTCA GATGTTTTGATGACCCAAACT 7071- GGACCTGGCCTGGTGGCGCCC CCACTCTCCCTGCCTGTCAGT 809F12- TCACAGAGCCTGTCCATCACTT CTTGGAGATCAGGCCTCCATC Ab1 GCACTGTCTCTGGGTTTTCGTT TCTTGCAGATCTAGTCAGAAC AAACAGAAATGGTGTACAGTG ATTGTACATAGTATTGGAAA GGTTCGCCAGCCTCCAGGAAA CACCTATTTAGAGTGGTACCT GGGTCTGGAGTGGCTGGGAGT GCAGAAACCAGGCCAGTCTC AATATGGCCTGGCGGAAGCAC CAAAGCTCCTGATCTACAAA AAATTGTAATTCGGCTCTCATG GTTTCCAACCGATTTTCTGGG TCCAGACTGAGCATCAGCAAA GTCCCAGACAGGTTCAGTGG GACAACTCCAAGAGTCAAGTT CAGTGGATCAGGGACAGATT TTCTTAAAAATGAACAGTCTGC TCACACTCAAGATCAGCAGA ACACTGATGACACAGGCATAT GTGGAGGCTGAGGATCTGGG ATTACTGTGCCAGAGTAGGGG AGTTTATTACTGCTTTCAAGG GTAACTACGTGTGGGACTATA TTCACATGTTCCGTACACGTT ATAACTACGCCTGGGGCCAAG CGGAGGGGGGACCAAGCTAG GGACTCTGGTCACTGTCTCTGC AAATAAGA A (SEQ ID NO: 159) (SEQ ID NO: 158)
TABLE-US-00011 TABLE 11 Amino acid sequences of the Heavy Chain and Light Chain Variable Domains (VH and VL) and their CDRs VH VH VH VL VL VL Antibody Hybridoma CDR CDR CDR CDR CDR CDR name code VH 1 2 3 VL 1 2 3 ACI- 631B2A2 EVQLQQSG EYSI GIN ES DVVMTQ KSS LVS WQ 7069- PELVKPGA H PDN TPLTLSV QSL KLD GTH 631B2- SVKISCKTS (SEQ GGT TIGQPASI LNS S FPH Ab1 GYTFTEYSI ID RYN SCKSSQS DGK (SEQ T HWVKQSH NO: QKF LLNSDGK TYL ID (SEQ GESLEWIG 11) KG TYLNWL N NO: ID GINPDNGG (SEQ LQRPGQS (SEQ 16) NO: TRYNQKFK ID PKRLIYL ID 17) GKATLTVD NO: VSKLDSR NO: KSSSTAYM 12) IPDRFTG 15) DLRSLTSE SGSGTDF DSAVYYC TLKISRV ARESWGQ EAEDLG GTTLTVSS VYYCWQ (SEQ ID NO: GTHFPHT 10) FGSGTKL ELK (SEQ ID NO: 14) ACI- 633B12C8 EVQLQQSG EYS GIN ES DVVMTQ KSS LVS WQ 7069- PELVKPGA MH PNN TPLTLSV QSL KLD GTH 633B12- SVKISCKTS (SEQ GGT TIGQPASI LHS S FPH Ab1 GFTFTEYS ID SYN SCKSSQS DGK (SEQ T MHWVKQS NO: QKF LLHSDGK TYL ID (SEQ HGKSLEWI 21) KG TYLNWL N NO: ID GGINPNNG (SEQ LQRPGQS (SEQ 16) NO: GTSYNQKF ID PKRLIYL ID 27) KGKATLTV NO: VSKLDSR NO: DKSSSTAY 22) IPDRFTG 25) MELRSLTS SGSGTDF EDSAVYYC TLKISRV ARESWGQ EAEDLG GTTLTVSS VYYCWQ (SEQ ID NO: GTHFPHT 20) FGAGTKL ELK (SEQ ID NO: 24) ACI- 634H10H7 EVQLQQSG DTY RID FYG DIKMTQS KAS YAT LQQ 7069- AELVKPGA MH PAN GSH PSSMYAS QDI SLA GES 634H10- SVRLSCTA (SEQ SNT WYF LGERVTI KSY D PYT Ab2 SGFNIKDT ID KFD DV TCKASQ LS (SEQ (SEQ YMIHWVKQ NO: PKF (SEQ DIKSYLS (SEQ ID ID RPEQGLEW 31) QG ID WYQHKP ID NO: NO: IGRIDPANS (SEQ NO: WKSPKA NO: 36) 37) NTKFDPKF ID 33) LIYYATS 35) QGKATITS NO: LADGVPS DTSSNTAY 32) RFSGSGS LQLSSLTSE GQDYSLT DTAVYYC ISSLESDD ARFYGGSH TATYYCL WYFDVWG QQGESPY AGTTVTVS TFGGGTK S LEIK (SEQ ID NO: (SEQ ID 30) NO: 34) ACI- 636E5B8 EVHLVESG NYG TISS DYG QLVLTQS TLS GSH GVG 7069- GDLVMPG MS GGK SGW SSASFSL SQH STG DTI 636E5- GSLKLSCA (SEQ YIN AWF GASAKLT STY D KEQ Ab1 ASGFTFSN ID YLD AY CTLSSQH TIE (SEQ FVY YGMSWVR NO: SLK (SEQ STYTIEW (SEQ ID V QTPDKRLE 41) G ID YQQQPL ID NO: (SEQ WVATISSG (SEQ NO: KPPKYV NO: 46) ID GKYINYLD ID 43) MELKKD 45) NO: SLKGRFTIS NO: GSHSTGD 47) RDNAKNTL 42) GIPDRFS YLQMSSLK GSSSGAD SEDTAMY RYLSISNI YCAKDYG QPEDEAI SGWAWFA YICGVGD YWGQGTL TIKEQFV VTVSA YVFGGG (SEQ ID NO: TKVTVL 40) (SEQ ID NO: 44) ACI- 641H1E7 QVQLKESG NYG LM YRT DVLMTQ RSS KVS FQG 7069- PGLVAPSQ VH WA GFA TPLSLPV QST NRF SHV 641H1- SLSITCTVS (SEQ GGS Y SLGDQAS VHT S PFT Ab2 GFSLTNYG ID TNY (SEQ ISCRSSQS IGN (SEQ (SEQ VHWVRQP NO: NSA ID IVHTIGN TYL ID ID PGKGLEWL 61) LMS NO: TYLEWY E NO: NO: GLMWAGG (SEQ 63) LQKPGQS (SEQ 66) 67) STNYNSAL ID PKLLIYK ID MSRLSISK NO: VSNRFSG NO: DNSKSQVF 62) VPDRFSG 65) LKMNSLQT SGSGTDF DDTAMYY TLKISRV CVIYRTGF EAEDLG AYWGQGT VYYCFQ LVTVSA GSHVPFT (SEQ ID NO: FGSGTKL 60) EIK (SEQ ID NO: 64) ACI- 642A10B1 QVQLQQPG KY EINP YMD DVVMTQ KSS LVS WQ 7069- 1 AELVKPGA WM SNG Y TPLTLSV QSL KLD GTH 642A10- SVKLSCKA H RTN (SEQ TIGQPASI FDR S FPW ab1 SGYTFTKY (SEQ YNE ID SCKSSQS DGK (SEQ T WMHWVK ID KFK NO: LFDRDG TYL ID (SEQ QRPGQGLE NO: S 73) KTYLNW N NO: ID WIGEINPSN 71) (SEQ LLQRPGQ (SEQ 16) NO: GRTNYNEK ID SPKRLIY ID 77) FKSKATLT NO: LVSKLDS NO: VDKSSSTA 72) GVPDRFT 75) YMQLSSLT GSGSGTD SEDSAVYY FTLKISR CARYMDY VEAEDL WGQGTSV GVYYCW TVSS QGTHFP (SEQ ID NO: WTFGGG 70) TKLEIK (SEQ ID NO: 74) ACI- 642D12B4 EVQLQQSG DPY RID FYG DIKMTQS KAS YAT LQQ 7069- AELVKPGA MH PAD SSH PSSMYAS QDI SLA GES 642D12- SVRLSCTA (SEQ GNT WYF LGERVTI KRY D PYT Ab1 SGFNIKDP ID KYD DV TCKASQ LS (SEQ (SEQ YMHWVRQ NO: PKF (SEQ DIKRYLS (SEQ ID ID RPKQGLE 81) QG ID WYQQKP ID NO: NO: WIGRIDPA (SEQ NO: WKSPKIL NO: 86) 87) DGNTKYDP ID 83) IYYATSL 85) KFQGKATL NO: ADGVPSR TADTSSNV 82) FSGTGSG AYLIALSSL QDYSLTI TSEDTAVY SSLESDD YCARFYGS VATYYC SHWYFDV LQQGESP WGAGTTV YTFGGGT TVSS KLEIK (SEQ ID NO: (SEQ ID 80) NO: 84) ACI- 646B7F7 QVQLKESG NFG IMW YKT DVLMTQ RSS KVS FQG 7069- PGLVAPSQ VH AGG GFA TPLSLPV QSI NRF SHV 646B7- SLSITCTVS (SEQ STN Y SLGDQAS VHA S PFT Ab1 GFSLTNFG ID YNS (SEQ ISCRSSQS IGN (SEQ (SEQ VHWVRQP NO: ALM ID IVHAIGN TYL ID ID PGKGLEWL 101) S NO: TYLEWY E NO: NO: GIMWAGG (SEQ 103) LQKPGQS (SEQ 106) 107) STNYNSAL ID PKLLIYK ID MSRLSISK NO: VSNRFSG NO: DNSKSQVF 102) VPDRFSG 105) LKMNSLQT SGSGTDF DDTAMYY TLKISRV CVIYKTGF EAEDLG AYWGQGT VYYCFQ LVTVSA GSHVPFT (SEQ ID NO: FGSGTKL 100) EIK (SEQ ID NO: 104) ACI- 712A6B10 QVQLQQSG EYTI WFH TSG DVVMTQ KSS LVS WQ 7071- AELVKPGT H PEN YGD IPLTLSITI QSL KLD GTH 712A6- SVKLSCKA (SEQ DNI Y GQPASIS LPS S FPPT Ab1 SAYTFTEY ID KYN (SEQ CKSSQSL DGK (SEQ (SEQ TIHWIKQK NO: ENF ID LPSDGKT TYL ID ID SGQGLEWI 121) KD NO: YLNWLL N NO: NO: GWFHPEN (SEQ 123) QRPGQSP (SEQ 16) 127) DNIKYNEN ID KRLIYLV ID FKDKATLT NO: SKLDSGV NO: ADRSSSTV 122) PDRFTGS 125) YMELSRLT GSGTDFT SEDSAVYF LKISRVE CAGTSGYG ADDLGV DYWGQGT YYCWQG TLTVSS THFPPTF (SEQ ID NO: GAGTKL 120) ELK (SEQ ID NO: 124) ACI- 809D9C2 QVQLQQSG DYS VIST YGN DIVMTQ RSS RMS MQH 7071- AELVRPGV MH YYG FPA AAPSIPV KSL NLA LEY 809D9- SVKISCKG (SEQ DTT SFS TPGESVSI LHS S PFT Ab2 SGYKFTDY ID YNQ Y SCRSSKS NGN (SEQ (SEQ SMHWVKQ NO: KFK (SEQ LLHSNGN TYL ID ID SHTKSLEW 141) G ID TYLYWF Y NO: NO: IGVISTYYG (SEQ NO: LQRPGQS (SEQ 146) 147) DTTYNQKF ID 143) PQLLIYR ID KGKATITV NO: MSNLAS NO: DKSSSTAY 142) GVPDRFS 145) MELARLTS GSGSGTA EDSAIYYC FTLRISR ATYGNFPA VEAEDV SFSYWGQG GVYYCM TLVTVSA QHLEYPF (SEQ ID NO: TFGSGTK 140) LEIK (SEQ ID NO: 144) ACI- 809F12D8 QVQLKESG RNG VIW VGG DVLMTQ RSS KVS FQG 7071- PGLVAPSQ VQ PGG NYV TPLSLPV QNI NRF SHV 809F12- SLSITCTVS (SEQ STN WD SLGDQAS VHS S PYT Ab1 GFSLNRNG ID CNS YNN ISCRSSQ IGN (SEQ (SEQ VQWVRQP NO: ALM YA NIVHSIG TYL ID ID PGKGLEWL 151) S (SEQ NTYLEW E NO: NO: GVIWPGGS (SEQ ID YLQKPG (SEQ 156) 157) TNCNSALM ID NO: QSPKLLI ID SRLSISKDN NO: 153) YKVSNRF NO: SKSQVFLK 152) SGVPDRF 155) MNSLHTD SGSGSGT DTGIYYCA DFTLKIS RVGGNYV RVEAEDL WDYNNYA GVYYCF WGQGTLV QGSHVP TVSA YTFGGGT (SEQ ID NO: KLEIR 150) (SEQ ID NO: 154)
Example 10: In Vivo Efficacy of ACI-7069-633B12-Ab1 (IgG2a Variant) in Transgenic Mouse Model of TDP-43 Proteinopathies
[0393] To evaluate the efficacy of ACI-7069-6331B12-Ab1 (IgG2a variant) in vivo, the ability of ACI-7069-6331B12-Ab1 (IgG2a variant) to reduce TDP-43 pathology in NEFH-tTA×hTDP-43ΔNLS bigenic mice (rNLS8 mice, Walker et al. 2015) was tested. The rNLS8 mice were injected weekly with ACI-7069-6331B12-Ab1 (IgG2a variant) (n=30) or vehicle (n=30) and at the end of dosing, molecular pathological markers such as phosphorylated and/or total insoluble TDP-43 were analyzed.
10.1 Animals
[0394] Prior to the initiation of the study, all animals were acclimated to the environment, examined, handled and weighed to ensure adequate health and to minimize non-specific stress associated with experimental manipulation. Mice were kept on chow diet which contained doxycycline (200 mg/kg) during breeding and until 8 weeks of age. At 8 weeks of age the diet was changed to a chow diet not containing doxycycline (DOX) to allow transgene expression. Throughout the study, light/dark cycles (12/12), room temperature (20-23° C.) and relative humidity (around 50%), were kept constant. Chow diet and water were provided ad libitum for the duration of the study. When mice started to display movement difficulties, the diet was changed to wet chow and hydrogel on the cage floor. All behavioral tests were performed during the animal's light cycle phase.
10.2. Compound Administration
[0395] On the day of injection, ACI-7069-633B12-Ab1 (IgG2a variant) (60 mg/kg) and vehicle were freshly prepared and administered i.p following a weekly dosing regimen throughout the study.
10.3. Collection of Brains
[0396] Brains were divided into two hemispheres. The left hemisphere was dissected to collect cortical brain areas. Mouse cortices and remaining brain tissue were flash-frozen for further biochemical analyses. The remaining right hemispheres were immersion-fixed directly after perfusion for 3 hours at room temperature and collected in freshly prepared 1×PBS containing 4% paraformaldehyde (PFA).
10.4. Imunohistochemistry
[0397] Immersion fixed right brain hemispheres were cut sagittally in a uniform, systematic random protocol on a Leica CM1950 cryotome at a section thickness of 10 microns. Systematic random sets of sagittal sections (7 sections from levels 2, 3, 4, 6, 8, 10 and 11 of the brain) per mouse were immunostained for TDP-43 and phosphorylated TDP-43. Iba1 staining was performed to quantify microglial numbers and morphology in brain. Antibody binding was visualized using fluorescently labeled secondary antibodies. Standard negative controls included wild type brain sections as well as sections from transgenic animals without the application of primary antibodies.
10.5. Imaging and Determination of Immunoreactivity
[0398] Mounted sections were imaged as a whole on an Axio.Scan Z1 slide scanner driven by ZEN software at 10× magnification using LED (Colibri2) illumination and a sensitive Orca Flash 4.0 monochromatic camera. Brain size was determined using separate delineation of the regions of interest in the cerebral cortex and dorsal striatum. Object density (OD) (in number of objects per mm.sup.2) was determined for all markers, labeled area percentage and OD relative to the region of interest size of a second delineation excluding any tissue artifacts (tissue folds, etc.).
10.6. Preparation of Protein Samples from Brain Cortex:
[0399] Tissues were thawed on ice and then sonicated in 5× v/w radioimmunoprecipitation assay buffer (RIPA, 50 mM Tris, 150 mM NaCl, 1% IGEPAL CA630, 5 mM EDTA, 0.5% sodium deoxycholate and 0.1% SDS, pH 8.0) containing 1 mM PMSF and a protease-/phosphatase inhibitor cocktail (Roche Applied Science). Samples were centrifuged at 4° C., 100,000 g for 30 minutes and the supernatant was considered as-soluble fraction. The pellet was washed by sonicating with RIPA and the supernatant was discarded. The RIPA-insoluble pellet was sonicated in 2× v/w urea buffer (7M urea, 2M thiourea, 4% CHAPS, and 30 mM Tris, pH8.5) and centrifuged at 22° C., 100,000 g for 30 minutes. This supernatant was considered as the RIPA-insoluble/urea-soluble fraction. Protein concentrations of the RIPA-soluble fractions were determined using BCA protein assay (Pierce).
10.7. Quantification of Insoluble TDP-43
[0400] Total TDP-43 levels in the RIPA insoluble fraction were analyzed by a commercial human TDP-43 AlphaLISA kit (Perkin Elmer, AL387HV).
10.8. Statistical Analysis
[0401] IHC and AlphaLISA data are presented as mean±SEM. Statistical differences between vehicle and ACI-7069-633B12-Ab1 (IgG2a variant) treated animals are analyzed by Welch's t-tests and are indicated by asterisks above respective bars (*p<0.05, **p<0.01, ****p<0.0001). Outliers in histological measurements were excluded either being Grubbs outlier in the group or level (single measurements), or due to technical reasons (image artifacts, tissue folds, etc.).
10.9. Results
[0402] Treatment with ACI-7069-633B12-Ab1 (IgG2a variant) reduces phosphorylated TDP-43 and insoluble TDP-43 in rNLS8 mice Overexpression of the DOX repressible form of K82A/R83A/K84A mutant human TDP-43 (hTDP-43ANLS) leads to a prominent accumulation and aggregation of TDP-43 in the cytoplasm of neurons in rNLS8 mice model. A pathological hallmark of this model are the deposition of insoluble and phosphorylated TDP-43 inclusions (pTDP-43). These small, spherical cytoplasmic inclusions are solely present in the transgenic animals and are entirely absent in WT or monogenic, transgenic tTA control mice. Moreover, pTDP-43 is widely absent during the 1st week of DOX absence, and accumulated with steep progression during the 3-4-weeks DOX removal (Walker et al., 2015). ACI-7069-633B12-Ab1 (IgG2a variant) treatment led to a statiscally significant reduction in the density of phosphorylated TDP-43 in both striatum and cerebral cortex (
10.10. Treatment with ACI-7069-633B12-Ab1 (IgG2a Variant) Reduces Insoluble TDP-43 in rNLS8 Mice
[0403] To confirm the reduction of TDP-43 pathology observed in immunohistochemistry readouts, the amount of total insoluble/aggregated TDP-43 in the brain, following biochemical fractionation, was quantified. RIPA-insoluble fractions were prepared from the cortex of left brain hemispheres containing insoluble/aggregated TDP-43. A significant reduction in the amount of insoluble TDP-43 was observed in mice treated with ACI-7069-633B12-Ab1 (IgG2a variant) compared to that of vehicle treated animals (
10.11. ACI-7069-633B12-Ab1 (IgG2a Variant) Treatment in rNLS8 Mice Increases Microglial Immunoreactive Area
[0404] Functional recovery in rNLS8 mice following suppression of transgene expression involves increase in microglial activity. Microglial cell body area increases in this phase and results in clearance of TDP-43 pathology and functional recovery of motor deficits suggesting a therapeutic paradigm in rNLS8 mouse model (Spiller K. J et al., Nature Neuroscience, 2018).
[0405] To evaluate the mode of action of ACI-7069-633B12-Ab1 in reducing TDP-43 pathology in rNLS8 mice, its effect on microglial activation was assessed. Iba1 staining was performed by immunohistochemistry to quantify the number and state of microglia in cerebral cortex of mice. Microgliosis was found in rNLS8 mice at terminal stage (5 weeks off Dox). ACI-7069-633B12-Ab1 treatment significantly increased Iba1 positive immunoreactive area in cortex compared to vehicle-treated control (
[0406] Next, the effect of ACI-7069-633B12-Ab1 on microglial morphology was evaluated. To correlate the increase in Iba1 immunoreactive area to changes in microglial activation states representing morphology, microglia were classified into three states based on their size and morphology (large hypertrophic, small ramifying and ramified resting). A significant increase in mean cell size was seen for large hypertrophic microglia in ACI-7069-633B12-Ab1 (IgG2a variant) treatment compared to vehicle-treated control (
Example 11: In Vitro Functionality of ACI-7069-633B12-Ab1 (IgG2a Variant) in Recombinant TDP-43 Aggregation Assay
[0407] To evaluate functionality of ACI-7069-633B12-Ab1 (IgG2a variant) in vitro, the ability of ACI-7069-633B12-Ab1 (IgG2a variant) to inhibit TDP-43 aggregation was tested. FL TDP-43 was fused at C-terminus to maltose binding protein (MBP) which was separated by a Tobacco Etch Virus (TEV) protease cleavage site and produced recombinantly. Aggregation of 2.5 μM TDP-43-TEV-MBP fusion protein in 30 mM Tris, 150 mM NaCl, pH 7.4 in the presence of 2.5 μM ACI-7069-633B12-Ab1 (IgG2a variant) or isotype control that does not bind to TDP-43 was induced by addition of TEV protease (AcTEV, Invitrogen) and absorbance was monitored in a μclear 96 well plate (Greiner) at 600 nm over 30 h. For evaluation, end points were normalized to isotype control and the percentage of aggregated TDP-43 was calculated for ACI-7069-633B12-Ab1. The antibody ACI-7069-633B12-Ab1 significantly inhibits TDP-43 aggregation by 98% compared to the isotype control (
Example 12: Detection and Quantification of TDP-43 in Biofluids with ACI-7069-633B12-Ab1
[0408] (IgG2a variant) and ACI-7071-809F12-Ab1 (IgG2a variant) Method: PerkinElmer's bead-based AlphaLISA immunoassay was established using ACI-7069-633B12-Ab1 (IgG2a variant) and ACI-7071-809F12-Ab1 (IgG2a variant). For CSF samples, dilution linearity was established in spike recovery experiments. The concentration of TDP-43 was then measured in diluted CSF samples. Samples were prepared in white Optiplate™-384 microplate and the emission at 615 nm was measured as raw AlphaLISA counts.
[0409] Results: Total TDP-43 in cerebrospinal fluid (CSF) samples from healthy control and FTLD-TDP (Semantic Dementia, C9orf72 or GRN) patients was quantified in this immunoassay (
Example 13: Binding to Pathological TDP-43 Assessed by Immunodepletion in FTD Brain Extracts
[0410] To evaluate the efficacy of antibodies in specifically binding TDP-43 aggregates in native state, immunodepletion experiments in brain extracts with enriched pathological TDP-43 were performed.
[0411] Method: Insoluble fractions from FTD type A (FTD-A) postmortem brains were prepared as described in Example 7. Immunodepletion was performed using Dynabeads™ magnetic beads, Protein G (Thermoscientific 10003D). After resuspension in the tube, 130 μl of beads were transferred to a 1.5 ml low binding tube. Beads were rinsed twice with PBS supplemented with 0.05% Tween-20 using a magnet to remove supernatant. Beads were split equally in three different low binding tubes. Antibodies (ACI-7069-633B12-Ab1 (IgG2a isotype), ACI-7069-642D12-Ab1 (IgG2a isotype), mouse IgG2a control) were diluted to 100 μg/ml and 100 μl was added to each tube after removing supernatant (using magnet). Antibody-beads mix was incubated at room temperature for 1 hour. The beads-antibodies complex were washed once with 500 μl PBS-0.05% Tween-20 and once with PBS, then resuspended in 250 μl PBS. Antibody-beads were split into two new tubes (120 μl per tube). Insoluble fractions were thawed on ice and sonicated for 30 seconds at amplitude 30 on ice. Thirty micrograms of brain material was added to each antibody-beads tube after removing supernatant and incubated at 4° C. overnight under continuous rotation. Tubes were placed on the magnet and the supernatant was collected as the immunodepleted fraction. Input and immunodepleted material were further analyzed by Western Blot. Western Blots were performed as described in example 7. Twenty μl of samples were loaded per lane. Immunoblotting was performed using the following antibodies: total TDP-43 (ACI-7069-633B12-Ab1 coupled to DyLight680), pTDP-43 (Biolegend, 829901) used at dilutions of 1:2000 and 1:1000 respectively. Goat anti-rat secondary antibody (catalog number 925-32219) was used at a dilution of 1:10000.
[0412] Results: ACI-7069-633B12-Ab1 and ACI-7069-642D12-Ab1 were able to specifically bind and deplete TDP-43 and pTDP-43 from sarkosyl insoluble fractions obtained from FTD type A brain tissue compared to isotype control antibody (
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